CN110862963B - Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility-related diseases - Google Patents
Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility-related diseasesDownload PDFInfo
- Publication number
- CN110862963B CN110862963BCN201911178836.XACN201911178836ACN110862963BCN 110862963 BCN110862963 BCN 110862963BCN 201911178836 ACN201911178836 ACN 201911178836ACN 110862963 BCN110862963 BCN 110862963B
- Authority
- CN
- China
- Prior art keywords
- decidua
- cells
- cell
- cd49a
- bright
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Reproductive Health (AREA)
- Wood Science & Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Hematology (AREA)
- Pharmacology & Pharmacy (AREA)
- Gynecology & Obstetrics (AREA)
- Endocrinology (AREA)
- General Chemical & Material Sciences (AREA)
- Pregnancy & Childbirth (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Virology (AREA)
- Epidemiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Translated fromChineseDescription
Technical Field
The invention belongs to the field of biological medicine, and relates to application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility-related diseases, a cell separation culture method and a pharmaceutical composition containing the same.
Background
The damage of endometrium is mainly damage of endometrium basal layer, the main reason of the damage is related to uterine curettage in pregnancy, and the endometrium basal layer in pregnancy is loose and is easier to be damaged compared with normal endometrium. Based on the national conditions and the current situation that the artificial abortion rate is gradually increased, the occurrence of damaged endometrium is not negligible. Damage to the basement membrane layer of the endometrium, possibly resulting in damage or loss of endometrial stem cells; meanwhile, local infection and aseptic inflammation of the damaged intima can destroy niche microenvironment of stem cells, so that epithelial and interstitial cell regeneration and repair are hindered, blood vessel formation is blocked, compact fibrous tissues are formed, and finally, the uterine cavity only covers a small amount of intima, even no intima exists, the gland is atrophied, and the normal form and function of the uterine cavity are lost. Further, disturbances of the immune status in the uterine cavity further mediate the risk of immunological abortion of the endometrium.
NK cells are an innate immune cell with the presence of endometrium. In the early stage of pregnancy, NK cells account for 40-70% of the total number of lymphocytes in the decidua, and are a type of lymphocyte with a high content in the normal decidua. Modern reproductive medicine research suggests that human decidua NK (dNK) cells have a unique CD56brightCD16-KIR+CD9+CD49a+Phenotype, unlike peripheral NK cells, is almost non-cytotoxic. The local involvement of decidua NK cells in decidua tissue promotes neovascularization, tissue remodeling, immunoregulation and placental formation.
Based on prior studies, no technical solution is disclosed or suggested for the preparation of products using decidua NK cells for the treatment of diseases, which is unknown and needs to be demonstrated. On the other hand, the decidua NK cell population itself is very heterogeneous, i.e., has CD56brightCD16-KIR+CD9+CD49a+NK cells in phenotype decidua tissues can be further divided into a plurality of groups according to different expression of surface markers of the NK cells, and the functions of the NK cells are different. The prior art does not disclose or suggest a technical scheme for preparing products for treating diseases by decidua NK cell subsets expressing specific markers.
Disclosure of Invention
The present invention is made to solve the above-mentioned problems, and an object of the present invention is to provide an NK cell, a use of a cell subset thereof, a method for preparing the same, and a pharmaceutical composition containing the same.
The first aspect of the invention provides the use of decidua NK cells and cell subsets thereof in the preparation of medicaments for treating diseases related to infertility.
The decidua NK cell refers to a surface marker CD56brightCD16-KIR+CD9+CD49a+NK cells within decidua tissue of phenotype. Decidua NK cell subset refers to the NK cell characteristic, and further adopts a cell marker CD39+，CD27+，CD160+Or TIGIT+Screening to obtain the product; decidua NK cell subset including a marker of CD39+，CD27+，CD160+Or TIGIT+Any one or more of the decidua NK subcellular cells of (a).
The infertility related diseases comprise endometrial growth disorder diseases and maternal and fetal immune tolerance disorder related diseases. Diseases associated with endometrial growth disorders include endometrial damage, premature ovarian failure, sex hormone disorders, polycystic ovary syndrome, pelvic inflammatory disease, decreased endometrial receptivity, endometritis, endometrial polyps, intrauterine adhesions, endometrial gland loss, endometrial fibrosis, amenorrhea, abnormal uterine bleeding, adenomyosis and endometriosis, reproductive system infections, uterine fibroids, and the like; the diseases related to the maternal-fetal immune tolerance disorder comprise recurrent spontaneous abortion, threatened abortion or failure of assisted reproductive technology treatment and the like.
Therefore, the medicine for treating the diseases related to the infertility is a medicine for treating the endometrial growth disorder diseases, a medicine for treating the diseases related to the maternal-fetal immune tolerance disorder or a medicine for improving the conception rate during the damage of the endometrium.
Specifically, the medicament for treating the endometrial growth disorder disease is any one or more of a combination of a medicament for promoting the increase of the thickness of endometrium, a medicament for enhancing the activity of endometrial stromal cells, a medicament for reducing damage of endometrial cells, a medicament for promoting the expression of VEGF (vascular endothelial growth factor) of endometrial stromal cells, a medicament for maintaining the dryness of endometrial stromal cells and a medicament for stimulating the proliferation; the medicine for treating the diseases related to maternal-fetal immune tolerance disorder is any one or combination of medicines for playing an immune tolerance effect, reducing spontaneous abortion and improving the level of T helper lymphocytes; the medicament for improving the conception rate when the endometrium is damaged is a medicament for reducing the fibrosis degree of the endometrium or improving the number of secretory glands.
More specifically, the medicine for maintaining the dryness of the endometrial stromal cells and stimulating the proliferation is a medicine for improving the positive rate of ALDH and Ki67 of the stromal cells; the drug playing a role in immune tolerance is a drug mediating the secretion of IL-10 by DC cells.
In a second aspect of the present invention, there is provided a method for preparing the decidua NK cells and cell subsets, comprising the steps of:
A. isolation of decidua NK cells and decidua NK cell subsets
(i) Separating decidua tissue cells: the decidua tissue was digested with collagenase IV (Sigma-Aldrich) and DNase I (Shanghai Sangon) to obtain cell suspension
(ii) Lymphocyte acquisition: percoll (GE healthcare) density gradient centrifugation.
(iii) Separating decidua NK cells by using a flow cytometer, firstly separating some impurity cells by using a general technology, then separating the decidua NK cells by using an anti-CD56 antibody, an anti-CD16 antibody and an anti-CD49a antibody and implementing a flow cytometer method or a magnetic bead method, wherein the marker of the decidua NK cells is CD56brightCD16-CD49a+。
(iv) The decidua NK cell subpopulation is further isolated by flow cytometry or magnetic bead method using anti-CD39 antibody, anti-CD27 antibody, anti-CD160 antibody, and anti-TIGIT antibody as necessary. The decidua NK cell subset marker is CD39+，CD27+，CD160+And TIGIT+Any one of and or a combination of at least two.
B. NK cell in vitro culture
Transferring NK cells or cell subsets into CTS AIM-V culture medium, 1640 culture medium or DMEM culture medium, and culturing at 37 deg.C with 5% CO2The culture time in the environment is 12-48 hours.
The third aspect of the invention provides a pharmaceutical composition for treating infertility-related diseases, which comprises decidua NK cells or cell subsets thereof and pharmaceutically acceptable excipients. Adjuvants help cells to exert therapeutic effects more stably, and these preparations can ensure the structural integrity and stability of the cells disclosed by the present invention, and protect the active functional groups of the cells to prevent the activity change.
In general, liquid formulations are stable for at least three months at 2 ℃ to 8 ℃ and frozen formulations are stable for at least six months at-30 ℃.
In the form of medicine, the pharmaceutical composition comprises tablets, pills, powder, injection, tincture, solution, extract, ointment and the like which are commonly used in the pharmaceutical field; also comprises a uterine mucosa administration preparation, such as an intrauterine drug release system and the like of films, suppositories, tablets, effervescent tablets, gels, stents and the like; mucosal absorption enhancers such as surfactants, chelating agents, fatty acids, fatty alcohols, fatty acid esters, cyclodextrin derivatives, protease inhibitors, and the like may also be included.
Compared with the prior art, the invention has the following technical effects:
the invention provides application of decidua NK cells and cell subsets thereof in preparation of medicaments for treating infertility related diseases. Experiments prove that the decidua NK cells and the cell subsets thereof can treat the endometrium growth disorder diseases by promoting the increase of the thickness of an endometrium, promoting the activity of endometrial cells, reducing the damage of endometrial cells, promoting VEGF expression, maintaining the dryness of endometrial stromal cells and stimulating proliferation, so that the success rate of conception of an endometrium damage model mouse is improved to 60-70% from 30%; the method can be used for treating diseases related to maternal-fetal immune tolerance disorder by playing a role in immune tolerance, reducing spontaneous abortion rate and improving T-helper lymphocyte level, and provides a new approach for treating infertility.
Detailed Description
The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the invention, and the examples do not include detailed descriptions of conventional methods.
Example 1 mulleryl NK cell candidate marker screening
The statistical calculation in this and the following embodiments calculates the p-value according to different statistical models of two-group comparison, multiple-group comparison, and rate comparison requirements selection software SPSS 22.0 version. P values less than 0.05 were considered statistically significant.
NK cells were first isolated using 10 healthy non-medical causes terminating the decidua of early pregnancy (normal group) and 5 decidua of early pregnancy in spontaneous abortion (abortion group), and their isolation and flow cytometric sorting can be referred to in the literature [ Fu B, et al. immunity,2017,47(6):1100-1113.e6.]. Examples are as follows: lymphocytes were obtained by 1mg/mL collagenase IV (Sigma-Aldrich) and 0.01mg/mL DNase I (Shanghai Sangon) digestion for 1h followed by Percoll (GE healthcare) density gradient centrifugation. The culture dish was incubated at 37 ℃ for 2h to remove stromal cells and macrophages, and then NK cells were isolated using a flow cytometer. First, screening the primary screening using CD56 antibody, CD3 antibody and CD14 antibodySorting NK cells, and further screening decidua NK cells by using CD16 antibodies and CD49a antibodies to obtain the cell with the phenotype of CD56brightCD16-CD49a+Decidua NK cell of (CD 56)brightCD16-CD49a+CD3-CD14-). And (3) cracking NK cells obtained from the normal group and the abortion group, and performing protein expression analysis on two groups of lysates by an iTRAQ-nano-HPLC-MS/MS method after the protein concentration is determined by a Bradford method, so as to confirm membrane surface markers with different contents in the two groups of NK cells. Methods reference [ Jiang, Hong-Lin, et al. cancer research 76.4(2016): 952-.]By analyzing the membrane surface markers through clustering statistics, the expression of CD39(UniProtKB: P49961), CD27(UniProtKB: P26842), CD160(UniProtKB: O95971) and TIGIT (UniProtKB: Q495A1) in normal decidua NK cells is remarkably higher than that of decidua cells in abortions (P is less than 0.05).
Example 2 preparation of uterine decidua NK cells and decidua NK cell subsets
Decidua NK cells are prepared by utilizing decidua tissues of early pregnancy terminated by healthy non-medical reasons and implemented according to the NK cell separation method described in example 1, and the method is briefly described as follows: lymphocytes were obtained by 1mg/mL collagenase IV (Sigma-Aldrich) and 0.01mg/mL DNase I (Shanghai Sangon) digestion for 1h followed by Percoll (GE healthcare) density gradient centrifugation. The culture dish was incubated at 37 ℃ for 2h to remove stromal cells and macrophages, and then NK cells were isolated using a flow cytometer. The phenotype of the obtained product is CD56brightCD16-CD49a+The decidua NK cell of (1). Further sorting of CD56 using antibody magnetic beadsbrightCD16-CD49a+CD39+Decidua NK cell population, CD56brightCD16-CD49a+CD27+Decidua NK cell population, CD56brightCD16-CD49a+CD160+Decidua NK cell population, CD56brightCD16-CD49a+TIGIT+Decidua NK cell population, and CD56brightCD16-CD49a+CD39+TIGIT+A population of decidua NK cells. The obtained cells can be directly tested, used or frozen. Collecting peripheral blood of the same volunteer according to a general methodNK cells were isolated as control cells.
Example 3 decidua NK cells and decidua NK cell subsets enhance endometrial cell viability, reduce endometrial cell damage, increase VEGF expression
After culturing non-pathological endometrial stromal cells (stroma cells) for 24 hours, each population of NK cells described in example 2, stromal cells: the NK cell number ratio was 10:1 and 5:1, respectively. The control group used the control NK cells described in example 2. NK cells were cleared after 48 hours of treatment and stromal cells and medium were sampled for analysis.
Stromal cells were tested for cell viability by the Presoblue method (Thermo Fisher Scientific) and measured 48 hours after treatment and expressed as the average of the controls normalized (tables 1 and 2).
TABLE 110: 1 relative cell viability of stromal cells under culture conditions
| Mean value of | SD | p value | |
| Blank (culture alone, no NK cells) | 100 | 6.72 | |
| Control cells | 97.45 | 8.34 | |
| CD56brightCD16-CD49a+Decidua NK cell of | 139.7 | 17.6 | p＜0.05 |
| CD56brightCD16-CD49a+CD39+Decidua NK cell population | 160 | 17.1 | p＜0.05 |
| CD56brightCD16-CD49a+CD27+Decidua NK cell population | 142.5 | 12.2 | p＜0.05 |
| CD56brightCD16-CD49a+CD160+Decidua NK cell population | 168.5 | 10.2 | p＜0.01 |
| CD56brightCD16-CD49a+TIGIT+Decidua NK cell population | 173.3 | 20.7 | p＜0.01 |
| CD56brightCD16-CD49a+CD39+TIGIT+Decidua NK cell population | 162.7 | 13.5 | p＜0.01 |
TABLE 25 relative cell viability of stromal cells under culture conditions
| Mean value of | SD | p value | |
| Blank (culture alone, no NK cells) | 100 | 11.3 | |
| Control cells | 94.36 | 6.5 | |
| CD56brightCD16-CD49a+Decidua NK cell of | 184.3 | 15.5 | p＜0.05 |
| CD56brightCD16-CD49a+CD39+Decidua NK cell population | 213.9 | 17.5 | p＜0.05 |
| CD56brightCD16-CD49a+CD27+Decidua NK cell population | 175.6 | 11.5 | p＜0.05 |
| CD56brightCD16-CD49a+CD160+Decidua NK cell population | 211.5 | 15.3 | p＜0.05 |
| CD56brightCD16-CD49a+TIGIT+Decidua NK cell population | 265.5 | 28.4 | p＜0.01 |
| CD56brightCD16-CD49a+CD39+TIGIT+Decidua NK cell population | 232.3 | 20.5 | p＜0.05 |
And (4) conclusion: the decidua NK cells and the cell subsets thereof have the capability of increasing the viability of uterine stromal cells and can be used as products for enhancing endometrial proliferation
Further, cellular damage at the stromal cell level was assessed: the cell damage was measured by colorimetry using a lactate dehydrogenase assay, enabling quantification of cell damage based on measurement of lactate dehydrogenase activity in damaged cells in the medium. The increase in cell membrane damage and cell lysis leads to an increase in lactate dehydrogenase activity, which is proportional to the number of lysed cells. After 48 hours of cell treatment, lactate dehydrogenase activity measurements were performed in the medium (Table 3).
TABLE 310 relative cell damage under culture conditions
| Mean value of | SD | p value | |
| Blank (culture alone, no NK cells) | 100 | 9.15 | |
| Control cells | 95.35 | 4.16 | |
| CD56brightCD16-CD49a+Decidua NK cell of | 61.50 | 4.55 | p＜0.05 |
| CD56brightCD16-CD49a+CD39+Decidua NK cell population | 72.21 | 5.22 | p＜0.05 |
| CD56brightCD16-CD49a+CD27+Decidua NK cell population | 59.83 | 8.95 | p＜0.05 |
| CD56brightCD16-CD49a+CD160+Decidua NK cell population | 49.72 | 3.32 | p＜0.05 |
| CD56brightCD16-CD49a+TIGIT+Decidua NK cell population | 37.7 | 4.9 | p＜0.05 |
| CD56brightCD16-CD49a+CD39+TIGIT+Decidua NK cell population | 35.53 | 2.52 | p＜0.01 |
And (4) conclusion: the decidua NK cells and the cell subsets thereof have the capability of reducing membrane damage and can be used as products for enhancing the activity of stromal cells.
Further, the potential enhancing effect of decidua NK cells and their cell subpopulations on endometrial stromal cell VEGF was investigated. The results are presented in table 4. After 48 hours of cell treatment, ELISA measurements of VEGF concentration were performed in the culture medium.
TABLE 410 VEGF expression under culture conditions
And (4) conclusion: the decidua NK cells and the cell subsets thereof have the function of promoting VEGF expression and enhancing endometrial angiogenesis.
Example 4 expression of decidua NK cells and decidua NK cell subsets into endometrial stromal cell markers
After culturing non-pathological endometrial stromal cells (stroma cells) for 24 hours, each population of NK cells described in example 2, stromal cells: the NK cell number ratio was 10:1 and 5:1, respectively. The control group used the control NK cells described in example 2. After 24 hours of culture in an incubator at 37 ℃ under 5% CO2, NK cells were post-cleared, and then the ALDH positive rate and Ki67 positive rate of stromal cells were examined by flow cytometry, the results of which are shown in Table 5.
TABLE 5 ALDH positivity and Ki67 positivity analysis
The result shows that decidua NK cells and the sub-population thereof have very strong effects of maintaining the dryness of stromal cells and stimulating proliferation.
Example 5 treatment of endometrial injury with decidua NK cells and decidua NK cell subsets
The thickness of the endometrium of the patient is less than 8mm due to factors such as artificial abortion, uterine curettage, infection and the like, and the clinical diagnosis is that the endometrium is thin. The administration to the patient is therapeutically ineffective against infection and the like. Administering a composition comprising the active ingredient decidua NK cells and sub-groups of decidua NK cells prepared in example 2, e.g. intravenous infusion of a composition comprising the active ingredient of cells, e.g. 2X 109. One or more intravenous administrations to the patient to promote an increase in endometrial thickness.
Example 6 effects of decidua NK cells and decidua NK cell subsets on decidua DC cells
Isolation of dendritic cell DC (CD1c positive) from decidua tissue terminating pregnancy for human non-medical reasons, isolation and screening methods equivalent to those of the literature (Guo P F, et al. blood,2010,116(12):2061-2069.) negative control groups (coculture with control NK cells described in example 2, stromal cell: NK cell ratio of 10:1), treatment groups (decidua NK cells and decidua NK cell subsets described in example 2, stromal cell: NK cell ratio of 10:1), LPS-treated groups (100 ng/ml), blank groups of culture alone, detection of interleukin 10(IL-10) and tumor necrosis factor alpha (TNF alpha) levels in the culture system after 48 hours, detection methods equivalent to those of the literature (Guo P F, et al. blood,2010,116(12):2061-2069.) showed that decidua NK cells and decidua NK cell subsets could significantly increase IL-10 secretion levels without increasing IL-10 secretion levels (Table 6-7 alpha) (Table 6-7) ). These results demonstrate that decidua NK cells and decidua NK cell subsets can exert an immune tolerogenic effect by DCs.
TABLE 6 analysis of IL-10 content
| IL-10(pg/ml) | SD | p value | |
| Blank (culture alone, no NK cells) | 79.69 | 7.60 | |
| Control cells | 88.35 | 7.15 | |
| CD56brightCD16-CD49a+Decidua NK cell of | 437.52 | 47.95 | p＜0.05 |
| CD56brightCD16-CD49a+CD39+Decidua NK cell population | 469.58 | 24.50 | p＜0.05 |
| CD56brightCD16-CD49a+CD27+Decidua NK cell population | 380.04 | 44.93 | p＜0.05 |
| CD56brightCD16-CD49a+CD160+Decidua NK cell population | 371.64 | 55.36 | p＜0.05 |
| CD56brightCD16-CD49a+TIGIT+Decidua NK cell population | 365.73 | 45.33 | p＜0.05 |
| CD56brightCD16-CD49a+CD39+TIGIT+Decidua NK cell population | 597.77 | 49.54 | p＜0.05 |
| LPS | 468.49 | 48.94 | p＜0.05 |
TABLE 7 analysis of TNF α content
| IL-10(pg/ml) | SD | p value | |
| Blank (culture alone, no NK cells) | 20.97 | 1.45 | |
| Control cells | 15.38 | 2.22 | |
| CD56brightCD16-CD49a+Decidua NK cell of | 19.56 | 2.06 | p＞0.05 |
| CD56brightCD16-CD49a+CD39+Decidua NK cell population | 28.48 | 3.08 | p＞0.05 |
| CD56brightCD16-CD49a+CD27+Decidua NK cell population | 18.32 | 1.18 | p＞0.05 |
| CD56brightCD16-CD49a+CD160+Decidua NK cell population | 20.14 | 2.73 | p＞0.05 |
| CD56brightCD16-CD49a+TIGIT+Decidua NK cell population | 29.42 | 3.71 | p＞0.05 |
| CD56brightCD16-CD49a+CD39+TIGIT+Decidua NK cell population | 15.04 | 1.43 | p＞0.05 |
| LPS | 1506.51 | 209.69 | p＜0.01 |
Example 7 therapeutic Effect of decidua NK cells and decidua NK cell subsets on spontaneous abortion models
CBA/J female mice and DBA/2J male mice are used for establishing a stress abortion model which is a classic maternal-fetal immune tolerance disorder research model, and establishing methods, experimental methods and observation time point equivalent documents (Blois S M, et al. Nature Medicine,2007,13(12): 1450-. Before the CBA/J female mice are combined into a cage, the female mice are divided into a negative control group, a model group, a stress pressure + control cell group and a treatment group. Treatment groups administered 1X 10 meridians separately5The decidua NK cells and the cell subsets thereof are administered once in 3 days for 3 times. The cages were combined 3 days after the first administration. Mice were caged immediately after vaginal emboli were established (effective n 10).
The experimental results show (table 8) that the abortion rate of the treatment group is significantly lower than that of the stress pressure abortion group, which indicates that the decidua NK cells and decidua NK cell subsets have good therapeutic effects.
TABLE 8 embryo uptake (abortion) analysis in groups of mice
Example 8 effects of decidua NK cells and decidua NK cell subgroups on T helper cells
The mice in the negative control group, model group, control group and treatment group of example 8 were isolated from periaortic lymph nodes and tested for levels of Foxp3 positive T helper lymphocytes. The isolation and detection methods are the same as in the literature (Kim B J, et al.. Proceedings of National Academy of sciences,2015,112(5): 1559. 1564. treatment of decidua NK cells and decidua NK cell subsets was shown to be effective in increasing levels of Foxp3 positive T helper lymphocytes (Table 9).
TABLE 9 groups of mice Foxp3+% expression analysis
Example 9 treatment of mouse endometrial injury models with decidua NK cells and decidua NK cell subsets
Firstly, constructing an animal endometrium injury model (C57 mouse), dividing 8-week-old female mice into groups, and constructing the endometrium injury model by adopting a double (infection + mechanical) injury method for 10 mice in each group, namely after anesthetizing the mice, taking a longitudinal incision with the length of about 2cm in the middle of the lower abdomen into the abdomen, making a 0.5cm longitudinal incision at the position 1/3 in the middle and lower uterus, scratching the uterine cavity at the upper section by adopting an endometrium spatula, stopping scraping when the concavo-convex feeling of the spatula entering and exiting the uterus disappears and the four walls feel rough, reserving lipopolysaccharide cotton threads in the uterine cavity after scraping, suturing the abdominal incision, and taking out the lipopolysaccharide cotton threads after 48 hours. After modeling is completed, a blank control group (a sham operation group) is set, and only physiological saline (a model) group is injected; control group, cell treated group. Treatment groups administered 1X 10 meridians separately5Decidua NK cells anddecidua NK cell subset was administered once on 3 days for a total of 3 times. Mice were mated with male mice 3 weeks after the mice were in estrus. Materials were taken 1 month later for evaluation of endometrial histology by HE staining and Masson staining, and mice were evaluated 3 months later for pregnancy results. As a result: tissue functional assessment at 1 month after surgery showed that the degree of fibrosis of decidua NK cells and decidua NK cell subgroups was significantly reduced compared to each control group; the number of secretory glands was higher than that of each control group. The pregnancy results evaluation showed that the pregnancy rates of decidua NK cells and decidua NK cell subgroups were higher than those of each control group, and the results are shown in table 10.
TABLE 10 pregnancy results analysis in groups of mice
| Group of | Rate of pregnancy | p value (vs control cells) |
| Negative control group | 100％ | |
| Model set (acellular) | 30％ | |
| Model group + control cells | 30％ | |
| CD56brightCD16-CD49a+Decidua NK cell of | 60％ | p＜0.05 |
| CD56brightCD16-CD49a+CD39+Decidua NK cell population | 60％ | p＜0.05 |
| CD56brightCD16-CD49a+CD27+Decidua NK cell population | 70％ | p＜0.05 |
| CD56brightCD16-CD49a+CD160+Decidua NK cell population | 70％ | p＜0.05 |
| CD56brightCD16-CD49a+TIGIT+Decidua NK cell population | 70％ | p＜0.05 |
| CD56brightCD16-CD49a+CD39+TIGIT+Decidua NK cell population | 70％ | p＜0.05 |
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. The application of decidua NK cells or cell subsets thereof in preparing medicaments for treating infertility related diseases is characterized in that:
the surface marker of the decidua NK cell is CD56brightCD16-CD49a+Said subpopulation of cells comprising CD56brightCD16-CD49a+CD39+Decidua NK cell population, CD56brightCD16-CD49a+CD27+Decidua NK cell population, CD56brightCD16-CD49a+CD160+Decidua NK cell population, CD56brightCD16-CD49a+TIGIT+Decidua NK cell population, and CD56brightCD16-CD49a+CD39+TIGIT+One or more of decidua NK cell population,
the medicine for treating the diseases related to the infertility is a medicine for treating endometrial injury, a medicine for treating diseases related to maternal and fetal immune tolerance disorder or a medicine for improving conception rate during endometrial injury.
2. Use of decidua NK cells or a cell subset thereof according to claim 1 for the preparation of a medicament for the treatment of a disease associated with infertility, characterized in that:
wherein the medicament for treating the endometrial injury is any one or more of a combination of a medicament for promoting the increase of the thickness of endometrium, a medicament for enhancing the activity of endometrial stromal cells, a medicament for reducing the injury of endometrial cells, a medicament for promoting the expression of VEGF in the endometrial stromal cells, a medicament for maintaining the dryness of the endometrial stromal cells and a medicament for stimulating the proliferation;
the medicine for treating the disease related to maternal-fetal immune tolerance disorder is any one or combination of medicines for maintaining or enhancing immune tolerance, medicines for reducing spontaneous abortion and medicines for increasing the level of T-helper lymphocytes;
the medicament for improving the conception rate when the endometrium is damaged is a medicament for reducing the fibrosis degree of the endometrium or improving the number of secretory glands.
3. Use of decidua NK cells or a cell subset thereof according to claim 2 for the preparation of a medicament for the treatment of a disease associated with infertility, characterized in that:
wherein, the medicine for maintaining the dryness of the endometrial stromal cells and stimulating the proliferation is a medicine for improving the positive rate of the stromal cells ALDH and Ki 67;
the medicament for maintaining or enhancing the immune tolerance effect is a medicament for mediating the IL-10 secretion of DC cells.
4. Use of decidua NK cells or cell subsets thereof according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of infertility-related diseases, wherein the cells and cell subsets are prepared by the following method:
A. isolation of decidua NK cells and decidua NK cell subsets
Digesting decidua tissue by enzymolysis of collagenase IV and DNase I to obtain cell suspension, then obtaining lymphocytes by a density gradient centrifugation method, and then separating a marker CD56 from the lymphocytes by utilizing anti-CD56 antibody, anti-CD16 antibody and anti-CD49a antibodybrightCD16-CD49a+The decidua NK cell of (a); then separating decidua NK cell subset by utilizing anti-CD39 antibody, anti-CD27 antibody, anti-CD160 antibody and anti-TIGIT antibody according to requirements;
B. NK cell in vitro culture
Transferring NK cells or cell subsets into CTS AIM-V culture medium, 1640 culture medium or DMEM culture medium, and culturing at 37 deg.C with 5% CO2The culture time in the environment is 12-24 hours.
5. Use of decidua NK cells or a cell subset thereof according to claim 1 for the preparation of a medicament for the treatment of a disease associated with infertility, characterized in that:
wherein the drug is decidua NK cells or a cell subset thereof as the only active ingredient or a pharmaceutical composition comprising decidua NK cells or a cell subset thereof,
the surface marker of the decidua NK cell is CD56brightCD16-CD49a+Said subpopulation of cells comprising CD56brightCD16-CD49a+CD39+Decidua NK cell population, CD56brightCD16-CD49a+CD27+Decidua NK cell population, CD56brightCD16-CD49a+CD160+Decidua NK cell population, CD56brightCD16-CD49a+TIGIT+Decidua NK cell population, and CD56brightCD16-CD49a+CD39+TIGIT+One or more of decidua NK cell population.
6. Use of decidua NK cells or a cell subset thereof according to claim 5 for the preparation of a medicament for the treatment of infertility-related diseases, characterized in that:
wherein, the pharmaceutical composition is an injection.
7. A pharmaceutical composition for treating infertility-related diseases is characterized by comprising decidua NK cells or cell subsets thereof and pharmaceutically acceptable auxiliary materials,
the surface marker of the decidua NK cell is CD56brightCD16-CD49a+Said subpopulation of cells comprising CD56brightCD16-CD49a+CD39+Decidua NK cell population, CD56brightCD16-CD49a+CD27+Decidua NK cell population, CD56brightCD16-CD49a+CD160+Decidua NK cell population, CD56brightCD16-CD49a+TIGIT+Decidua NK cell population, and CD56brightCD16-CD49a+CD39+TIGIT+One or more of decidua NK cell population.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911178836.XACN110862963B (en) | 2019-11-27 | 2019-11-27 | Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility-related diseases |
| PCT/CN2020/130939WO2021104215A1 (en) | 2019-11-27 | 2020-11-23 | Use of decidual nk cells and cell subsets thereof in preparation of drug for treating infertility-related diseases |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911178836.XACN110862963B (en) | 2019-11-27 | 2019-11-27 | Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility-related diseases |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110862963A CN110862963A (en) | 2020-03-06 |
| CN110862963Btrue CN110862963B (en) | 2021-08-27 |
Family
ID=69656650
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911178836.XAActiveCN110862963B (en) | 2019-11-27 | 2019-11-27 | Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility-related diseases |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN110862963B (en) |
| WO (1) | WO2021104215A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110862963B (en)* | 2019-11-27 | 2021-08-27 | 沣潮医药科技（上海）有限公司 | Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility-related diseases |
| CN111607564A (en)* | 2020-04-30 | 2020-09-01 | 浙江大学 | A method to assess the secretory function of decidual NK cells |
| CN112708653B (en)* | 2020-11-26 | 2023-06-16 | 中国科学技术大学 | Detection method for predicting recurrent abortion and/or diagnosing cause of recurrent abortion by menstrual blood |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017072165A1 (en)* | 2015-10-30 | 2017-05-04 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for monitoring the immunological profile of a subject |
| CN107029233A (en)* | 2016-02-04 | 2017-08-11 | 复旦大学附属妇产科医院 | Purposes of the interleukins-8 antibody in endometriosis medicine composition is prepared |
| CN107144687A (en)* | 2017-05-03 | 2017-09-08 | 首都医科大学附属北京妇产医院 | Detection method and kit for decidua lymphocyte subtype and application thereof |
| CN107850596A (en)* | 2015-07-24 | 2018-03-27 | 先天制药公司 | Method for detecting tissue infiltration's NK cells |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701677A (en)* | 2015-11-18 | 2017-05-24 | 深圳爱生再生医学科技有限公司 | In-vitro expansion culture method of NK cells |
| CN110721196B (en)* | 2019-11-27 | 2021-07-06 | 沣潮医药科技（上海）有限公司 | Use of decidual NK cells and exosomes derived from cell subsets in the preparation of infertility-related disease drugs and adjuvant therapeutics |
| CN110862963B (en)* | 2019-11-27 | 2021-08-27 | 沣潮医药科技（上海）有限公司 | Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility-related diseases |
- 2019
- 2019-11-27CNCN201911178836.XApatent/CN110862963B/enactiveActive
- 2020
- 2020-11-23WOPCT/CN2020/130939patent/WO2021104215A1/ennot_activeCeased
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107850596A (en)* | 2015-07-24 | 2018-03-27 | 先天制药公司 | Method for detecting tissue infiltration's NK cells |
| WO2017072165A1 (en)* | 2015-10-30 | 2017-05-04 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for monitoring the immunological profile of a subject |
| CN107029233A (en)* | 2016-02-04 | 2017-08-11 | 复旦大学附属妇产科医院 | Purposes of the interleukins-8 antibody in endometriosis medicine composition is prepared |
| CN107144687A (en)* | 2017-05-03 | 2017-09-08 | 首都医科大学附属北京妇产医院 | Detection method and kit for decidua lymphocyte subtype and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Decidual natural killer cells: key regulators of placental development (a review);B. Anne Croy等;《Journal of Reproductive Immunology》;20021231;第151–168页* |
| 原因不明复发性流产患者蜕膜NK细胞亚群特征研究;杜冀晖等;《国际检验医学杂志》;20121231;第33卷(第3期);第2865-2866页* |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2021104215A1 (en) | 2021-06-03 |
| CN110862963A (en) | 2020-03-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110721196B (en) | Use of decidual NK cells and exosomes derived from cell subsets in the preparation of infertility-related disease drugs and adjuvant therapeutics | |
| CN110862963B (en) | Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility-related diseases | |
| CN105769916B (en) | Application of the source for mesenchymal stem cells excretion body in preparation treatment preeclampsia drug or preparation | |
| CN110946878B (en) | Application of uterine cavity fluid source exosome in preparation of medicine and adjuvant therapy agent for treating infertility related diseases | |
| US20230256021A1 (en) | Compositions for preventing or treating diabetic skin disease comprising exosome-derived from thrombin-treated stem cell | |
| KR102312937B1 (en) | Pharmaceutical composition for preventing and treating optic nerve disease | |
| CN112870228A (en) | Multifunctional microenvironment protection exosome hydrogel and preparation method and application thereof | |
| Xu et al. | Autocross-linked hyaluronic acid gel and adipose-derived mesenchymal stem cell composites for the treatment intrauterine adhesions | |
| WO2024027673A1 (en) | Use of nocardia rubra cell wall skeleton in treatment of chronic cervicitis | |
| KR20220029634A (en) | Composition for treating asherman syndrome comprising perivascular stem cell medium or cyclophilin a | |
| CN114984059A (en) | Anti-colorectal cancer probiotic preparation and preparation method and application thereof | |
| CN117982543A (en) | Application of Lactobacillus plantarum AR501 in the preparation of medicine for preventing and treating premature ovarian insufficiency | |
| KR101617912B1 (en) | Pharmaceutical compositions for preventing or treating containing peripheral blood mononuclear cells as an effective ingredient | |
| CN113827618B (en) | Use of stem cell conditioned media in the preparation of drugs for the treatment of inflammatory skin | |
| CN114099545B (en) | Action of exosomes in thoracic aortic dissection/aneurysm | |
| WO2023217247A1 (en) | Use of cd177 anti-inflammatory neutrophil in treatment of cerebral ischemic stroke | |
| WO2023246293A1 (en) | Use of nocardia rubra cell wall skeleton in treatment of cervical lesions | |
| CN111419848A (en) | A pharmaceutical composition for inducing chondrogenic differentiation of stem cells and its application | |
| CN114984028A (en) | Application of astragaloside | |
| CN116004527A (en) | Mesenchymal stem cell apoptosis vesicle delivery system, construction method and application thereof | |
| CN111135193A (en) | Method for recovering endometrium by using stem cells | |
| Mei et al. | Human umbilical cord mesenchymal stem cells (hUCMSCs) promotes the regeneration of severe endometrial damage in a rat model | |
| US11413313B2 (en) | Pharmaceutical composition for preventing or treating atopic dermatitis comprising clonal stem cells | |
| CN116920013A (en) | Traditional Chinese medicine composition for treating or preventing preeclampsia | |
| CN120585848A (en) | Composition and application thereof in treating inflammatory bowel disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right | Effective date of registration:20250615 Address after:201400 No. 324, Building 1, Shunfu Road, A3 Industrial Park, Guangming, Fengxian District, Shanghai China Patentee after:FENGCHAO MEDICAL TECHNOLOGY (SHANGHAI) Co.,Ltd. Country or region after:China Patentee after:PLA Naval Military Medical University Address before:201400 No. 324, Building 1, Shunfu Road, A3 Industrial Park, Guangming, Fengxian District, Shanghai China Patentee before:FENGCHAO MEDICAL TECHNOLOGY (SHANGHAI) Co.,Ltd. Country or region before:China |