Cell preparation for treating viral pneumoniaTechnical Field
The invention belongs to the field of biotechnology and biological medicine, and relates to a method for treating viral pneumonia, in particular to a method for treating viral pneumonia by using a cell preparation prepared from mesenchymal stem cells, a cell preparation prepared from mesenchymal stem cells and application of the cell preparation in treating viral pneumonia.
Background
Pneumonia, especially viral pneumonia, is a lung inflammation caused by upper respiratory virus infection and spreading downwards. The disease can occur all the year round, but is mostly seen in winter and spring, and can be outbreak or sporadic and epidemic. Clinically, it is mainly manifested as fever, headache, general soreness, dry cough and lung infiltration. Viral pneumonia occurs in association with virulence of the virus, the route of infection, the age of the host, the immune function status, and the like. The incidence rate of children is generally higher than that of adults.
In acute respiratory tract infection, the virus infection accounts for 90%, while the virus infection is mainly the upper respiratory tract, and comprises common cold, pharyngitis, laryngotracheobronchitis, bronchiolitis, infant herpangina, epidemic chest pain, etc. Viruses causing pneumonia are not common, among which influenza viruses are common, others are parainfluenza viruses, cytomegaloviruses, adenoviruses, rhinoviruses, coronaviruses and certain enteroviruses, such as coxsackie, eka viruses, etc., and viruses such as herpes simplex, varicella-zoster, rubella, measles, etc. Infants are also frequently given pneumonia by respiratory syncytial virus infection. Viral pneumonia occurs in winter and spring, and can develop epidemic or outbreak. In non-bacterial pneumonia, viral infection accounts for 25% -50%, most patients are children, and adults are relatively rare.
In recent years, because immunosuppressive drugs are widely used for tumor and organ transplantation, and the number of people suffering from AIDS is increased year by year, herpes simplex virus, varicella-zoster virus, cytomegalovirus and the like can cause serious pneumonia. Viral pneumonia is an inhalational infection, is mainly caused by downward spreading of upper respiratory tract virus infection through human-to-human droplet infection, often accompanies tracheobronchitis, livestock such as horses, pigs and the like sometimes carry some influenza virus, and is occasionally infected by contact. Faeces are orally infected with enteroviruses and respiratory syncytial viruses are transmitted by dust. Cases of organ transplantation can be caused by multiple blood transfusions, even with donor organs. Viral pneumonia with bleeding is not accompanied by tracheobronchitis.
Viral pneumonia is generally lighter in clinical manifestations, similar to symptoms of mycoplasma pneumonia. Slow onset of disease, headache, debilitation, fever, cough with little sticky sputum. Signs are often deficient. X-ray examination shows that the lung inflammation is in the form of spots, flakes or uniform shadows. The total number of leukocytes may be normal, reduced or slightly increased. The course of the disease is generally 1-2 weeks. In immunocompromised patients, viral pneumonia is often severe, with persistent hyperpyrexia, palpitations, shortness of breath, cyanosis, extreme failure, with concomitant shock, heart failure and azotemia. Respiratory distress syndrome can occur in severe cases due to alveolar interstitium and intra-alveolar edema. The physical examination can be performed by using a wet-sound-producing method. X-ray examination showed diffuse nodular infiltration, which is most commonly seen in the two lower 2/3 lung fields.
Viral pneumonia is diagnosed based on clinical symptoms and X-ray changes, excluding pneumonia caused by other pathogens. The diagnosis depends on etiology examination, including virus isolation, serological examination, and detection of viral antigens. Inclusion of the nucleus in the respiratory secretions may indicate viral infection, but not necessarily from the lungs, requiring further collection of lower respiratory secretions or lung biopsy specimens for culture isolation of the virus. The usual methods for serological examination are detection of specific IgG antibodies, such as complement fixation assays, hemagglutination inhibition assays, neutralization assays, but are only available as retrospective diagnostics and are of no early diagnostic value.
The existing treatment methods for viral pneumonia are still limited, so that the research and development of safe and effective new clinical treatment schemes are always hot spots and difficult problems in the field.
Mesenchymal stem cells (mesenchymal stem cells, MSCs) are a heterogeneous group of cells derived from a matrix, available from most tissues in humans. A large number of experimental researches show that the mesenchymal stem cells have the differentiation potential of the epidermal cells and can promote the healing of the wounded skin. The marrow-derived mesenchymal stem cells have the hidden trouble of virus pollution, and the cell number and the expansion and differentiation capacity of the marrow-derived mesenchymal stem cells obviously decrease along with the age of a donor, so that the marrow-derived mesenchymal stem cells are not suitable for batch preparation. While the mesenchymal stem cells of human umbilical cord, placenta or amniotic membrane successfully avoid the limitations of embryo stem cell source deficiency, allograft rejection, ethics and the like; they have self-renewal, tissue repair, immunomodulation, can differentiate towards mesodermal lineages, for example into adipocytes, osteocytes, chondrocytes, etc., and, in addition, towards cells of other germ lineages, for example into epidermal cells, vascular endothelial cells; in addition, the cells are easy to expand in vitro, and the differentiation capacity and proliferation capacity after expansion are kept stable, so that the method is suitable for large-scale preparation. In recent years, preliminary research results at home and abroad show that MSCs have biological effects of treating viral pneumonia, which may bring about great transformation for clinical treatment of viral pneumonia.
Techniques for obtaining stem cells from placenta or umbilical cord sources are well established. Numerous methods for preparing mesenchymal stem cells from human placenta or umbilical cord have been disclosed in the prior art, for example, CN109481466a (chinese application No. 201811568010X) describes a detailed placental mesenchymal stem cell acquisition method, CN102660497B (chinese patent No. ZL 2012101599162) describes a detailed umbilical cord mesenchymal stem cell acquisition method, the entire contents of which are incorporated herein by reference.
Although attempts to treat viral pneumonia using mesenchymal stem cells have been reported, these methods still have their limitations. Accordingly, it would be highly desirable to provide a new method of treating viral pneumonia using mesenchymal stem cells more effectively.
Disclosure of Invention
The present invention aims to provide a mesenchymal stem cell preparation prepared from peritock tissue-derived mesenchymal stem cells, particularly placenta-derived mesenchymal stem cells, and the preparation of the mesenchymal stem cells has been unexpectedly found to be capable of significantly improving physiological changes of the lung by intravenous injection of the mesenchymal stem cell preparation into a virus-infected viral pneumonia mouse. The present invention has been completed based on such findings.
To this end, the first aspect of the present invention provides a cell preparation comprising placental mesenchymal stem cells and a pharmaceutically acceptable vehicle.
The cell preparation according to the first aspect of the present invention, wherein the placental mesenchymal stem cells are prepared by a method comprising the steps of:
(1) Treatment of placenta lobules: placing placenta in a white porcelain dish, washing with tissue washing liquid to remove placenta blood stasis, shearing 20g placenta lobule tissue in a steel cup, washing twice with tissue washing liquid, soaking for 5min, and weighing 15g better tissue in a 100mm glass dish; adding 10ml of tissue washing liquid, and cutting the leaflets to 0.2cm3 Adding 100ml of tissue washing liquid, stirring, filtering with 300 mesh sieve, and repeating the operation to wash twice with tissue washing liquid to remove blood cells; the tissue cleaning solution is 0.9% physiological saline containing 1% diabody;
(2) Mixed enzyme digestion and termination: adding the washed lobule tissue into 15-30 ml of mixed enzyme digestion solution preheated at 37 ℃ for fully and uniformly mixing, shaking and digesting for 30min at 100rpm at 37 ℃ by a shaking table, and adding 2ml of FBS into the tissue solution after digestion is finished to terminate digestion;
(3) Collecting primary cells: adding 50ml of tissue washing liquid into the tissue liquid obtained in the previous step, uniformly mixing, filtering with 300 meshes, and collecting cell liquid; washing the digested tissue repeatedly twice, combining the two filtrates into a centrifuge tube, and centrifuging at 1500rpm for 8min; removing the supernatant, adding a proper amount of tissue washing liquid for resuspension, supplementing to 200ml, and centrifuging at 1500rpm for 8min; removing the supernatant, re-suspending the cell sediment to 30ml by adding DMEM-F12, filtering by a 100um filter screen, and then flushing the filter screen by 10ml of DMEM-F12 to obtain 40ml of cell suspension serving as primary cells;
(4) Primary cell cryopreservation: centrifuging at 1800rpm for 10min, collecting cell precipitate and 5ml of the lower liquid, slowly adding 10ml of frozen stock solution after re-suspending, and shaking; subpackaging the obtained cell suspension into 9 2ml freezing tubes, wherein each tube is 1.5ml, placing the tube into a precooled program cooling box, using a program cooling instrument to carry out program cooling, and transferring the cells into a liquid nitrogen storage tank for freezing;
(5) Cell resuscitation: taking 2 tubes of frozen cells, quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, and adding 8ml of complete culture medium for drip recovery; centrifuging at 1200rpm for 5min, removing supernatant, and adding 5ml of complete culture medium for resuspension; each tube of cells was inoculated into 1T 75 flask, supplemented with complete medium to 30ml, and placed in a CO2 incubator at 37℃with 5% CO2 Culturing under saturated humidity; the whole culture medium is used for changing liquid once every 3-4 days, and after 12 days of recovery, the liquid is counted according to the clone formation condition until the cell density is not less than 3000 cells/cm2 The subsequent passage can be carried out; the complete medium is DMEM-F12 medium containing 10% fbs;
(6) Cell passage: taking P0 generation cells, washing with PBS, adding 2ml pancreatin to digest for 2-5min until most of the cells fall off, adding 5ml complete culture medium to stop digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min, discarding the supernatant, adding 5ml complete culture medium to resuspension, counting and inoculating to a culture bottle, wherein the cell density is 8000-12000 cells/cm2 Placing in a CO2 incubator at 37deg.C with 5% CO2 Culturing under saturated humidity until the cell density reaches more than 90%, and completing the passage of cells from the generation P0 to the generation P1; and repeating the operations in sequence to respectively carry out the passage of the cells from the generation P1 to the generation P2, the generation P2 to the generation P3, the generation P3 to the generation P4 and the generation P4 to the generation P5, so as to obtain the mesenchymal stem cells of each generation.
The cell preparation according to the first aspect of the present invention, wherein in the step (2), the mixed enzyme digestion solution comprises: 15-30 volumes of Hank' S balanced salt solution, 0.2-0.6 volumes of Liberase MNP-S enzyme and 0.2-2 volumes of DNA I type enzyme.
The cell preparation according to the first aspect of the present invention, wherein in the step (4), the formula of the frozen stock solution is: 65% of DMEM-F12, 15% of human serum albumin and 20% of DMSO.
The cell preparation according to the first aspect of the present invention, wherein in the process of preparing mesenchymal stem cells, further comprising:
(7) Detecting at least one of the following items for the placenta mesenchymal stem cells obtained in the step (6): cell activity, cell contamination, genetic disease, HLA-ABC/DR ligand.
The cell preparation according to the first aspect of the present invention, wherein in the process of preparing mesenchymal stem cells, further comprising:
(8) Freezing and storing the placenta mesenchymal stem cells of each generation after the passage obtained in the step (6) in liquid nitrogen; and
(9) Creating a database of placental stem cells comprising the information detected in step (7) above, and correlating the database with the cryopreserved cells of step (8).
The cell preparation according to the first aspect of the present invention, wherein the cell purity of each generation of placental mesenchymal stem cells obtained in the process of preparing mesenchymal stem cells is greater than 90%.
The cell preparation according to the first aspect of the present invention, wherein the Hank's balanced salt solution consists of: the pH was adjusted to 7.4 with 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4.7H2O, 0.1g/L MgSO2.6H2O, 0.06g/L Na2HPO4.2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35g/L NaHCO3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide.
The cell preparation according to the first aspect of the present invention, wherein the mixed enzyme digestion solution comprises Hank' S balanced salt solution, liberase MNP-S enzyme, and DNA type I enzyme, and 0.2-0.3 g/L zinc chloride is added.
The cell preparation according to the first aspect of the present invention, wherein the pharmaceutically acceptable vehicle is water.
The cell preparation according to the first aspect of the invention is a preparation for use in an injection mode.
The cell preparation according to the first aspect of the present invention, wherein the placental mesenchymal stem cells are cells subcultured to 1 to 20 passages, for example, to 1 to 10 passages, for example, to 1 to 5 passages.
The cell preparation according to the first aspect of the present invention, wherein the concentration of the placental mesenchymal stem cells is (0.5 to 10) ×106 And each ml.
The cell preparation according to the first aspect of the present invention, wherein the concentration of the placental mesenchymal stem cells is (0.5 to 5). Times.106 And each ml.
The cell preparation according to the first aspect of the present invention, wherein the concentration of the placental mesenchymal stem cells is (1 to 3) ×106 And each ml.
The cell preparation according to the first aspect of the present invention, further comprising sodium chloride.
The cell preparation according to the first aspect of the present invention further comprises sodium chloride at a concentration of 7 to 10mg/ml.
The cell preparation according to the first aspect of the present invention further comprises sodium chloride at a concentration of 7.5 to 9.5mg/ml.
The cell preparation according to the first aspect of the present invention further comprises sodium chloride at a concentration of 8 to 9mg/ml.
The cell preparation according to the first aspect of the present invention, further comprising chlorogenic acid.
The cell preparation according to the first aspect of the present invention further comprises chlorogenic acid at a concentration of 0.2 to 2mg/ml.
The cell preparation according to the first aspect of the present invention further comprises chlorogenic acid at a concentration of 0.3 to 1.5mg/ml.
The cell preparation according to the first aspect of the present invention further comprises chlorogenic acid at a concentration of 0.5 to 1mg/ml.
The cell preparation according to the first aspect of the present invention, further comprising sorbitol.
The cell preparation according to the first aspect of the present invention further comprises sorbitol at a concentration of 2 to 10mg/ml.
The cell preparation according to the first aspect of the present invention further comprises sorbitol at a concentration of 2.5 to 7.5mg/ml.
The cell preparation according to the first aspect of the present invention further comprises sorbitol at a concentration of 3 to 5mg/ml.
The cell preparation according to the first aspect of the invention, having a formulation according to any one of the embodiments of the invention.
In the present invention, sodium chloride, chlorogenic acid, sorbitol, etc. may be referred to as pharmaceutical excipients.
The cell preparation according to the first aspect of the invention is prepared according to a method comprising the steps of: dissolving sodium chloride (and pharmaceutical excipients such as chlorogenic acid and sorbitol) with proper amount of water to obtain saline solution; transferring the mesenchymal stem cells obtained by subculture into a centrifuge tube, centrifuging (for example, centrifuging at 2000rpm for 5 min), discarding the supernatant, and adding saline solution to resuspend the cells to a specified cell concentration to obtain a cell preparation.
The cell preparation according to the first aspect of the present invention is a cell preparation for use in the treatment of pneumonia, such as viral pneumonia.
Further, the second aspect of the present invention relates to the use of a cell preparation comprising placental mesenchymal stem cells and a pharmaceutically acceptable vehicle for the manufacture of a medicament for the treatment of pneumonia, e.g. viral pneumonia.
The use according to the second aspect of the present invention, wherein said placental mesenchymal stem cells are prepared by a process comprising the steps of:
(1) Treatment of placenta lobules: placing placenta in a white porcelain dish, washing with tissue washing liquid to remove placenta blood stasis, shearing 20g placenta lobule tissue in a steel cup, washing twice with tissue washing liquid, soaking for 5min, and weighing 15g better tissue in a 100mm glass dish; adding 10ml of tissue washing liquid, and cutting the leaflets to 0.2cm3 Adding 100ml of tissue washing liquid, stirring, filtering with 300 mesh sieve, and repeating the operation to wash twice with tissue washing liquid to remove blood cells; the tissue cleaning solution is 0.9% physiological saline containing 1% diabody;
(2) Mixed enzyme digestion and termination: adding the washed lobule tissue into 15-30 ml of mixed enzyme digestion solution preheated at 37 ℃ for fully and uniformly mixing, shaking and digesting for 30min at 100rpm at 37 ℃ by a shaking table, and adding 2ml of FBS into the tissue solution after digestion is finished to terminate digestion;
(3) Collecting primary cells: adding 50ml of tissue washing liquid into the tissue liquid obtained in the previous step, uniformly mixing, filtering with 300 meshes, and collecting cell liquid; washing the digested tissue repeatedly twice, combining the two filtrates into a centrifuge tube, and centrifuging at 1500rpm for 8min; removing the supernatant, adding a proper amount of tissue washing liquid for resuspension, supplementing to 200ml, and centrifuging at 1500rpm for 8min; removing the supernatant, re-suspending the cell sediment to 30ml by adding DMEM-F12, filtering by a 100um filter screen, and then flushing the filter screen by 10ml of DMEM-F12 to obtain 40ml of cell suspension serving as primary cells;
(4) Primary cell cryopreservation: centrifuging at 1800rpm for 10min, collecting cell precipitate and 5ml of the lower liquid, slowly adding 10ml of frozen stock solution after re-suspending, and shaking; subpackaging the obtained cell suspension into 9 2ml freezing tubes, wherein each tube is 1.5ml, placing the tube into a precooled program cooling box, using a program cooling instrument to carry out program cooling, and transferring the cells into a liquid nitrogen storage tank for freezing;
(5) Cell resuscitation: taking 2 tubes of frozen cells, quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, and adding 8ml of complete culture medium for drip recovery; centrifuging at 1200rpm for 5min, removing supernatant, and adding 5ml of complete culture medium for resuspension; each tube of cells was inoculated into 1T 75 flask, supplemented with complete medium to 30ml, and placed in a CO2 incubator at 37℃with 5% CO2 Culturing under saturated humidity; the whole culture medium is used for changing liquid once every 3-4 days, and after 12 days of recovery, the liquid is counted according to the clone formation condition until the cell density is not less than 3000 cells/cm2 The subsequent passage can be carried out; the complete medium is DMEM-F12 medium containing 10% fbs;
(6) Cell passage: washing P0 generation cells with PBS, adding 2ml pancreatin for digestion for 2-5min until most of the cells fall off, adding 5ml complete medium for stopping digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min, discarding the supernatant, and adding 5ml complete mediumCounting and inoculating to culture flask after suspending, and cell density is 8000-12000 cells/cm2 Placing in a CO2 incubator at 37deg.C with 5% CO2 Culturing under saturated humidity until the cell density reaches more than 90%, and completing the passage of cells from the generation P0 to the generation P1; and repeating the operations in sequence to respectively carry out the passage of the cells from the generation P1 to the generation P2, the generation P2 to the generation P3, the generation P3 to the generation P4 and the generation P4 to the generation P5, so as to obtain the mesenchymal stem cells of each generation.
The use according to the second aspect of the present invention, wherein in the step (2) of preparing the mesenchymal stem cells, the mixed enzyme digestion solution comprises: 15-30 volumes of Hank' S balanced salt solution, 0.2-0.6 volumes of Liberase MNP-S enzyme and 0.2-2 volumes of DNA I type enzyme.
The use according to the second aspect of the present invention, wherein in the step (4) of preparing the mesenchymal stem cells, the formulation of the cryopreservation solution is: 65% of DMEM-F12, 15% of human serum albumin and 20% of DMSO.
The use according to the second aspect of the present invention, wherein in the process of preparing mesenchymal stem cells, further comprising:
(7) Detecting at least one of the following items for the placenta mesenchymal stem cells obtained in the step (6): cell activity, cell contamination, genetic disease, HLA-ABC/DR ligand.
The use according to the second aspect of the present invention, wherein in the process of preparing mesenchymal stem cells, further comprising:
(8) Freezing and storing the placenta mesenchymal stem cells of each generation after the passage obtained in the step (6) in liquid nitrogen; and
(9) Creating a database of placental stem cells comprising the information detected in step (7) above, and correlating the database with the cryopreserved cells of step (8).
According to the use of the second aspect of the present invention, wherein in the process of preparing the mesenchymal stem cells, the cell purity of each generation of the placental mesenchymal stem cells obtained is more than 90%.
The use according to the second aspect of the invention, wherein the Hank's balanced salt solution consists of: the pH was adjusted to 7.4 with 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4.7H2O, 0.1g/L MgSO2.6H2O, 0.06g/L Na2HPO4.2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35g/L NaHCO3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide.
The use according to the second aspect of the present invention, wherein the mixed enzyme digestion solution comprises Hank' S balanced salt solution, liberase MNP-S enzyme, DNA type I enzyme, and zinc chloride in an amount of 0.2-0.3 g/L.
The use according to the second aspect of the invention, wherein the pharmaceutically acceptable vehicle in the cell preparation is water.
The use according to the second aspect of the invention, wherein the cell preparation is a preparation for use by injection.
The use according to the second aspect of the present invention, wherein said placental mesenchymal stem cells are cells subcultured to 1 to 20 passages, for example to 1 to 10 passages, for example to 1 to 5 passages.
The use according to the second aspect of the present invention, wherein the concentration of said placental mesenchymal stem cells in said cell preparation is (0.5-10) ×106 And each ml.
The use according to the second aspect of the present invention, wherein the concentration of said placental mesenchymal stem cells in said cell preparation is (0.5-5) ×106 And each ml.
The use according to the second aspect of the present invention, wherein the concentration of said placental mesenchymal stem cells in said cell preparation is (1-3) ×106 And each ml.
The use according to the second aspect of the invention, wherein the cell preparation further comprises sodium chloride.
The use according to the second aspect of the present invention, wherein the cell preparation further comprises sodium chloride at a concentration of 7 to 10mg/ml.
The use according to the second aspect of the invention, wherein the cell preparation further comprises sodium chloride at a concentration of 7.5 to 9.5mg/ml.
The use according to the second aspect of the present invention, wherein the cell preparation further comprises sodium chloride at a concentration of 8 to 9mg/ml.
The use according to the second aspect of the invention, wherein chlorogenic acid is also comprised in the cell preparation.
The use according to the second aspect of the present invention, wherein the cell preparation further comprises chlorogenic acid in a concentration of 0.2 to 2mg/ml.
The use according to the second aspect of the present invention, wherein the cell preparation further comprises chlorogenic acid in a concentration of 0.3 to 1.5mg/ml.
The use according to the second aspect of the present invention, wherein the cell preparation further comprises chlorogenic acid in a concentration of 0.5 to 1mg/ml.
The use according to the second aspect of the invention, wherein sorbitol is also comprised in the cell preparation.
The use according to the second aspect of the present invention, wherein the cell preparation further comprises sorbitol in a concentration of 2 to 10mg/ml.
The use according to the second aspect of the invention, wherein the cell preparation further comprises sorbitol in a concentration of 2.5-7.5 mg/ml.
The use according to the second aspect of the present invention, wherein the cell preparation further comprises sorbitol in a concentration of 3 to 5mg/ml.
The use according to the second aspect of the invention, wherein the cell preparation has a formulation according to any one of the embodiments of the invention.
The use according to the second aspect of the invention, wherein the cell preparation is prepared according to a method comprising the steps of: dissolving sodium chloride (and pharmaceutical excipients such as chlorogenic acid and sorbitol) with proper amount of water to obtain saline solution; transferring the mesenchymal stem cells obtained by subculture into a centrifuge tube, centrifuging (for example, centrifuging at 2000rpm for 5 min), discarding the supernatant, and adding saline solution to resuspend the cells to a specified cell concentration to obtain a cell preparation.
Of the various operating steps described above, although specific steps are described herein as being distinguished in some details or language description from those described in the preparation examples of the detailed description section below, those skilled in the art can readily generalize the method steps described above based on the detailed disclosure of the invention as a whole.
Any of the embodiments of any of the aspects of the invention may be combined with other embodiments, provided that they do not contradict. Furthermore, in any of the embodiments of any of the aspects of the present invention, any technical feature may be applied to the technical feature in other embodiments as long as they do not contradict. The present invention is further described below.
All documents cited herein are incorporated by reference in their entirety and are incorporated by reference herein to the extent they are not inconsistent with this invention. Furthermore, various terms and phrases used herein have a common meaning known to those skilled in the art, and even though they are still intended to be described and explained in greater detail herein, the terms and phrases used herein should not be construed to be inconsistent with the ordinary meaning in the sense of the present invention.
In the present invention, the term "placental mesenchymal stem cells" refers to placental derived mesenchymal stem cells. Thus, in the present invention, and in particular in relation to the present invention, the term "placental mesenchymal stem cells" may be used interchangeably with "placental stem cells", "mesenchymal stem cells", unless specifically indicated otherwise.
In the present invention, the term "PBS buffer" or "PBS" refers to phosphate buffer. The general formulation and method of formulation of the PBS used in the context of the present invention and their general properties such as pH or pH range are well known to those skilled in the art, and these PBS buffers are typically commercially available pre-formulations (or pre-powders), e.g., PBS used in the field of the present invention is typically a commercial buffer of pH7.4 (±0.1), e.g., hyClone brand PBS buffer; the typical PBS buffer composition used in this field includes 137mM sodium chloride, 2.7nM potassium chloride and 10mM phosphate, and the composition of the PBS used in this invention is that described herein unless otherwise specified.
In the present invention, the term "placenta" refers to a neonatal placenta, in particular to a placenta within 4 hours after delivery.
The mesenchymal stem cells are adult stem cells with self-replication and multidirectional differentiation potential, have the advantages of easy separation, culture and amplification, low immunogenicity, no expression of a type II main tissue compatibility complex (MHC), can be used in a variant, have strong migration and immunoregulation capacity, promote tissue injury repair and regeneration in a paracrine mode, and are ideal seed cells for regenerative medicine.
The viral pneumonia is described in detail in Han Xudong (Han Xudong, et al, viral pneumonia, journal of doctor's repair (department edition), 2004, 27 (2): 12). Viral pneumonia is often present as an inhalation infection, which is transmitted by droplets and intimate contact, and may be caused by the downward spread of upper respiratory viral infection, and may also be secondary to eruptive viral infection, often accompanied by tracheal-bronchial infection. Influenza virus is the most common pathogen of adult and elderly viral pneumonia, and infant viral pneumonia is often caused by respiratory syncytial virus infection. Other viruses such as parainfluenza virus, cytomegalovirus, coronaviruses, adenoviruses, rhinoviruses and certain enteroviruses such as coxsackie, epstein barr virus and the like can also cause viral pneumonia. In the non-bacterial pneumonia, the viral pneumonia accounts for 25% -50%, is frequently generated in winter and spring, can be sporadic or epidemic, and is frequently seen in infants, the elderly and patients with the original chronic cardiopulmonary diseases. In recent years, because immunosuppressive drugs are widely applied to organ transplantation patients and the number of people suffering from AIDS is increased, the incidence rate of viral pneumonia is gradually increased, and the epidemic of SARS makes the viral pneumonia particularly important. The clinical manifestations of general viral pneumonia are mostly slight, similar to mycoplasma pneumonia symptoms, and the disease course is 1-2 weeks. Severe pneumonia may be associated with persistent hyperpyrexia, palpitations, shortness of breath, dyspnea, cyanosis, and may also be accompanied by shock and respiratory failure. In viral pneumonia, in addition to direct damage to the body caused by viruses, autoimmune damage also plays an important role in viral diseases. The invasion of viruses into bronchiole epithelium can cause bronchiolitis, infection, and pneumonia caused by pulmonary interstitial and alveoli. Congestion and bleeding of lesion parts; a strong inflammatory response involving monocytes occurs. Fibrin-containing monocytes, and occasionally polymorphonuclear leukocytes, may be present in the alveoli, and in severe cases the hyaline membrane may be present, resulting in a severe impairment of alveolar diffuse function. Adenovirus, cytomegalovirus, respiratory syncytial virus or varicella-zoster virus can see characteristic intracellular viral inclusion bodies. After absorption of the lesions, fibrosis or nodular calcification may remain. The lung and immune organs are the main target organs for SARS virus attack. Early changes in the lung are desquamative alveolar inflammatory changes, and convalescence is an organized glomerular pneumonitis change; after absorption of the lesions, lung fibrosis of varying degrees may be left behind.
The present invention uses classical animal tests in the art to verify the biological effects of the cell preparations of the present invention in the use of pneumonia, such as viral pneumonia, and to present satisfactory results, which provide a solid basis for the effectiveness of clinical applications.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein.
CN109481466a (chinese application No. 201811568010X), the entire contents of which are incorporated herein by reference, describes a detailed placental mesenchymal stem cell acquisition method. The reagents for the chemical reagents such as chlorogenic acid used in the present invention are commercially available.
Example 1, placental whole cell treatment:
1. pre-preparing mixed synthase digestion liquid: 22ml of HBSS (Hank' S balanced salt solution) containing calcium and magnesium ions, 0.4ml of Liberase MNP-S enzyme (for example, from Xibao organism, cat# 5578582001) and 0.7ml of DNA type I enzyme were respectively removed from the tube, zinc chloride (added at a concentration of 0.2g/L, 0.25g/L or 0.3 g/L) was added to the tube, and the mixture was homogenized, and the mixture was preheated at 37℃for 20 minutes or more. The Hank's balanced salt solution consists of: the pH was adjusted to 7.4 with 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4.7H2O, 0.1g/L MgSO2.6H2O, 0.06g/L Na2HPO4.2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35g/L NaHCO3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide.
2. Preparation of placenta lobules: placenta was taken out of the collection bag and placed in a white porcelain dish, after washing with tissue washing solution, the placenta blood stasis was removed, and a small amount of placenta lobular tissue (about 20 g) was cut out in a steel cup. Tissue washing liquid (0.9% normal saline and double antibody (the double antibody is the green streptomycin and the content is 1%) is used for washing twice, and after soaking for 5min, 15 g+/-1 g of better tissue is weighed in a 100mm glass dish.
3. Removal of blood cells: adding 10ml of tissue washing liquid, and cutting the leaflets to 0.2cm3 Adding 100ml tissue washing liquid to the left and right sizes, stirring, filtering with 300 mesh filter screen, and washing twice with tissue washing liquid (after each time, moving lobule tissue into steel cup, adding 100ml tissue washing liquid, stirring, filtering with 300 mesh).
4. Mixed enzyme digestion and termination: the washed leaflet tissue was added to the preheated 23ml of the mixed enzyme digestion solution and thoroughly mixed, and then the leaflet tissue was digested by shaking at 37℃of a shaker at 100rpm for 30 minutes. After digestion, interstitial fluid+2 ml FBS was terminated.
5. Primary cell collection:
diluting and mixing the tissue fluid with 50ml tissue washing liquid, filtering with 300 meshes, collecting cell fluid, washing digested tissue twice (50 ml tissue washing liquid is used each time), mixing the filtrate into 1 250ml centrifuge tube, centrifuging at 1500rpm for 8min (acceleration 9, deceleration 7);
Removing the supernatant, adding a proper amount of tissue washing liquid to resuspend and supplement to 200ml, and centrifuging at 1500rpm for 8min (acceleration 9, deceleration 7);
removing the supernatant, re-suspending the cell sediment to 30ml by adding DMEM-F12, filtering by a 100um filter screen, and then flushing the filter screen by 10ml of DMEM-F12 to obtain 40ml of cell suspension serving as primary cells; 1ml of the suspension is taken and used for cell counting by a symex blood analyzer, and the purity of the primary cells is high and the content of the mesenchymal stem cells is about 60-70 percent.
6. Primary cell cryopreservation:
the formula of the existing frozen stock solution is as follows: 65% DMEM-F12, 15% Human Serum Albumin (HSA), 20% DMSO such as WAK brand DMSO, ready to use;
centrifuging the cell suspension at 1800rpm for 10min (acceleration 9 and deceleration 7), collecting cell precipitate and 5ml of the supernatant (10 ml of the supernatant is used for sample), slowly adding the prepared frozen stock solution after re-suspending, and shaking uniformly while adding;
the cell suspension was dispensed into 9 2ml cryopreservation tubes, 1.5ml per tube (pre-chilled with a temperature programmed box). Remaining cell suspension + leave-on supernatant for sterile detection;
and (3) performing program cooling by using a program cooling instrument, transferring the cells into a liquid nitrogen storage tank, and freezing the obtained primary cells.
Through the whole placenta cell treatment process of the above example 1, a full-term placenta sample is selected, 15g of placenta lobular tissue at a specific position is cut, digested by the mixed enzyme digestive juice system to obtain cells, and purified to obtain a group of purer primary mesenchymal stem cells (CD 73 is expressed to be more than 60% and CD45 is not expressed), wherein the content of the mesenchymal stem cells in the primary cells reaches 60% -70%, and the number of the primary cells obtained per gram of placenta lobular tissue can reach (2.4-2.8) multiplied by 107 And the yield is stable, and the sample specificity is greatly reduced.
The cell yield after placenta tissue treatment was very stable in this example, and typical data for some experiments are shown in Table 1 of CN 109481466A.
In addition, the results of the flow phenotype identification of the primary cells obtained after the placenta treatment show that the expression of CD73 is up to more than 60%, and CD45 is not expressed, which indicates that the primary cells are a group of relatively pure mesenchymal stem cells and do not contain blood cells. Typical flow phenotyping results can also be seen in figure 1 of CN109481466 a.
EXAMPLE 2 Primary cell resuscitationAnd subculture
1. Cell resuscitation:
taking 2 tubes of frozen cells, quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, and adding 8ml of complete culture medium for drip recovery; as used herein, complete medium is DMEM-F12 medium containing 10% fbs, unless otherwise specified;
Then, after centrifugation at 1200rpm for 5min (acceleration 9, deceleration 7), the supernatant was removed and 5ml of complete medium was added for resuspension; each tube of cells was inoculated into 1T 75 flask, supplemented with complete medium to 30ml, and incubated in a CO2 incubator (37 ℃,5% CO2, saturated humidity); the whole culture medium is used for changing liquid once every 3-4 days, and after 12 days of recovery, the liquid is counted according to the clone formation condition until the cell density is not less than 3000 cells/cm2 The subsequent passage can be carried out;
2. cell passage: washing the recovered P0 generation cells with PBS, adding 2ml pancreatin to digest for 2-5min until most of the cells fall off, adding 5ml complete culture medium to stop digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min (acceleration 9 and deceleration 7), discarding the supernatant, adding 5ml complete culture medium to resuspension, counting and inoculating to a culture flask, wherein the cell density is 8000-12000 cells/cm2 Culturing in a CO2 incubator (37 ℃ C., 5% CO2, saturated humidity) until the cell density reaches more than 90% (usually about 5 days), and completing the passage of cells from the generation P0 to the generation P1;
and repeating the passage operation from the generation P0 to the generation P1 in sequence to respectively carry out the passage of the cells from the generation P1 to the generation P2, the generation P2 to the generation P3, the generation P3 to the generation P4 and the generation P4 to the generation P5, so as to obtain the mesenchymal stem cells of each generation.
In this example the primary seed density was about 5X 105 cells/cm2 The inoculation has many adherent cells in 4 days of microscopic examination field, and is in spindle shape. The seeds can be transferred to the generation P1 after 10 days of inoculation. The cell growth speed is high, the quantity is large, the morphology is fusiform, and the cell is full. Typical results of cell counts for some experiments during passage from P0 to P1 are shown in table 2 and fig. 2 of CN109481466 a.
In addition, during the P1-P5 inoculation passage, the culture is usually carried out for 4-5 days, i.e. the harvest and passage to the next generation.
In this experiment, the P1-P5 generation flow phenotype was identified, and the results showed that positive expression of CD73, CD90 and CD105 was all >98%, while CD34, CD45, CD19 and HLA-DR were identified, and typical results are shown in Table 3 and FIG. 4 of CN109481466A, which demonstrated that isolated and cultured cells in placenta were mesenchymal stem cells and were high in purity.
In addition, the growth cycle was measured on P5 generation cells of some samples, and the results showed that cells in G2 phase <1%, cells in S phase >10%, demonstrated that these cells were strong in proliferation capacity, did not enter into the division phase, and typical results are shown in table 4 and figures of CN109481466 a.
Example 3 biological characterization of placental MSCs
Biological characterization of placental mesenchymal stem cells was performed by reference to the methods [0062] to [0089] of the already-issued patent CN102676451a (chinese patent application No. 201210044648. X), and the results show that MSCs isolated by the method of the present invention have the ability to differentiate into osteoblasts, adipocytes, chondrocytes, confirming that MSCs obtained by the method of the present invention have stem cell characteristics. For example, the induction differentiation test of P5 generation cells is exemplified, and the results show that these cells have the ability to differentiate into osteoblasts, adipogenic cells, chondrogenic cells. A micrograph of typical adipogenic differentiation, osteogenic differentiation, and chondrogenic differentiation is shown in FIG. 6 of CN 109481466A.
In the case of the cell preparation of the present invention, the amount per 1ml is not less than 50ml per batch in actual preparation, unless otherwise specified. In the examples of the present invention for preparing cell preparations, stem cells are obtained by the methods of examples 1-2 herein, unless otherwise specified. In the context of the present invention, the preparation of mesenchymal stem cells (i.e. the cell preparation) is carried out under aseptic conditions.
EXAMPLE 11a preparation of mesenchymal Stem cells
Mesenchymal stem cells (P4 generation): 2X 106 The number of the two-dimensional space-saving type,
sodium chloride: 8.5mg of the total weight of the composition,
chlorogenic acid: 0.75mg of the total amount of the components,
sorbitol: 4mg of the total weight of the composition,
water, in an amount of 1ml.
The preparation method comprises the following steps: dissolving sodium chloride, chlorogenic acid and sorbitol with proper amount of water to obtain saline solution; the mesenchymal stem cells obtained by passaging the cells in step 2 of example 2 were transferred into a centrifuge tube, centrifuged at 2000rpm for 5min, the supernatant was discarded, and the cells were resuspended to a prescribed cell concentration by adding a saline solution to prepare a cell preparation.
Example 11a1 preparation of mesenchymal Stem cells
The cell preparation of this example 11a1 was obtained with reference to the formulation and preparation of example 11a, except that sorbitol was not added.
Example 11a2 preparation of mesenchymal Stem cells
The cell preparation of example 11a2 was obtained by referring to the formulation and preparation of example 11a, except that chlorogenic acid was not added.
Example 11a3 preparation of mesenchymal Stem cells
The cell preparation of this example 11a3 was obtained with reference to the formulation and preparation of example 11a, except that neither sorbitol nor chlorogenic acid was added.
EXAMPLE 11b preparation of mesenchymal Stem cell preparation
Mesenchymal stem cells (P5 generation): 1X 106 The number of the two-dimensional space-saving type,
sodium chloride: 9mg of the extract of the plant,
chlorogenic acid: 1.5mg of the total amount of the,
sorbitol: 2.5mg of the total weight of the composition,
water, in an amount of 1ml.
The preparation method comprises the following steps: dissolving sodium chloride, chlorogenic acid and sorbitol with proper amount of water to obtain saline solution; the mesenchymal stem cells obtained by passaging the cells in step 2 of example 2 were transferred into a centrifuge tube, centrifuged at 2000rpm for 5min, the supernatant was discarded, and the cells were resuspended to a prescribed cell concentration by adding a saline solution to prepare a cell preparation.
Example 11c preparation of mesenchymal Stem cell preparation
Mesenchymal stem cells (P3 generation): 3X 106 The number of the two-dimensional space-saving type,
sodium chloride: 8.5mg of the total weight of the composition,
chlorogenic acid: 0.3mg of the total amount of the,
sorbitol: 7.5mg of the total weight of the powder,
water, in an amount of 1ml.
The preparation method comprises the following steps: dissolving sodium chloride, chlorogenic acid and sorbitol with proper amount of water to obtain saline solution; the mesenchymal stem cells obtained by passaging the cells in step 2 of example 2 were transferred into a centrifuge tube, centrifuged at 2000rpm for 5min, the supernatant was discarded, and the cells were resuspended to a prescribed cell concentration by adding a saline solution to prepare a cell preparation.
EXAMPLE 11d preparation of mesenchymal Stem cell preparation
Mesenchymal stem cells (P1 generation): 1.5X106 The number of the two-dimensional space-saving type,
sodium chloride: 8mg of the extract, which is obtained by mixing,
chlorogenic acid: 1mg of the extract of the plant,
sorbitol: 3mg of the total amount of,
water, in an amount of 1ml.
The preparation method comprises the following steps: dissolving sodium chloride, chlorogenic acid and sorbitol with proper amount of water to obtain saline solution; the mesenchymal stem cells obtained by passaging the cells in step 2 of example 2 were transferred into a centrifuge tube, centrifuged at 2000rpm for 5min, the supernatant was discarded, and the cells were resuspended to a prescribed cell concentration by adding a saline solution to prepare a cell preparation.
Example 11e preparation of mesenchymal Stem cell preparation
Mesenchymal stem cells (P2 generation): 2.5X106 The number of the two-dimensional space-saving type,
sodium chloride: 9mg of the extract of the plant,
chlorogenic acid: 0.5mg of the total amount of the components,
sorbitol: 5mg of the extract of the plant,
water, in an amount of 1ml.
The preparation method comprises the following steps: dissolving sodium chloride, chlorogenic acid and sorbitol with proper amount of water to obtain saline solution; the mesenchymal stem cells obtained by passaging the cells in step 2 of example 2 were transferred into a centrifuge tube, centrifuged at 2000rpm for 5min, the supernatant was discarded, and the cells were resuspended to a prescribed cell concentration by adding a saline solution to prepare a cell preparation.
Test example 1: determination of cell viability:
cell viability assays are widely used in a number of scientific fields, such as drug screening, cell proliferation assays, drug toxicity assays, tumor drug sensitivity assays, and the like. Currently, the most commonly used method for detecting cell viability is MTT colorimetric [ Fischer D, li Y, ahlemeyer B, et al in vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis [ J ]. Biomaterials,2003,24 (7): 1121-1131], CCK-8 colorimetry [ Chen CL, lin CF, chang WT, et al Ceramide products p38 MAPK and JNK activation through a mechanism involving a thioredoxin-interacting protein-mediated path [ J ]. Blood,2008,111 (8): 4365-4374], trypan blue staining exclusion [ Louis KS, siegel ac. Cell viability analysis using trypan blue: manual and automated Methods [ J ]. Methods Mol,2011,740:7-12], etc.
This test example uses MTT colorimetry to determine the cell viability of the various cell preparations prepared herein. Specifically, the cell concentration of each cell preparation at 0 was measured, and then the cells were left in the dark at 2 to 6℃for 48 hours, and the cell concentration at 48 hours was measured, and the average value was obtained by measuring 5 times for each cell preparation in the same manner, and the percentage obtained by dividing the cell concentration at 48 times by the cell concentration at 0 and multiplying the divided value by 100% was used as the cell viability.
Results: example 11a=95.6%, example 11a1=93.3%, example 1a2=96.1%, example 1a3=94.4%, example 11b=93.8%, example 11c=95.1%, example 11d=96.2%, example 11e=94.9%. From these results, cell preparations of different formulations were substantially identical in terms of cell viability after 48 hours, showing no differences.
Test example 2: test of effectiveness of mesenchymal Stem cells in treating viral pneumonia
The lung index is the percentage of the body's lung mass to the body mass, and the magnitude of the lung index value is often used to indicate the severity of pneumonic lesions. Influenza virus infection causes toxic pneumonia in mice, inflammatory infiltration increases lung mass, and the greater the lung index value, the more severe the degree of pneumopathy. The lung index is used as an index for judging the action of the medicine, has a quantitative concept, and can objectively reflect the change degree of the pneumonic lesions.
The experimental example is carried out by the method carried out by Nanjing university of Chinese medicine journal, 2010, 26 (4): 315 referring to Zhou Kunfu (Zhou Kunfu, et al) for the influence of 3 methods on the pulmonary index and the pulmonary index inhibition rate of mice infected with influenza virus.
This test example 2 was conducted using the cell preparations obtained in examples 11a, 11a1, 11a2 and 11a 3.
Influenza virus A mouse lung adapted strain, namely FM1 strain, is provided by the national academy of sciences of Chinese traditional medicine and stored at-80 ℃. After serial passage 2 times in allantoic cavity of 10-day-old chick embryo (Boringer John Viton Co.) after resuscitation, the blood coagulation titer was measured to be 1:512, and the half-lethal dose LD50 for mice was 10-4.28 . Male ICR mice weighing 18-22 g were purchased from the university of Chinese medicine laboratory animal center in Nanjing and bred on a regular basis. Ribavirin tablets are commercially available (100 mg/tablet, chongqing Kerui H20073882).
Test mice were randomly grouped, 15 per group, and: normal, model, positive (ribavirin), example 11a1, example 11a2, example 11a 3.
And (3) testing: each group of animals was fed in a conventional standard manner under equivalent conditions to the SPF class animal house;
the animals in the normal group are routinely raised during the whole test period, and the animals are not detoxified or dosed;
Each cell preparation was treated with 1X 10 cells daily on day 0 and day 1, respectively5 The dose of individual/animals was injected into the mice of the 4 example groups via the tail vein;
mice in model, positive and 4 example groups were infected nasally with viral allantoic droplets under shallow ether anaesthesia at day 3, 15 times LD50 challenge per mouse;
the model group does not perform any treatment after virus attack;
ribavirin 75mg/kg was given daily for 5 consecutive days following challenge with the positive group virus;
on day 8, 10 mice in each group were weighed, sacrificed by cervical vertebrae removal, dissected to obtain the lungs, weighed accurately, and lung index inhibition were calculated.
And (3) data processing:
the lung index and lung index inhibition were calculated as follows:
lung index = mouse lung mass/mouse mass
Pulmonary index inhibition = (model group pulmonary index mean-treatment group pulmonary index mean)/model group pulmonary index mean x 100%
All data are expressed as mean ± standard deviation, and are compared using t-test.
In addition, in this example, the serum interleukin 4 level of mice was also detected using an ELISA method, as follows: 24h after the last virus excitation, randomly selecting 5 mice from each group, taking 1mL of blood by adopting an eyeball removal method after full anesthesia, performing heparin anticoagulation, centrifuging for 5min at 800r/min, taking upper serum, and detecting the level of interleukin 4 in the serum strictly according to ELISA kit specifications.
The results are shown in the following table:
| group of | Lung index (. Times.0.001) | Lung index inhibition rate | Interleukin 4 levels |
| Normal group | 833±86 | - | 22.84±6.41ng/L |
| Model group | 1673±186△△ | - | 79.36±7.83ng/L△△ |
| Positive group | 1086±169** | 35.09％ | 29.17±7.22ng/L** |
| Example 11a group | 921±174** | 44.95％ | 31.43±5.93ng/L** |
| Example 11a1 group | 1326±189*# | 20.74％ | 45.52±8.14ng/L**# |
| Example 11a2 group | 1292±216*# | 22.77％ | 48.37±6.57ng/L*## |
| Example 11a3 group | 1347±233*# | 19.49％ | 44.64±7.68ng/L**# |
Note that: in comparison with the normal group, deltaDeltaP <0.01; comparing with the model group, P <0.05, P <0.01; compared to example 11a group, #p <0.05, #p <0.01.
The result shows that the lung index of the model group is obviously different from that of the normal group (P < 0.01), the influenza virus can cause the increase of the lung index of the normal mice, and the modeling is successful; the lung index of the 4 cell treatment groups, the positive group and the model group are obviously different (P <0.01, P < 0.05), which indicates that the lung index value of the mice infected by the influenza virus can be reduced by the 4 cells and the positive medicament; in addition, the biological effects of the 4 cell preparations were significantly different from the group of example 11a, the group of example 11a1, the group of example 11a2, and the group of example 11a3 (P < 0.05). In addition, satisfactory differences between groups in terms of changes in interleukin 4 levels were also presented.
Furthermore, in combination with the above measurement results regarding cell viability, it was shown that chlorogenic acid and sorbitol added in the cell preparation do not directly act on stem cells, but act on the animal body together with the stem cells, i.e., it was unexpectedly found that the simultaneous addition of both sorbitol and chlorogenic acid in the cell preparation can significantly improve the biological effects of the cell preparation.
The above-described embodiments are merely preferred embodiments for fully explaining the present application, and the scope of the present application is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present application, and are intended to be within the scope of the present application. The protection scope of the application is subject to the claims.