CN111484972B

CN111484972B - Method for obtaining mesenchymal stem cells with multidirectional differentiation potential and immunoregulation function from children foreskin culture

Info

Publication number: CN111484972B
Application number: CN202010485741.9A
Authority: CN
Inventors: 马廉
Original assignee: Shenzhen Childrens Hospital
Current assignee: Shenzhen Childrens Hospital
Priority date: 2020-06-01
Filing date: 2020-06-01
Publication date: 2022-07-08
Anticipated expiration: 2040-06-01
Also published as: CN111484972A

Abstract

The invention provides a method for obtaining mesenchymal stem cells with multidirectional differentiation potential and immunoregulation function from foreskin culture of children, which comprises the following steps: cleaning foreskin cut by circumcision with sterile PBS, and placing in a culture dish; longitudinally slicing the foreskin to diffuse the tissue, separating the epidermis from the dermis, and then cutting the dermis into small pieces to form a homogenate; uniformly inoculating the tissue homogenate into a culture bottle, horizontally placing the culture bottle to ensure that tissue blocks are uniformly distributed on the whole bottom surface as much as possible, and adding a culture medium for primary culture; and finally subculturing to the 3 rd generation to establish a mesenchymal stem cell bank. The culture method has the advantages of high speed, high yield, rich raw material sources, high purity of the cultured mesenchymal stem cells, multidirectional differentiation potential and immune regulation and control functions, capability of expressing more immunosuppressive related genes and chemotactic genes, and capability of inhibiting inflammatory response and treating various sepsis (infectivity (bacteria and viruses) and immunity).

Description

Method for obtaining mesenchymal stem cells with multidirectional differentiation potential and immunoregulation function from children foreskin culture

Technical Field

The invention relates to the field of stem cell culture, in particular to a method for obtaining mesenchymal stem cells with multidirectional differentiation potential and immunoregulation function from children foreskin culture.

Background

Before this, the staged results of stem cell therapy for sepsis have been obtained at home and abroad, the safety of MSCs for sepsis therapy is preliminarily confirmed through human clinical research, and certain curative effect is shown. Research has found that MSCs also play an active role in treating virus-related diseases such as Human Immunodeficiency Virus (HIV) immune abnormality, Hepatitis B Virus (HBV) chronic hepatitis, influenza virus Acute Lung Injury (ALI) and the like.

Mesenchymal Stem Cells (MSCs) are multipotent stromal cells present in various tissues and can be differentiated into different types of cells including osteoblasts, adipocytes, chondrocytes, myocytes, nerve cells, etc., and MSCs have become a hotspot in clinical cell therapy due to their great tissue regeneration potential, support of hematopoiesis, and immune regulation properties. MSCs were originally isolated from Bone Marrow (BM) and in recent years have been successfully obtained from other tissues, including adult and fetal human tissues. In recent years, skin tissue has been considered as a reservoir of regenerative cells, and there are approximately 20 different cell types of skin tissue, including classical fibroblasts, stromal cells, progenitor/precursor cells, and various types of stem cells, which have extremely high therapeutic value. Wherein, Foreskin (FSK) is a biological waste, is mainly obtained after infant circumcision, can be preserved, and is used as an indication for skin transplantation, and foreskin-derived fibroblast-like stromal cells have MSCs phenotype and multidirectional differentiation potential and meet International Society for Cell Therapy (ISCT) standard. Skin stromal cells isolated from human neonatal foreskin have also been reported to differentiate into endothelial cell lines, providing a new source of cells for tissue regeneration and vascularization of engineered tissues. The discarded infant foreskin tissue has rich and cheap sources, does not relate to ethical problems, and can be used as a substitute and a key source for cell therapy.

Research proves that although the different MSC populations have the same basic characteristics and characteristics, the immune characteristics and the responsiveness to inflammation have some differences, and the heterogeneity of the immune regulation function of the MSCs seriously influences the treatment effect of the MSCs, so that the clinical application of the MSCs is limited, and the problem to be solved is urgent at present.

At present, adipose tissues and human umbilical cord-derived MSCs are applied to clinical cell therapy and achieve certain effects, but the curative effect is not satisfactory. In addition, the adipose tissues are obtained by invasive extraction, and the yield of adipose tissue stem cells is low; the umbilical cord is the source of mesenchymal stem cells, but can only be obtained at birth, the time limitation is strong, and the yield and purity of the adipose tissue and human umbilical cord-derived MSCs mesenchymal stem cells cannot meet the standard of multiple clinical use dosage. However, foreskin diseases such as phimosis of children and the like have high incidence, and the foreskin is the utilization of surgical waste, so the sources of the foreskin are rich, and the problem of ethics is not involved.

Therefore, the invention aims to provide a method for obtaining mesenchymal stem cells with multidirectional differentiation potential and immunoregulation function from foreskin culture of children.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for obtaining mesenchymal stem cells (FSK-MSCs) with multidirectional differentiation potential and immunoregulation function from the culture of foreskin of children, wherein the mesenchymal stem cells are cultured by utilizing waste tissues-the tissue of the foreskin of children with high yield and high purity, the obtained cells have the phenotype of MSCs and the multidirectional differentiation potential, meet the International Society for Cell Therapy (ISCT) standard and meet the multiple clinical treatment dosage, and the genomics expresses more immunosuppressive related genes and chemotactic genes than the MSCs of other sources such as umbilical cord and the like, is more suitable for the treatment of various sepsis (infectious, bacteria, viruses and immunity), can more effectively inhibit inflammatory factor storm, and thus treat the sepsis.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a method for obtaining mesenchymal stem cells with multidirectional differentiation potential and immune regulation function from foreskin culture of children, which comprises the following steps:

step one, cleaning foreskin cut by circumcision with sterile PBS, and placing the foreskin in a culture dish;

step two, longitudinally slicing the foreskin to diffuse tissues, separating epidermis and dermis, and then cutting the dermis into small blocks to form tissue homogenate;

step three, uniformly inoculating the tissue homogenate into a culture bottle, horizontally placing the culture bottle to ensure that tissue blocks are uniformly distributed on the whole bottom surface as much as possible, and adding a culture medium to carry out primary culture;

and step four, carrying out primary culture until the liquid is changed once in a total amount for 5-10 days, carrying out subculture when the adherent cells are cultured to reach a sub-fusion state, and culturing until the 3 rd generation establishes a mesenchymal stem cell bank.

Further, the medium in step three is DMEM/f12 medium containing FBS (fetal bovine serum) or mesenchymal stem cell serum-free medium containing penicillin/streptomycin.

Further, the concentration of FBS in the above DMEM/f12 medium was 10%.

Further, the concentration of penicillin/streptomycin in the serum-free culture medium of the mesenchymal stem cells is 50U/mL.

Further, in step one the foreskin may be placed in sterile phosphate buffered saline containing penicillin/streptomycin prior to treatment.

Further, the above culture conditions were 37 ℃ and 5% CO by volume fraction₂The constant temperature and humidity incubator.

Further, subculture in step four was digested with trypsin.

The second aspect of the present invention is to provide the mesenchymal stem cells cultured by the above method.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

(1) high speed: the FSK-MSCs cultured by the invention have higher growth speed and proliferation speed compared with the MSCs from the umbilical cord, and the foreskin tissue and the Wharton's jelly tissue with equal weight of 0.2 g are taken and cultured and subcultured by the same operation under the same sample condition so as to obtain 2.86 hundred million 2 nd generation cells, the umbilical cord needs 26 days, and the foreskin only needs 20 days.

(2) High yield: the foreskin excised from the child phimosis operation is only 0.5-1 g, the invention subcultures to 3 rd generation for cell counting by high speed and high yield culture method per gram of foreskin, the 3 rd generation obtains 21.45 hundred million cells, the fifth generation obtains 193.05 hundred million cells, while the 5 th generation mesenchymal stem cells are selected for clinical treatment, the dosage is exemplified by the therapeutic dosage of COVID-19 of 300 ten thousand/Kg, assuming that the patient body weight is 50Kg, the therapeutic dosage is 1500 ten thousand cells per time, so the cell amount of 128.7 therapeutic dosages can be generated per gram of foreskin. While umbilical cord-derived MSCs produced only 16.45 million generation 3 cells and 148.05 million generation 5 cells per gram of wharton's jelly.

(3) High purity: detecting the expression conditions of MSCs phenotype CD73, CD90, CD105, CD45, CD34, CD14, CD19 and HLA-DR of the FSK-MSCs by using a single cell sequencing technology, comparing the expression conditions with umbilical cord source MSCs, and verifying that the FSK-MSCs have the MSCs phenotype and multidirectional differentiation potential by using a flow cytometer, and verifying the result again, thereby indicating that the FSK-MSCs do have the MSCs phenotype and accord with International Society for Cell Therapy (ISCT) standards.

(4) Multidirectional differentiation potential: detecting the expression of adipocyte markers ADIPOR1, ADIPOR2, BSCL2, CD106, CD137, CD29, CD34, CD44, CD45, CHC22 and CIDEA by using a single cell sequencing technology; neuronal markers PDGFD, RYR3, SDK2, STK32B, TRPC 3; primordial germ cell markers ITGB1, KIT, NR6a1, ID4, FOXO1, UCHL1, MEGAA4, POU5F1, PRMT 5; bone cell markers SOST, PFN1, EFNB2, PDPN, FGF 23; the epithelial cell marker KRT7 shows that FSK-MSCs have multidirectional differentiation potential and meet the International Society for Cell Therapy (ISCT) standard.

(5) The biological characteristics of the FSK-MSCs cultured by the invention can inhibit inflammatory reaction, and the genomics expresses more immunosuppressive related genes and chemotactic genes compared with MSCs from other sources such as umbilical cords.

(6) Compared with MSCs from other sources such as umbilical cords, the cultured FSK-MSCs have stronger immune regulation function, secrete more inflammatory factors, particularly inhibit related inflammatory factors through immunosuppression, and inhibit inflammatory factor storm more strongly.

(7) Compared with MSCs from other sources such as umbilical cords, the FSK-MSCs cultured by the invention are more suitable for treating various sepsis (infectivity: bacteria, virus and immunity).

In conclusion, the method for obtaining the mesenchymal stem cells with the multidirectional differentiation potential and the immunoregulation function from the foreskin of the children provided by the invention has the advantages of high speed, high yield and rich raw material sources, and the mesenchymal stem cells obtained by the method have high purity, the multidirectional differentiation potential and the immunoregulation function, can express more immunosuppressive related genes and chemotactic genes, and can be used for treating various sepsis (infectious diseases (bacteria and viruses) and immunity) caused by inflammatory reaction.

Drawings

FIG. 1 is a diagram of FSK-MSCs in phase contrast microscopy according to an embodiment of the present invention;

FIG. 2 is a typical flow cytometry histogram of positive cells from the clusters of differentiation of the MSCs markers CD73, CD90, and CD105 and their negative cells to the human leukocyte antigens HLA-DR, CD45, CD19, CD14, and CD34 in one embodiment of the present invention; wherein fig. 2a1 is a positive cell of MSCs marker cluster of differentiation CD73, fig. 2a2 is a positive cell of MSCs marker cluster of differentiation CD90, fig. 2A3 is a positive cell of MSCs marker cluster of differentiation CD105, fig. 2a4 is a negative cell of MSCs marker cluster of differentiation HLA-DR, fig. 2a5 is a negative cell of MSCs marker cluster of differentiation CD45, fig. 2a6 is a negative cell of MSCs marker cluster of differentiation CD19, fig. 2a7 is a negative cell of MSCs marker cluster of differentiation CD14, and fig. 2A8 is a negative cell of MSCs marker cluster of differentiation CD 34;

FIG. 3 is a graph showing the results of single cell sequencing for detecting expression of phenotype-associated genes in MSCs according to one embodiment of the present invention;

FIG. 4 is a diagram showing the result of single cell sequencing to detect the expression of sternness genes in FSK-MSCs according to one embodiment of the present invention;

FIG. 5 is a graph showing the results of single cell sequencing technology detecting the expression of adipocytes markers ADIPOR1, ADIPOR2, BSCL2, CD34, CD44 and CIDEA by FSK-MSCs in accordance with one embodiment of the present invention;

FIG. 6 is a graph showing the results of detecting FSK-MSCs expressing neuronal markers PDGFD, RYR3, SDK2, STK32B, TRPC3 according to the single cell sequencing technique of the present invention;

FIG. 7 is a graph of the results of single cell sequencing technology detecting FSK-MSCs expressing primary germ cell markers ITGB1, KIT, NR6A1, ID4, FOXO1, UCHL1, POU5F1, and PRMT5 in one embodiment of the present invention;

FIG. 8 is a graph showing the results of single cell sequencing technology detecting FSK-MSCs expressing bone cell markers PFN1, EFNB2, and PDPN in accordance with one embodiment of the present invention;

FIG. 9 is a graph showing the results of single cell sequencing technology detecting FSK-MSCs expressing lung-associated markers SCGB3A1, SFTPD, GPRC5A, ABCA3 in accordance with one embodiment of the present invention;

FIG. 10 is a graph showing the results of single cell sequencing technology detecting FSK-MSCs expressing the epithelial cell marker KRT7 in accordance with one embodiment of the present invention;

FIG. 11 shows the detection of HuMSCs expressing immune related genes by single cell sequencing in an embodiment of the present invention: graphs of results for TGFB1, TNFAIP6, IL10, HMOX1, IDO1, LIF, IL6, CD274, PTGS 1;

FIG. 12 shows the detection of expression chemotaxis-associated markers for HuMSCs by single cell sequencing in accordance with an embodiment of the present invention: CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CXCL12, PDGFRA, PDGFRB;

FIG. 13 shows the detection of FKMSCs expressing immune-related genes by single cell sequencing according to an embodiment of the present invention: results plots for TGFB1, TNFAIP6, IL10, HMOX1, IDO1, LIF, IL6, CD274, PTGS 1;

FIG. 14 shows the detection of FKMSCs expression chemotaxis-associated markers by single cell sequencing according to an embodiment of the present invention: CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CXCL12, PDGFRA, PDGFRB;

FIG. 15 is a bar graph of single cell sequencing measurements of the percentage of HuMSCs, FKMSCs expressing TGFB1, TNFAIP6 in accordance with one embodiment of the present invention;

FIG. 16 is a bar graph of the percentage of cells in HuMSCs, FKMSCs that simultaneously express chemokines, TGFB1, and TNFAIP6 detected by single cell sequencing in accordance with one embodiment of the present invention;

FIG. 17 is a graph showing the analysis of the results of the immunoregulation of FSK-MSCs and human umbilical cord-derived MSCs in an ELISA assay according to an embodiment of the present invention.

Detailed Description

The invention provides a method for obtaining mesenchymal stem cells with multidirectional differentiation potential and immunoregulation function from foreskin culture of children, which comprises the following steps:

In a preferred embodiment of the invention, the culture medium in step three is DMEM/f12 medium containing FBS or mesenchymal stem cell serum-free medium containing penicillin/streptomycin.

In a preferred embodiment of the present invention, the concentration of FBS in the DMEM/f12 medium is 10%.

In a preferred embodiment of the present invention, the concentration of penicillin/streptomycin in the serum-free culture medium of mesenchymal stem cells is 50U/mL.

In a preferred embodiment of the invention, the foreskin in step one may be placed in sterile phosphate buffered saline containing penicillin/streptomycin prior to treatment.

In a preferred embodiment of the present invention, the above-mentioned culture conditions are 37 ℃ and 5% CO by volume₂The constant temperature and humidity incubator.

In a preferred embodiment of the invention, the subculture in step four is digested with trypsin.

The invention also provides the mesenchymal stem cells cultured by the method.

The present invention will now be described in detail and specifically with reference to the following examples so as to provide a better understanding of the present invention, but the following examples are not intended to limit the scope of the present invention.

Example 1

The embodiment provides a method for obtaining mesenchymal stem cells with multidirectional differentiation potential and immunoregulation function from foreskin culture of children, which comprises the following specific steps:

(1) after circumcision, foreskin samples were collected in a sterile sample container containing sterile phosphate buffered saline (PBS, Lonza) supplemented with penicillin/streptomycin (Lonza).

(2) After reaching the laboratory, washed with sterile PBS and transferred to petri dishes. FSK was then longitudinally sliced to diffuse the tissue, the epidermis and dermis were separated, and the dermis was then cut into small pieces to obtain a tissue homogenate.

(3) Uniformly inoculating the tissue homogenate into a T75 culture bottle, horizontally placing the culture bottle to ensure that tissue blocks are uniformly distributed on the whole bottom surface as much as possible, adding a mesenchymal stem cell serum-free culture medium (Lonza) added with 50U/mL penicillin/streptomycin (Lonza), wherein the culture medium forms a complete culture medium for subsequent culture, placing the complete culture medium at 37 ℃, and placing the complete culture medium with the volume fraction of 5% CO₂The carbon dioxide constant temperature and humidity incubator performs primary culture until cells climb out of the periphery of the tissue block.

(4) And carrying out primary culture for 5-10 days, and completely changing the liquid once, wherein non-adherent cells are removed when the liquid is changed. When sub-confluency (80-90%) of the adherent cells was achieved, they were digested with trypsin (Gibco, life technologies) and passaged, and subcultured repeatedly to passage 3 to establish various cell banks. In the culture process, the morphology and cell size of FSK-MSCs under a phase contrast microscope are shown in FIG. 1. As can be seen from FIG. 1, FSK-MSCs are typically fibroblast-like, in elongated, adherent and fusiform form.

Example 2

(1) after circumcision, the foreskin sample is collected in a sterile sample container containing sterile phosphate buffered saline (PBS; Lonza) supplemented with penicillin/streptomycin (Lonza).

(2) After reaching the laboratory, washed with sterile PBS and transferred to petri dishes. FSK was then longitudinally sliced to diffuse the tissue, the epidermis and dermis were separated, and the dermis was then diced to give a tissue homogenate.

(3) Uniformly inoculating the tissue homogenate into a T75 culture bottle, horizontally placing the culture bottle to make the tissue blocks uniformly distributed on the whole bottom surface as much as possible, adding DMEM/f12 culture medium (Lonza) added with 10% FBS (Sigma-Aldrich) to form a complete culture medium for subsequent culture, placing at 37 deg.C, and adding 5% CO by volume₂The carbon dioxide constant temperature and humidity incubator performs primary culture until cells climb out of the periphery of the tissue block.

(4) And carrying out primary culture for 5-10 days, and completely changing the liquid once, wherein non-adherent cells are removed when the liquid is changed. When sub-confluency (80-90%) of the adherent cells was achieved, they were digested with trypsin (Gibco, life technologies) and passaged, and subcultured repeatedly to passage 3 to establish various cell banks.

Verification example 1

This example investigated the yields of the methods of example 1 and example 2, and the specific operating procedures and results were as follows:

and (3) culturing 0.5 g of foreskin according to the culture scheme, subculturing to the 3 rd generation, and counting cells, wherein the cell amount obtained in the 3 rd generation is 10.725 hundred million cells, and the cell amount obtained in the fifth generation is 96.525 hundred million cells.

Verification example 2

In this example, the mesenchymal stem cells provided in the first example were subjected to cell phenotype identification by flow cytometry and single cell sequencing, and the specific steps and results are as follows:

one, flow cytometry

The experimental steps are as follows:

step 1: taking 1 branch of flow tube, and adding 100 mu l of FSK-MSC cell suspension; (cell number about 1.5X10⁵)

Step 2: the cells were washed with 1ml of incubation buffer, centrifuged at 300g for 5min and the supernatant removed. Repeating for 2 times

And step 3: adding 200 microliters of incubation buffer solution, resuspending, and dividing into 1 and 2 tubes

And 4, step 4: sealing at room temperature for 5 min.

And 5: isotype control antibody was added to tube 1; and adding antibodies of CD73/CD90/CD105/CD34/CD 45/CD 19/HLA-DR into the tube 2, and incubating for 1h in the dark.

Step 6: in the same step, washing with an incubation buffer solution, centrifugally recycling cells, adding 1ml of the incubation buffer solution for re-suspension, uniformly mixing, centrifuging for 5min at 500g, and when removing the supernatant for the second time, leaving about 200 mu l of liquid at the bottom for uniformly mixing; mixing gently, and loading on the machine within 1h, with the result shown in FIG. 2.

As can be seen in fig. 2, the establishment of cell phenotype by flow cytometry using a panel of surface markers, showing positive cells for the MSCs marker cluster of differentiation CD73, CD90, and CD105 and their negative cells for CD45, CD34, CD14, CD19, and the human leukocyte antigen HLA-DR, indicates that FSK-MSCs do have MSCs phenotype, meeting International Society for Cell Therapy (ISCT) criteria.

Second, single cell sequencing

By utilizing a 10x Genomics chromeum system, Gel Bead with sequence labels, a sample and reagent premixed liquid and oil are loaded to respective sample introduction channels, and a double-cross system formed by a micro-fluid channel network is used for finally forming single-cell micro-reaction system GEMs wrapped by oil drops. The method comprises the following specific steps:

step 1: cell preparation

Performing quality inspection and counting on the single cell suspension, generally requiring the cell survival rate to be more than 80%, washing and re-suspending the qualified cells to prepare cells with proper cell concentration of 700-1200 cells/mu l for 10x Genomics chromosome^TMAnd operating the system on the computer.

Step 2: GEM generation and tagging

According to the expected number of obtained target cells, constructing GEMs (gel Bead in emulsion) for single cell separation, and after the GEMs are normally formed, collecting the GEMs and carrying out reverse transcription in a PCR instrument to realize labeling.

And step 3: post GEM-RT purification and cDNA amplification

And (3) carrying out oil breaking treatment on the GEMs, purifying and enriching a single-strand cDNA by using magnetic beads, and then carrying out cDNA amplification and quality inspection.

And 4, step 4: library construction and quantification

And (3) carrying out second-generation sequencing library construction on the cDNA qualified for quality inspection, and finally carrying out quantitative quality inspection on the library through the experimental processes of fragmentation, connection of sequencing joints, sample Index PCR and the like.

And 5: sequencing on machine

Sequencing the constructed library, using an Illumina Hiseq or Novaseq platform, adopting a PE150 sequencing mode, and recommending the sequencing quantity to reach 50k reads/cell or above, wherein the result is shown in figures 3-9.

As can be seen in FIG. 3, the phenotype-associated genes of MSCs were expressed; as can be seen from FIG. 4, the sternness gene expression of FSK-MSCs; as can be seen from fig. 5, FSK-MSCs expressed adipocyte markers ADIPOR1, ADIPOR2, BSCL2, CD34, CD44, CIDEA; as can be seen in FIG. 6, FSK-MSCs express neuronal markers PDGFD, RYR3, SDK2, STK32B, TRPC 3; as can be seen in fig. 7, FSK-MSCs express the primitive germ cell markers ITGB1, KIT, NR6a1, ID4, FOXO1, UCHL1, POU5F1, PRMT 5; as can be seen from FIG. 8, FSK-MSCs express osteocyte markers PFN1, EFNB2, PDPN; as can be seen in FIG. 9, FSK-MSCs expressed lung-associated markers SCGB3A1, SFTPD, GPRC5A, ABCA 3; as can be seen in FIG. 10, FSK-MSCs expressed the epithelial cell marker KRT 7.

Verification example 3

In this example, the study of immunological characteristics of the mesenchymal stem cells provided in example 1 was performed by single cell sequencing and enzyme-linked immunosorbent assay, and the specific steps and results were as follows:

one, single cell sequencing and results

The single cell sequencing method was the same as that performed in validation example 2, and the results are shown in FIGS. 10-15.

As can be seen in fig. 11, HuMSCs express immune-related genes: TGFB1, TNFAIP6, IL10, HMOX1, IDO1, LIF, IL6, CD274, PTGS 1.

As can be seen in fig. 12, HuMSCs express chemotaxis-associated markers: CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CXCL12, PDGFRA, PDGFRB.

As can be seen in fig. 13, FSK-MSCs express immune-related genes: TGFB1, TNFAIP6, IL10, HMOX1, IDO1, LIF, IL6, CD274, PTGS 1.

As can be seen in fig. 14, FSK-MSCs express chemotaxis-associated markers: CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CXCL12, PDGFRA, PDGFRB.

As can be seen from FIGS. 15-16, HuMSCs, FSK-MSCs express the percentage of TGFB1, TNFAIP 6: FKMSCs high expression anti-inflammatory factor TGF-beta and TSG-6 genes, which are 79% and 26% respectively; the HuMSCs account for 64 percent and 6 percent respectively, the proportion of FKMSCs simultaneously expressing TGF-beta and TSG-6 genes reaches 22 percent, and the proportion of HuMSCs simultaneously expressing TGF-beta and TSG-6 genes is 6 percent.

Second, ELISA adsorption experiments and results

Preparation work before detection:

1. please remove the kit from the refrigerator 20 minutes in advance to equilibrate to room temperature.

2. The 20 Xconcentrated wash was diluted to 1 Xworking solution with double distilled water. The unused material was returned to 4 ℃.

3. And (5) adding the standard substance and specimen universal diluent into the freeze-dried standard substance, standing for 15 minutes, and gently mixing (the concentration is 1000pg/ml) after the standard substance and specimen universal diluent are fully dissolved. And then diluted as necessary. (the following concentrations: 200, 100, 50, 25, 12.5, 6.25, 3.125, 0pg/ml were used for the standard curve for recommendation). The reconstituted standard stock solution (1000pg/ml) was discarded unless it was used up.

4. Biotinylated antibody working solution: 30 Xconcentrated biotinylated antibody was diluted to 1 Xworking solution with biotinylated antibody diluent 20 minutes prior to use, as needed for the current experiment. It is used on the same day.

5. Enzyme conjugate working solution: 30 Xconcentrated enzyme conjugate was diluted to 1 Xworking solution with enzyme conjugate diluent 20 minutes prior to use, as required for the current experiment. It is used on the same day.

The method comprises the following operation steps:

1. the panels required for the test were removed from the sealed bag which had been allowed to equilibrate to room temperature, the unused panels and desiccant were returned to the aluminum foil bag to compact the self-sealing strips, the bag was sealed and returned to 4 ℃.

2. The blank wells are added with standard products and general diluent for the standard products, the other corresponding wells are added with the standard products or the standard products with different concentrations (100 mu L/well), the reaction wells are sealed by sealing plate gummed paper, and the incubator is incubated for 90 minutes at 37 ℃.

3. Biotinylated antibody working solutions were prepared 20 minutes earlier.

4. The plate was washed 5 times.

5. The biotinylated antibody dilution was added to the blank wells and the biotinylated antibody working solution (100. mu.L/well) was added to the remaining wells. The reaction wells were sealed with new sealing plate gummed paper and incubated at 37 ℃ for 60 minutes.

6. The enzyme conjugate working solution was prepared 20 minutes earlier. Protected from light and placed at room temperature (22-25 ℃).

7. The plate was washed 5 times.

8. The blank wells were loaded with enzyme conjugate dilutions and the remaining wells were loaded with enzyme conjugate working solution (100. mu.L/well). The reaction wells were sealed with new sealing plate gummed paper, incubated at 37 ℃ for 30 minutes in the dark.

9. And (4) turning on a power supply of the microplate reader, preheating the instrument, and setting a detection program.

10. The plate was washed 5 times.

11. Adding 100 mu l/hole of chromogenic substrate (TMB), incubating at 37 ℃ in the dark, and incubating for 15 minutes in the dark.

12. Add 100. mu.l/well of the reaction terminator and measure the OD450 value immediately after mixing (within 3 minutes). The reading results are saved in the machine and a paper result is printed, the result being shown in figure 17.

13. And after the experiment is finished, putting the unused reagent back to the refrigerator for storage according to the specified storage temperature.

As can be seen from fig. 17, FSK-MSCs showed stronger immunomodulatory properties than human umbilical cord-derived MSCs: the contents of inflammatory inhibitory factors IL-10, TGF-beta and the like secreted by the FSK-MSCs are higher than that of HuMSCs, and the effect of the FSK-MSCs in down-regulating proinflammatory factors IL-1 and TNF-alpha in the inflammatory reaction process is more obvious than that of the HuMSCs.

In conclusion, the invention provides a method for obtaining mesenchymal stem cells with multidirectional differentiation potential and immunoregulation function from foreskin culture of children, wherein the mesenchymal stem cells are cultured by utilizing waste tissues-foreskin tissues of children with high yield and high purity, and the obtained cells have MSCs phenotype and multidirectional differentiation potential, meet the international cell therapy society (ISCT) standard, meet multiple clinical treatment dosage and can more effectively inhibit inflammatory factor storm so as to treat sepsis.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Claims

1. The application of the mesenchymal stem cells obtained from the culture of foreskin of children in the preparation of the medicine for treating sepsis is characterized in that the method for obtaining the mesenchymal stem cells comprises the following steps:

step three, uniformly inoculating the tissue homogenate into a culture bottle, horizontally placing the culture bottle to ensure that tissue blocks are uniformly distributed on the whole bottom surface as much as possible, and adding a culture medium to perform primary culture;

2. The use according to claim 1, wherein the medium in step three is DMEM/f12 medium containing FBS or mesenchymal stem cell serum-free medium containing penicillin/streptomycin.

3. The use according to claim 2, wherein the concentration of FBS in DMEM/f12 medium is 10%.

4. The use of claim 2, wherein the concentration of penicillin/streptomycin in the serum-free medium of mesenchymal stem cells is 50U/mL.

5. Use according to claim 1, wherein in step one the foreskin is placed in sterile phosphate buffered saline containing penicillin/streptomycin prior to treatment.

6. The use according to claim 1, wherein the culture conditions in step three are 37 ℃ and 5% CO by volume₂The constant temperature and humidity incubator.