CN102586182A - Preparation and application of human-stem-cell-secreted bioactive factor and lysis solution - Google Patents

Abstract

The invention discloses preparation and application of human-stem-cell-secreted bioactive factors and a lysis solution. The process comprises the following steps of: extracting stem cells of the umbilical cord or the placenta tissue of a health human person; performing in-vitro culture and amplification; and preparing high-purity and high-activity stem-cell-secreted bioactive factors and bioactive substances in the stem cell (namely, high-purity stem cell lysis solution) by the modern biological technologies of low-temperature separation, purification and the like. According to the invention, the bioactive factors or the substances do not contain any animal component, and can be developed into a product with a cell biological activity; after acting on the human body, the product can activate the stem cell of the organism and repair or replace damaged, lesion and old cells; and the product has the functions of regulating the cell metabolism function of the organism, enhancing the immunity of the organism, delaying the aging and the like. The human bioactive factors or the substances have great application prospect in the fields of medical science, healthcare and beauty.

Description

The preparation and the application of people's derived stem cell secretion biologically active factors and lysate

Technical field

The invention belongs to biological technical field; Be specifically related to from the umbilical cord tissue of healthy subjects or placenta tissue, extract stem cell; Through vitro culture, amplification, extract excretory biologically active factors and interior biologically active substance and the application of stem cell in stem cell cultivation, the amplification procedure then.

Background technology

Stem cell (stem cell) is one type and has self, the highly propagation and the cell colony of multidirectional differentiation potential, is " forefathers' cell ", " the omnipotent cell " that forms the various tissues of human body, organ.Embryonic stem cell and adult stem cell can be divided into according to its etap, myeloid-lymphoid stem cell, multipotential stem cell and unipotent stem cell can be divided into again according to the differentiation potential size.

Mescenchymal stem cell (mesenchymal stem cells; MSCs) be one type of tissue stem cell with multidirectional differentiation potential; Can from multiple tissues such as marrow, peripheral blood, fat and skin, obtain; As one type of multipotential stem cell, it can be to direction proliferation and differentiation such as skeletonization, cartilage, muscle, nerve and insulin-like cell.At present MSCs is mainly derived from adult's marrow, but adult's bone marrow MSCs cell quantity and proliferation and differentiation potential descends with the increase of donor, and viral infection rate is higher, and gathers and have wound, makes its source limited.Recently, developed from umbilical cord or placenta tissue and extracted MSCs, it also is one type of stem cell with multidirectional differentiation potential; Increasing research shows that it has the exploitation potential quality; Compare with bone marrow MSCs and to have remarkable advantages: (1) source is sufficient, draws materials conveniently, and is with low cost; (2) mescenchymal stem cell content is abundant, and comparatively original, differentiation capability is strong, can separate, cultivate external, and amplification is rapid, and biological character is stable, and repeatedly going down to posterity amplification still can keep vigorous function, and competent cell source can be provided; (3) immunogenicity is extremely low, and the probability of immunological rejection is extremely low, and also has certain immune suppression function; (4) can hematopoiesis support with promote portability function; (5) the paracrine ability is strong, in the proliferate process, can produce the large number of biological active factor.

Stem cell not only can produce or secrete a large amount of biologically active factorss; And also contain abundant biologically active substance in the stem cell; These stem cell biological active factores or material can effectively be regulated and control the conduction of body cell signal, activating human body stem cell, and then the cell of physiological reparation or alternative body injury, pathology and aging etc.For example stem cell can produce stem cell factor (SCF), NGFF (NGF), stroma cell source property growth factor (SDF), vascular endothelial growth factor (VEGF), Prostatropin (bFGF), rhIGF-1 (IGF), Urogastron (EGF), interleukin-6 and interleukin-7 (IL-6 and IL-7), megakaryocyte colony stimulating factor (M-CSF), tumour necrosis factor (TNF), the Interferon, rabbit factors such as (IFN), and these cytokines have functions such as the cell proliferation of promotion, differentiation, anti-apoptosis; Stem cell can also produce natural immunity albumen, and for example IgG, IgA, IgM, IgD, IgE etc. can resist or repair the damage that self body cell is caused with extraneous factor.

Research shows human bone marrow, blood vessel, fatty tissue, Skelettmuskel, heart and brain, and the last intracutaneous that is also included within lung, liver, pancreas, digestive tube, skin, retina, breast, ovary, prostate gland and testis is all separable to corresponding stem cell.In theory, stem cell can be repaired the tissue defect at any position of health, can utilize stem cell to go to treat genetic diseases, malignant disease and degenerated disease etc.On October 10th, 2007; One piece of RP of international artery cell authority's serial publication of magazine " Cell " " Cell-Stem Cell " discloses the stem cell function reduction can cause the mankind aging, and proof stem cell aging course receives external and the common regulation and control of internal factor.

Therefore; The biologically active factors or the biologically active substance in the stem cell that utilize stem cell secretion to produce activate the body stem cell; And then through self stem cell physiological reparation or substitute body injury, pathology and old and feeble cell, have broad application prospects in diseases prevention and treatment and health and beauty field.Stem cell biological active factor that utilizes in the present reality or material are not enough below existing mostly: (1) directly utilizes tissue extraction such as placenta, and it is low to cause the stem cell biological active factor or the interior biologically active substance purity of stem cell that obtain; (2) utilize the nutrient solution that contains the animal source composition to carry out stem cell and (for example cultivate amplification; Use foetal calf serum); Inevitably contain the composition of animal-origin even contain in the secretory substance that extracts and be present in the intravital specific virus of animal etc., if be applied to clinical or then there is bigger potential safety hazard in health and beauty; (3) embryonic stem cell etc. is wayward in the proliferation and differentiation process; Tumorigenicity is big; Extract in the secretory substance of this type of stem cell and possibly contain the factor that makes the normal stem cell vicious transformation; And the differentiation of stem cells degree of tissue such as marrow is higher, begins to express the relevant people HLA, and directly application may produce immunological rejection; (4) stem cell contains abundant biologically active substance; Proliferation and differentiation of stem cell etc. is played an important role; But be not secreted into the extracellular at this type of proliferation and differentiation process material, do not occur extracting the patented technology of biologically active substance in the mescenchymal stem cell in the reality as yet.

In view of last; We extract stem cell at research and development from people's umbilical cord or placenta tissue; Utilize serum-free mescenchymal stem cell substratum (not containing the animal source composition) to carry out vitro culture, amplification; Extract excretory biologically active factors and the interior biologically active substance of stem cell in stem cell cultivation, the amplification procedure then, utilize this type biologically active factors or biologically active substance human activin self stem cell to come physiological reparation or alternative body injury, pathology and old and feeble histocyte.

Summary of the invention

The objective of the invention is: 1, provide can activation equipment soma cell biologically active factors crowd and the lysate (its staple is biologically active substance crowd in the stem cell) of stem cell; 2, the preparation method of the biologically active factors and the stem cell lysate of stem cell secretion is provided; 3, the purposes of the biologically active factors and the stem cell lysate of stem cell secretion is provided.

Technical scheme of the present invention is following:

From the umbilical cord of healthy subjects or placenta tissue, extract stem cell, utilize serum-free mescenchymal stem cell substratum (not containing the animal source composition) to carry out vitro culture, amplification, and keep high proliferation ability and the stable character of stem cell.Collect umbilical cord or placenta stem-cell and cultivate the waste liquid of changing in the amplification procedure, and place-80 ℃ of refrigerators to preserve; Umbilical cord or placenta stem-cell be external to be passaged to for the 5th generation at most, and the stem cell of collecting this moment not only output is high, active strong, and biological character is stable.With the waste liquid of collecting through thawing, low ternperature separation process, ultrafiltration with concentrate and can obtain containing the compsn that enriches biologically active factors; The stem cell of collecting is adjusted to suitable concn to be sealed in the frozen pipe; Through very low temperature liquid nitrogen multigelation to cytoclasis; Through modern biotechnology means such as low ternperature separation process, purifying, can obtain interior biologically active substance crowd of highly purified stem cell or compsn and be highly purified stem cell lysate again.

The whole range request of crossing does not have pollutions such as bacterium, fungi, virus, chlamydozoan and mycoplasma.

The stem cell that the present invention obtains is a mescenchymal stem cell; The biologically active factors or the material of preparation are rich in STEMCELLFACTOR crowd, polypeptide, natural immunity albumen and nucleic acid etc.; This biologically active factors or material crowd can promote body stem cells hyperplasia, differentiation; Activation equipment soma cell improves body immunity and anti-aging effects.This stem cell biological active factor or material group compound can be made into injection, paint, paste, creme or pulvis etc., at medical science, health care and beauty treatment fields splendid application prospect are arranged.

The present invention and currently available products or compared with techniques have clear superiority: first; Effective biologically active substance purity that prior art is directly extracted, output and active not high shortcoming have been overcome from tissues such as umbilical cord or placenta; The present invention cultivates, increases from the stem cell of umbilical cord or placenta tissue extraction; Collect high yield, highly purified stem cell; Utilize that modern technologies such as very low temperature are extracted, separation, purifying etc., thereby obtain high purity, high yield and highly active stem cell biological active factor or material; Second; Overcome and utilized the deficiency that contains animal source composition substratum in the prior art; Because utilizing the substratum that contains the animal source composition to carry out stem cell cultivates, (for example increases; Use foetal calf serum), inevitably contain the composition of animal-origin in the secretory substance of extraction even contain and be present in the intravital specific virus of animal etc., if be applied to clinical or then there is bigger potential safety hazard in health and beauty; The 3rd; Source of human stem cell of the present invention is umbilical cord or placenta tissue mescenchymal stem cell, and the proliferation and differentiation ability is strong, is easy to control; Biological character is stable; And the stem cell that the present invention utilized was strict controlled in for 5 generations with interior (comprising for the 5th generation), and the improper differentiation of cell is controlled in the lowest range, thereby had guaranteed the stable of quality product; The 4th; The present invention is broken through the physical property cracking to stem cell; Extracted the biologically active substance in the stem cell, because stem cell is not secreted into the extracellular at a lot of biologically active substances of proliferation and differentiation process, and these materials have vital role to the proliferation and differentiation of stem cell etc.

Embodiment

Embodiment 1, the preparation of umbilical cord tissue stem cell

Extract stem cell from the umbilical cord tissue of healthy subjects, this stem cell is mainly mescenchymal stem cell, and the stem cell of acquisition carries out vitro culture, amplification.Primary cell is collected the back by the cultivation of going down to posterity of 1: 2 ratio, when cell about 80% converges, utilizes pancreatin to digest, collect; Ratio by identical goes down to posterity; Going down to posterity at most was cultured to for the 5th generation, collected for 5 generations with the cell of interior (comprising for the 5th generation), and it is stable that the stem cell biological of collecting this moment is learned proterties; Competence for added value is strong, collects the useless nutrient solution of changing in cultivation, the amplification procedure simultaneously and in-80 ℃ of refrigerators, preserves.Detailed process is following:

1. gather the umbilical cord of healthy subjects;

2. remove blood vessel and outer membrane in the umbilical cord;

3. umbilical cord tissue is cut into 1～2mm³The fritter of size;

4. will shear the back fritter and plant in the petridish that is added with serum-free mescenchymal stem cell nutrient solution (diameter is 100mm), place 37 ℃, saturated humidity, 5%CO₂Cultivate in the incubator;

5. amount was changed liquid in per 3～4 days half, cultivated 14～16 days cells and grew and about 80% converge, and collect the useless nutrient solution of changing and in-80 ℃ of refrigerators, preserve;

6. when cell about 80% converges, utilize 0.25% pancreatin pair cell to digest, with 1～3 * 10⁵Cells/ml is inoculated in the cultivation of going down to posterity in the petridish, and collects the useless nutrient solution of changing and in-80 ℃ of refrigerators, preserve;

7. when cell about 80% converges, utilize 0.25% pancreatin pair cell to digest, collect the 1st generation cell, go down to posterity by 1: 2 ratio, place 37 ℃, saturated humidity, 5%CO₂Cultivate in the incubator, and collect the useless nutrient solution of changing and in-80 ℃ of refrigerators, preserve;

8. if need repeatedly go down to posterity, 7. repeating step gets final product;

9. separation, purifying obtain the high purity mescenchymal stem cell, utilize its mescenchymal stem cell purity of FACS Calibur flow cytometry analysis.

Embodiment 2, the preparation of placenta tissue stem cell

Extract stem cell from the placenta tissue of healthy subjects, this stem cell is mainly mescenchymal stem cell, and the stem cell of acquisition carries out vitro culture, amplification.Primary cell is collected the back by the cultivation of going down to posterity of 1: 2 ratio, when cell about 80% converges, utilizes pancreatin to digest, collect; Ratio by identical goes down to posterity; Going down to posterity at most was cultured to for the 5th generation, collected for 5 generations with the cell of interior (comprising for the 5th generation), and it is stable that the stem cell biological of collecting this moment is learned proterties; Competence for added value is strong, collects the useless nutrient solution of changing in cultivation, the amplification procedure simultaneously and in-80 ℃ of refrigerators, preserves.Detailed process is following:

1. gather the placenta amnion undertissue or the chorion tissue of healthy subjects;

2. PBS washes repeatedly and removes bloodstain;

3. placenta amnion undertissue or chorion tissue are cut into 1～2mm³The fritter of size;

4. will shear the back fritter and plant in the petridish that is added with serum-free mescenchymal stem cell nutrient solution (diameter is 100mm), place 37 ℃, saturated humidity, 5%CO₂Cultivate in the incubator;

5. amount was changed liquid in per 3～4 days half, cultivated 14～16 days cells and grew and about 80% converge, and collect the useless nutrient solution of changing and in-80 ℃ of refrigerators, preserve;

6. when cell about 80% converges, utilize 0.25% pancreatin pair cell to digest, with 1～3 * 10⁵Cells/ml is inoculated in the cultivation of going down to posterity in the petridish, and collects the useless nutrient solution of changing and in-80 ℃ of refrigerators, preserve;

7. when cell about 80% converges, utilize 0.25% pancreatin pair cell to digest, collect the 1st generation cell, go down to posterity by 1: 2 ratio, place 37 ℃, saturated humidity, 5%CO₂Cultivate in the incubator, and collect the useless nutrient solution of changing and in-80 ℃ of refrigerators, preserve;

8. if need repeatedly go down to posterity, 7. repeating step gets final product;

9. separation, purifying obtain the high purity mescenchymal stem cell, utilize its mescenchymal stem cell purity of FACS Calibur flow cytometry analysis.

The preparation of the biologically active factors of embodiment 3, umbilical cord or placenta tissue stem cell secretion

Stem cell is cultivated the liquid waste melt of collecting in the amplification procedure; Mixing, filter through 0.25 micron filter, is the ultrafiltration of 50KD Hollow Fiber Ultrafiltration post through molecular weight cut-off again; Liquid after the ultrafiltration is concentrated, can obtain containing the compsn that enriches the stem cell biological active factor.

The preparation of biologically active substance in embodiment 4, umbilical cord or the placenta tissue stem cell

Umbilical cord or placenta tissue stem cell concentration that vitro culture is obtained are adjusted into 5 * 10⁶～1 * 10⁷Individual/milliliter is sealed in the frozen pipe; Through very low temperature liquid nitrogen multigelation 4～5 times to cytoclasis; Through modern biotechnology means such as low ternperature separation process, purifying, just can obtain interior biologically active substance crowd of highly purified stem cell or compsn and be highly purified stem cell lysate again.

This biologically active substance crowd is mainly from mescenchymal stem cell; Comprise but be not limited to stem cell factor (SCF), NGFF (NGF), stroma cell source property growth factor (SDF), vascular endothelial growth factor (VEGF), Prostatropin (bFGF), rhIGF-1 (IGF), Urogastron (EGF), interleukin-6 and interleukin-7 (IL-6 and IL-7), megakaryocyte colony stimulating factor (M-CSF), tumour necrosis factor (TNF), the Interferon, rabbit factor, thymus nucleic acid (DNA) and Yeast Nucleic Acid (RNA), active polypeptide and immune protein IgG, IgA, IgM, IgD, IgE or the like such as (IFN); These materials can promote body stem cell growth, propagation; Play the effect of activation stem cell, fundamentally regulate human physiological functions.

The activity of biologically active substance checking in embodiment 5, umbilical cord or placenta tissue stem cell secretion biologically active factors and the stem cell

Biologically active substance in stem cell biological active factor or the stem cell is joined in the substratum by 15 mcg/ml concentration; And with the culture medium culturing of this kind concentration normally be passaged to the 8th generation umbilical cord or placenta mesenchyma stem cell (this in generation the multiplication capacity of cell weaken; The active reduction); The growth conditions of microscopically observation of cell, and in the 3rd day collecting cell, compare with control group; Add the group of biologically active substance in stem cell biological active factor or the stem cell, the mescenchymal stem cell multiplication capacity significantly strengthens.

The application of biologically active substance in medical science in embodiment 6, umbilical cord or placenta tissue stem cell secretion biologically active factors and the stem cell

Biologically active substance in stem cell biological active factor or the stem cell is joined in the nutrient solution by 10～50 mcg/ml concentration,, can promote the proliferation and differentiation of vitro culture stem cell, play the effect of activation stem cell function because of being rich in the STEMCELLFACTOR crowd.

The application of biologically active substance in health care and beauty treatment fields in embodiment 7, umbilical cord or placenta tissue stem cell secretion biologically active factors and the stem cell

Biologically active substance in stem cell biological active factor or the stem cell was processed healthcare products in 1: 1000～1: 10000 in the ratio adding specific food products; But get into activation equipment soma cell behind body; Repair or substitute impaired with old and feeble cell; Improve metabolism, improve Abwehrkraft des Koepers, delay senility.

Biologically active substance in stem cell biological active factor or the stem cell is joined in the makeup with 10～20 mcg/ml; Be used for skin outward; But activation skin progenitor cell; Promote skin progenitor cell propagation, differentiation, repair or alternative old and feeble skin cells, fundamentally change the physiological function of skin.

Claims (12)

1. the preparation and the application of people's derived stem cell secretion biologically active factors and lysate; It is characterized in that stem cell secretion biologically active factors and lysate derive from the stem cell of umbilical cord tissue or placenta tissue; Be rich in materials such as STEMCELLFACTOR crowd, active polypeptide, immune protein and nucleic acid in it; The useless nutrient solution that its preparation method is cultivated for 1. extracting people's derived stem cell → vitro culture, amplification → collections, change in the amplification procedure, and be stored in-80 ℃ of refrigerator → thawing give up nutrient solution also mixing → low ternperature separation process, ultrafiltration and concentrate → obtain the biologically active factors crowd or the compsn of high purity stem cell secretion; 2. extract people's derived stem cell → vitro culture, amplification → collection stem cell through very low temperature liquid nitrogen multigelation to cytoclasis → low ternperature separation process, purifying → obtain in the stem cell biologically active substance crowd or compsn be high purity stem cell lysate, its both purposes are the application of medical science, health care and beauty treatment fields.

2. stem cell according to claim 1 is characterized in that deriving from the umbilical cord tissue or the placenta tissue of healthy subjects, obtains through vitro culture, amplification.

3. stem cell according to claim 2 is characterized in that stem cell is a mescenchymal stem cell.

4. stem cell according to claim 2 is characterized in that umbilical cord or placenta tissue come derived stem cell to go down to posterity by 1: 2 ratio, is cultured to for the 5th generation at most, promptly collects for 5 generations with the cell of interior (comprising for the 5th generation), and its cytoactive height and proterties are stable.

5. stem cell according to claim 2, the used defined medium that it is characterized in that cultivating, increasing are serum-free mescenchymal stem cell substratum (not containing the animal source composition).

6. stem cell secretion biologically active factors according to claim 1 is characterized in that umbilical cord or placenta tissue come excretory biologically active factors in derived stem cell cultivation, the amplification procedure.

7. stem cell lysate according to claim 1 is characterized in that umbilical cord or placenta tissue come derived stem cell through interior biologically active substance.

8. the preparation method of stem cell secretion biologically active factors according to claim 1 is characterized in that useless nutrient solution is through low ternperature separation process, the post ultrafiltration of 50KD Hollow Fiber Ultrafiltration and concentrated obtaining.

9. the preparation method of stem cell lysate according to claim 1, it is characterized in that stem cell through very low temperature liquid nitrogen multigelation 4～5 times to cytoclasis, obtain through modern biotechnology means such as low ternperature separation process, purifying again.

10. the medical usage of stem cell secretion biologically active factors according to claim 1 and stem cell lysate; It is characterized in that biologically active substance can promote the stem cells hyperplasia differentiation in stem cell secretion biologically active factors and the stem cell; Through activation equipment soma cell, repair or substitute impaired, pathology and old and feeble cell.

11. the purposes of the healthcare field of stem cell secretion biologically active factors according to claim 1 and stem cell lysate; It is characterized in that biologically active substance is processed healthcare products with 1: 1000～1: 10000 ratio in stem cell secretion biologically active factors and the stem cell; Can improve Abwehrkraft des Koepers after getting into human body, delay senility.

12. the purposes of the beauty treatment fields of stem cell secretion biologically active factors according to claim 1 and stem cell lysate; It is characterized in that biologically active substance joins in the makeup with 10～20 mcg/ml concentration in emiocytosis biologically active factors and the stem cell; The activation skin progenitor cell fundamentally changes the physiological function of skin.