CN111759864B

CN111759864B - Application of amniotic fluid stem cells in preparation of medicine for treating lupus nephritis

Info

Publication number: CN111759864B
Application number: CN202010672436.0A
Authority: CN
Inventors: 陈香美; 白雪源; 张俊铭
Original assignee: Chinese PLA General Hospital
Current assignee: Chinese PLA General Hospital
Priority date: 2020-07-14
Filing date: 2020-07-14
Publication date: 2022-09-13
Anticipated expiration: 2040-07-14
Also published as: CN111759864A

Abstract

The invention provides an application of amniotic fluid stem cells in preparing a medicament for treating lupus nephritis. The amniotic fluid stem cell can down-regulate the expression of proinflammatory factors IL-17 and IL-12p40 and up-regulate the expression of an inflammation factor IL-1ra, and can down-regulate proinflammatory M1 type macrophages, dendritic cells, eosinophilic granulocytes and up-regulate inflammatory Th2 cells so as to play a therapeutic effect. The amniotic fluid cells are easy to obtain and have no ethical disputes; the culture is simple; strong differentiation capability, low immunogenicity and the like. The invention provides a new strategy for treating the systemic lupus erythematosus and the complication lupus nephritis.

Description

Application of amniotic fluid stem cells in preparation of medicine for treating lupus nephritis

Technical Field

The invention relates to a new application of amniotic fluid stem cells, in particular to an application of amniotic fluid stem cells in preparing a medicine for treating lupus nephritis.

Background

Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease involving multiple organs, and various factors are closely related to the onset of SLE, including genetic, pharmaceutical, environmental, infection, endocrine and mental factors, which cause immune dysfunction and immune tolerance abnormality of the body, and a large amount of autoantibodies such as anti-dsDNA antibodies and antinuclear antibodies are produced, and thus, various organs are involved, and great harm is brought to the health of people. SLE is common in female patients (with a ratio of about 1:9 for men and women), especially in women of childbearing age. Although less common, male patients have more severe disease activity and clinical symptoms than female patients. SLE incidence and prevalence are higher in asian populations than in european populations, in terms of genetic background.

Lupus Nephritis (LN) is one of the most common and serious complications of SLE, and it is currently considered that LN is a complex immune complex-mediated immune Nephritis, and its possible pathogenesis is that the immune complex deposits in kidney nephron, thereby activating body's complement system and causing a series of immune injury reactions, but its specific pathogenesis has not been fully elucidated. From an epidemiological perspective, asian and hispanic populations are more susceptible to LN and more severe than are european populations. Currently, the first line drugs of lupus nephritis are hormones and immunosuppressants, but are not effective for some refractory lupus nephritis patients, and can cause serious side effects in long-term use. Therefore, the search for safe and effective therapeutic drugs is urgent.

Stem cells are important research hotspots in the life science field in recent years, and besides the capacity of self-renewal and differentiation into specific cells under certain conditions, the stem cells also have an important capacity, namely, the stem cells can play a role in immune regulation and control in an autocrine/paracrine mode, and belong to immune cells to a certain extent, and the remarkable characteristic opens up a new field for the treatment of immune diseases. Mesenchymal stem cells are a research hotspot in the field of stem cells, are generally recognized to have the function of immune regulation and can be used for treating immune diseases. At present, animal experiments and clinical applications prove that mesenchymal stem cells derived from bone marrow, umbilical cord and the like can delay the progress of lupus nephritis through immune regulation, but the use of mesenchymal stem cells of bone marrow, fat and the like for treating diseases has two non-negligible defects: firstly, mesenchymal stem cells can cause damage to donors in different degrees when the materials are taken; secondly, the number, proliferation and differentiation capacity of mesenchymal stem cells decreases with the age of the donor.

The amniotic fluid stem cells are emphasized by researchers due to the characteristics of wide sources, simple separation operation and low immunogenicity, and the research reports that the amniotic fluid stem cells progress in treating various diseases of a nervous system, a cardiovascular system and a urinary system at present; meanwhile, Hauser et al found through studies on acute kidney injury models that amniotic fluid stem cell transplantation could improve The self-repair ability of kidneys after acute kidney injury [ Hauser P V, De Fazio R, Bruno S, et al.Stem cells derived from human immunological fluid recovery [ J ]. The American journel of technology, 2010,177(4):2011-21 ]. However, whether amniotic fluid stem cells can play a role in treating lupus nephritis is still unknown at present.

Disclosure of Invention

The invention aims to provide a new application of amniotic fluid stem cells, and particularly relates to an application of the amniotic fluid stem cells in preparation of a medicine for treating lupus nephritis.

The amniotic fluid stem cells are successfully separated, cultured and identified from the amniotic fluid in the middle pregnancy period of the human, and the amniotic fluid stem cells are found to be capable of reducing the urinary protein level of lupus mice, improving the pathological damage of the kidney, reducing the production of proinflammatory factors (IL-17 and IL-12p40) and increasing the production of inflammation-inhibiting factors (IL-1ra) in the treatment of lupus nephritis, so that the amniotic fluid stem cells are proved to be effective in treating lupus nephritis. The invention further discovers that the amniotic fluid stem cell treatment obviously relieves the pathological damage of lupus nephritis and effectively treats the lupus nephritis by regulating down the mechanism of inflammatory M1 type macrophages, dendritic cells and eosinophils and regulating up inflammatory Th2 cells.

Therefore, in one aspect, the invention provides application of amniotic fluid stem cells in preparation of a medicine for treating lupus nephritis.

On the other hand, the invention provides the application of the amniotic fluid stem cells in preparing the medicine for treating the systemic lupus erythematosus.

According to a specific embodiment of the present invention, the amniotic fluid stem cells of the present invention have the ability to differentiate into adipocytes and/or osteocytes.

According to a specific embodiment of the present invention, the amniotic fluid stem cells of the present invention are HLA-ABC positive for MHC class I molecules and HLA-DR negative for MHC class II molecules.

According to a particular embodiment of the invention, the amniotic fluid stem cells of the invention are derived from human amniotic fluid in the middle of gestation.

According to a specific embodiment of the present invention, the amniotic fluid stem cells of the present invention are isolated by differential adherence and mechanical separation. In some embodiments of the invention, the amniotic fluid stem cells in amniotic fluid at the middle stage of pregnancy are separated by differential adherence and a mechanical separation method, the amniotic fluid stem cells are separated smoothly, and after epithelial-like cells are scraped by a cell scraper for about 3 times and collected, fibroblast-like cells with uniform shapes are obtained and are arranged in a radial shape and in a tight arrangement. Can be induced to differentiate into fat cells and bone cells under appropriate conditions, and flow cytometry detection of surface markers thereof proves that the surface markers express mesenchymal stem cell markers (CD29, CD44, CD73, CD90, CD105) and pluripotent stem cell markers (SSEA-4), do not express hematopoietic stem cell markers (CD34, CD45, CD133) and do not express MHC class II antigen HLA-DR so as to prove that the surface markers have low immunogenicity.

According to the specific embodiment of the invention, the amniotic fluid stem cells can reduce the urinary protein level of lupus mice and improve the kidney pathological damage of lupus individuals. Namely, the medicine is used for reducing the urinary protein level of lupus individuals and improving the pathological damage of the kidney of the lupus individuals.

According to a specific embodiment of the invention, the amniotic fluid stem cells in the invention can down-regulate the expression of proinflammatory factors IL-17 and IL-12p 40; up-regulating the expression of inflammation-inhibiting factor IL-1 ra.

According to a particular embodiment of the invention, the amniotic fluid stem cells of the invention are capable of down-regulating pro-inflammatory macrophages of type M1, dendritic cells and eosinophils.

M1 type macrophages are pro-inflammatory and secrete proinflammatory factors IL-6, IL-1 beta and the like. The proportion of M1 type macrophage infiltration in the kidney of patients with lupus nephritis is significantly higher than that of normal people. Dendritic cells can infiltrate into the kidney for antigen presentation, so as to activate lymphocytes and amplify inflammatory expression; eosinophils are cells that are extremely important in immune response inflammation and can release the contents of the granules to cause tissue damage and promote the progression of inflammation.

According to a specific embodiment of the present invention, the amniotic fluid stem cells of the present invention can up-regulate inflammatory Th2 cells. Th2 cell belongs to anti-inflammatory cell, and secretes anti-inflammatory factor (IL-4, IL-10).

In some embodiments of the invention, the urinary protein decreased by 24 hours (P <0.05) in the AF group and UC group compared to CON group after treatment with amniotic fluid stem cells of the invention; the morphological expression of PAS stained kidney tissue is observed under a microscope, and CON group mainly shows diffuse mesangial hyperplasia, basement membrane thickening accompanied by glomerular sclerosis, and heavier renal interstitial inflammation infiltration and fibrosis degree. Compared with the CON group, the pathological results of the AF group and the UC group show that the proliferation of the glomerular mesangium is reduced, and the infiltration and the fibrosis degree of interstitial inflammation cells are lighter. The protein chip screens 22 proteins with difference between the AF group and the CON group, 14 proteins are up-regulated after treatment, and 8 proteins are down-regulated after treatment, wherein proinflammatory factors IL-17 and IL-12p40 are down-regulated, and inflammation inhibiting factors IL-1ra are up-regulated.

In some embodiments of the invention, MRL/lpr mouse kidney immune cells were divided into 22 subpopulations by mass cytometry using 42 markers. From the change of 22 immune cell subsets, 7 subsets with significant difference between the amniotic fluid stem cell group and the model control group are provided. After the treatment by using the amniotic fluid stem cells, compared with a model control group, the proportion of the proinflammatory M1 type macrophages (subgroup 3) in the amniotic fluid stem cell group is reduced, the proportion of dendritic cells (subgroup 6) playing a role in antigen presentation is reduced, the proportion of eosinophils (subgroup 9) reflecting an inflammatory state is reduced, and the proportion of inflammatory Th2 cells (subgroup 16) is inhibited to be increased. The result shows that the inflammatory state of the kidney of the lupus mouse is improved after the amniotic fluid stem cell treatment. From the change in the proportion of major immune cell types, CD4 following stem cell therapy ⁺ T cell, CD8 ⁺ There was an increase in T cells and a decrease in each myeloid cell line (including DCs, monocytes macrophages, MDSCs and eosinophils). Of these variations, CD4 ⁺ T cells are obviously increased in the amniotic fluid stem cell group compared with the model control group, and the rest changes are not significant.

In addition, the invention provides an amniotic fluid stem cell product, wherein the amniotic fluid stem cell is positive for MHC class I molecule HLA-ABC and negative for MHC class II molecule HLA-DR; express CD29, CD44, CD73, CD90, CD105 and SSEA-4, and do not express CD34, CD45, CD 133. In the present invention, the expression "positive" or "expression" of a certain factor or marker means that the expression rate is 70% or more, preferably 80% or more, and more preferably 90% or more; the expression "negative" or "not expressed" for a certain factor or marker means that the expression rate is 5% or less, preferably 3% or less, more preferably 1% or less. In some embodiments of the invention, the amniotic fluid stem cell preparation of the invention has an expression rate of CD34 of the amniotic fluid stem cell of less than 1%, preferably less than 0.5%; the expression rate of CD45 is below 3%, preferably below 2.5%; the expression rate of CD133 is less than 1%, preferably less than 0.5%; the expression rate of CD29 is more than 90%, preferably more than 95%; the expression rate of CD44 is more than 95%, preferably more than 99%; the expression rate of CD73 is more than 95%, preferably more than 99%; the expression rate of CD90 is more than 95%, preferably more than 99%; the expression rate of CD105 is more than 90%, preferably more than 95%; the expression rate of HLA-ABC is more than 90%, preferably more than 95%; an HLA-DR expression rate of 1% or less, preferably 0.5% or less; the expression rate of SSEA-4 is 70% or more, preferably 80% or more.

According to some embodiments of the invention, the amniotic fluid stem cell preparation is isolated by differential adherence and mechanical separation. In some more specific embodiments of the present invention, the amniotic fluid stem cell preparation is prepared according to the following method: filtering amniotic fluid in middle stage of human pregnancy to remove relatively large tissue debris, centrifuging to collect cell precipitate, resuspending the cells in complete culture medium (DMEM/F12 medium containing 20% FBS, 1% streptomycin and 10ng/ml bFGF), and adding 5% CO at saturated humidity and 37 deg.C ₂ Culturing in an incubator until scattered adherent cells appear, changing a fresh complete culture medium, continuously culturing the adherent cells until the adherent cells grow like colonies, scraping the epithelioid cell colonies, and culturing the fibroblast-like cells. The obtained fibroblast-like cells with basically uniform cell colony morphology are the amniotic fluid stem cell product, and can be further subjected to passage, amplification and freezing storage.

In conclusion, the amniotic fluid stem cells are successfully separated, cultured and identified from the amniotic fluid at the middle of pregnancy of a human, and the treatment of the amniotic fluid stem cells can reduce the urinary protein level of lupus mice, improve the pathological damage of the kidney of the lupus mice, reduce the production of proinflammatory factors IL-17 and IL-12p40 and increase the production of an inflammation-inhibiting factor IL-1ra, so that the treatment of lupus nephritis by the amniotic fluid stem cells is proved to be effective. The amniotic fluid stem cell treatment obviously relieves the pathological damage of lupus nephritis and effectively treats the lupus nephritis by down-regulating inflammatory M1 type macrophages, dendritic cells and eosinophilic granulocytes and up-regulating and inhibiting the mechanism of inflammatory Th2 cells. Compared with embryonic stem cells and adult mesenchymal stem cells, the amniotic fluid stem cells have the following advantages: (1) easy acquisition, no ethical dispute: can be obtained by amniocentesis during prenatal diagnosis, has little damage to a mother body and a fetus, and avoids the ethical problem caused by using embryonic stem cells; (2) the in vitro culture is simple and rapid: the in vitro amplification is very rapid, and one-time cloning can be completed within 24-48 hours; (3) strong induced differentiation capability: the amniotic fluid stem cells are derived from fetuses and accessories thereof, have the capacity of differentiating towards an inner germ layer, a middle germ layer and an outer germ layer, and can be induced and differentiated into various cells; (4) low immunogenicity: MHCII molecules are not expressed or weakly expressed, so that the probability of immunological rejection of organisms can be avoided. The invention provides a new treatment strategy for treating two diseases of lupus nephritis and systemic lupus erythematosus.

Drawings

FIGS. 1A-1C show the results of amniotic fluid stem cell isolation, culture and passage. Wherein FIG. 1A is a graph of primary amniotic cells; FIG. 1B is a photograph of amniotic fluid stem cells of passage 2; FIG. 1C is a diagram of passage 5 amniotic fluid stem cells.

FIG. 2 shows the expression of 5 th generation amniotic fluid stem cell surface markers. Wherein the positive expression rates of the following molecules are respectively CD 340.1%, CD 452.1%, CD 1330.3%, CD 2998.5%, CD 4499.7%, CD 7399.8%, CD 9099.2%, CD 10597.7%, HLA-ABC 97.8%, HLA-DR 0.1% and SSEA-482.6%.

FIGS. 3A and 3B show the results of the differentiation potential of amniotic fluid stem cells. Wherein, fig. 3A is the result of the adipogenic differentiation of the amniotic fluid stem cells, and fig. 3B is the result of the osteogenic differentiation of the amniotic fluid stem cells.

Fig. 4A-4C are PAS staining patterns of kidney pathology. Wherein, FIG. 4A is CON group; FIG. 4B is the AF group; fig. 4C shows UC panels.

FIG. 5 is a graph showing the changes in the levels of urine protein in MRL/lpr mice.

Figure 6 is a differential protein volcano plot. Wherein, the red spot FALSE represents the protein without difference, the blue spot TRUE represents the protein with difference, the selection standard is P <0.05, and the fold difference (fold change) > 1.2 or < 0.83.

Figure 7 is a heatmap of the differential protein. Wherein, X1, X2 and X3 represent 3 samples of group A, X4, X5 and X6 represent 3 samples of group B, red represents up-regulation, and blue represents down-regulation.

Fig. 8A-8C are 42 markers dividing three groups of mouse kidney immune cells into 22 subpopulations (heat maps). Among them, fig. 8A: a model control group; FIG. 8B: amniotic fluid stem cell group: FIG. 8C: umbilical cord mesenchymal stem cell group.

Fig. 9A-9C show that 42 markers divided three groups of mouse kidney immune cells into 22 subpopulations (viSNE). Among them, fig. 9A: a model control group; FIG. 9B: amniotic fluid stem cell group: FIG. 9C: umbilical cord mesenchymal stem cell group. Different colors represent different subpopulations, and different scatter densities represent the number of cells occupied by each subpopulation of cells.

FIG. 10 is a graph showing the difference in the ratio of 22 immune cell subsets among three groups.

Fig. 11 shows the 7 subpopulations that were significantly different between the amniotic fluid stem cell group and the model control group.

Fig. 12A-12D are graphs of the differences in major immune cell subpopulations between three groups, wherein fig. 12A: model control, fig. 12B: amniotic fluid stem cell group, fig. 12C: umbilical cord mesenchymal stem cell group, fig. 12D: quantitative comparison among the three groups.

Detailed Description

The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.

The procedures not specified in the examples are carried out according to the conventional methods in the art or according to the operating conditions recommended by the equipment manufacturers.

Example 1 sorting, culturing and identification of amniotic fluid Stem cells

In the experiment, amniotic fluid stem cells in amniotic fluid in the middle of pregnancy are sorted and purified by a method of differential adherence and mechanical separation, and then the amniotic fluid stem cells are identified by a method of detecting a surface antibody by flow cytometry and osteogenic induced differentiation of adipogenic so as to be convenient for the next step of research by using the amniotic fluid stem cells.

1.1 Experimental materials

1.1.1 Experimental samples

The amniotic fluid sample in the middle pregnancy stage is obtained by extracting 10-15ml of amniotic fluid by amniocentesis in the middle pregnancy stage (from 14 weeks to 27 weeks of pregnancy) on the basis of removing fetal deformities and maternal diseases, and the acquisition of the amniotic fluid sample is approved by informed consent of patients and family members and ethical committee of hospitals.

1.1.2 Experimental reagents

DMEM/F12 medium (Hyclone, USA); fetal bovine serum (israel BI); human bFGF cytokine (Peprotech, usa); 0.25% pancreatin (Sigma-Aldrich, usa); dimethylsulfoxide (DMSO) (Sigma-Aldrich, usa); penicillin streptomycin mixed solution (Hyclone, usa); CD29-APC mouse monoclonal antibody (Miltenyi Biotec, USA); CD90-PE mouse monoclonal antibody (Miltenyi Biotec, USA); SSEA-4 mouse monoclonal antibody (Miltenyi Biotec, USA); CD133-PE mouse monoclonal antibody (Miltenyi Biotec, USA); CD73-PE mouse monoclonal antibody (Miltenyi Biotec, USA); HLA-DR-PE mouse monoclonal antibody (Miltenyi Biotec, USA); HLA-ABC mouse monoclonal antibody (Miltenyi Biotec, USA); CD105-FITC mouse monoclonal antibody (Miltenyi Biotec, USA); CD34-APC mouse monoclonal antibody (Miltenyi Biotec, USA); CD44-FITC mouse monoclonal antibody (Miltenyi Biotec, USA); CD45-FITC mouse monoclonal antibody (Miltenyi Biotec, USA); recombinant antibody PE-IgG isotype control of genetic engineering (Miltenyi Biotec, USA); recombinant antibody FITC-IgG isotype control of genetic engineering (Miltenyi Biotec, USA); recombinant antibody PC7-IgG isotype control of genetic engineering (Miltenyi Biotec, USA); recombinant antibody APC-IgG isotype control of genetic engineering (Miltenyi Biotec, USA); mesenchymal stem cell osteogenic induction differentiation medium kit (Cyagen, china); mesenchymal stem cell adipogenic induction differentiation medium kit (Cyagen, china).

1.2 Experimental methods

1.2.1 Collection, culture and purification of amniotic fluid Stem cells

(1) 10-15ml of amniotic fluid sample is filtered by sterile 200-mesh filter gauze, so that larger tissue fragments can be removed;

(2) transferring the amniotic fluid sample into a 15ml centrifuge tube, centrifuging at the room temperature at the rotating speed of 1500rpm/min for 10 minutes, removing the supernatant, and collecting cell precipitate;

(3) after the cells were loosened, 10ml of complete medium (20% FBS, 1% streptomycin and 10ng/ml bFGF in DMEM/F12 medium) was added to the cells to resuspend the cells;

(4) blowing, mixing, inoculating to 100mm culture dish, and culturing at saturated humidity, 37 deg.C and 5% CO ₂ Culturing in an incubator;

(5) after primary amniotic fluid cells are cultured for 7 days, scattered adherent cells are observed to appear under a mirror, the first liquid change is carried out, a liquid transfer gun is used for sucking out a culture solution containing nonadherent cells and impurities, and 10ml of fresh complete culture medium is changed (the DMEM/F12 culture medium contains 20% of FBS, 1% of streptomycin and 10ng/ml of bFGF);

(6) after the adherent cells are continuously cultured for 3 days, the cell morphology is observed under an inverted phase contrast microscope, the adherent cells grow in a colony-like manner, two different morphologies in the adherent cell colony can be seen, one form is epithelial-like cells, the other form is fibroblast-like cells, the growth range of the fibroblast-like cell colony is marked at the bottom of the culture dish by using a black marker pen, then the epithelial-like cell colony is repeatedly scraped out of a calibration area by using a sterile cell scraper, then the cell colony is washed for 3 times by using a PBS (phosphate buffered saline) solution containing penicillin and streptomycin (the cell clusters in the calibration area are not washed away), and then 10ml of complete culture medium is added into the culture dish for continuous culture;

(7) changing the liquid every 3 days later, and repeating the operation when epithelial-like cell aggregation is observed again;

(8) after scraping by a cell scraper for about 3 times, the epithelial-like cell colonies are basically and completely scraped, only a few scattered epithelial-like cells are left, the rest cell colonies are uniform in shape, and the fibroblasts are arranged in a vortex shape, so that the primarily purified fibroblasts are obtained.

1.2.2. Passage and expansion of amniotic fluid cells

(1) When the fibroblast grows to 80% confluence, the ratio of 1: 3, proportional passage;

(2) the supernatant was discarded, 1.5ml of 0.25% pancreatin-digested cells were added, and the mixture was digested at 37 ℃ for about 1.5 minutes in an incubator;

(3) when observed under an inverted phase contrast microscope, the cell mass on the culture dish shows cytoplasmic retraction and cell gaps become larger, and digestion is stopped by adding 1.5ml of complete medium (DMEM/F12 medium contains 20% FBS, 1% streptomycin and 10ng/ml bFGF);

(4) blowing and beating the cells till the cells are completely loosened and shed, and transferring the cells into a 15ml centrifuge tube by using a pipette;

(5) centrifuging at the room temperature at the rotating speed of 1300rpm/min for 5 minutes, removing supernatant, and loosening cells;

(6) adding a complete culture medium, uniformly mixing by blowing, and inoculating the single cell suspension into a 100mm culture dish for continuous culture;

(7) according to the growth rate of the cell population, the passage is continued when the cells grow to 80% confluence, and the passage is performed every 2-3 days, which is the same as the above.

1.2.3 cryopreservation and recovery of amniotic fluid Stem cells

(1) The supernatant was discarded, 1.5ml of 0.25% pancreatin-digested cells were added, and the mixture was digested at 37 ℃ for about 1.5 minutes in an incubator;

(2) when observed under an inverted phase contrast microscope, the cell mass on the culture dish shows cytoplasmic retraction and cell gaps become larger, and digestion is stopped by adding 1.5ml of complete medium (DMEM/F12 medium contains 20% FBS, 1% streptomycin and 10ng/ml bFGF);

(3) centrifuging at the room temperature at the rotating speed of 1300rpm/min for 5 minutes, removing supernatant, and loosening cells;

(4) adding 1ml of cryopreservation solution (90% FBS + 10% DMSO) into the precipitate, blowing, beating, mixing uniformly, transferring into a cryopreservation tube, sealing, and marking;

(5) firstly, storing in a refrigerator at 4 ℃ for half an hour, then placing in a programmed cooling box, storing at-20 ℃ for 2 hours, and then transferring to an ultra-low temperature refrigerator at-80 ℃ for long-term storage;

(6) the speed of cell recovery is high, the principle of 'slow freezing and instant dissolution' is followed, the cells to be recovered are taken out and directly placed in a 37-degree constant-temperature water bath kettle, and the cells are immediately taken out after being observed to be completely melted;

(7) transferring the cell suspension into a centrifuge tube containing 9ml of incomplete culture medium (without FBS), blowing and uniformly mixing, and centrifuging at the rotating speed of 1300rpm/min for 5 minutes;

(8) the supernatant was discarded, the cells were fluffed, 10ml of complete medium was added to resuspend the cells, and the cells were inoculated into a 100mm petri dish for further culture.

1.2.4 flow cytometry detection of amniotic fluid stem cell surface markers

(1) Taking 5 th generation amniotic fluid stem cells, digesting the cells with 0.25% pancreatin, adding a complete culture medium to terminate, transferring the cells into a 15ml centrifuge tube, and centrifugally collecting the cells (1300rpm/min, 5 minutes);

(2) cells were washed 3 times with sterile PBS centrifugation (1300rpm/min, 5 min), after the last wash, approximately 100 μ l of supernatant was left and cells were resuspended;

(3) adjusting the cell concentration to 1 × 10 ⁶ -10 ⁷ In the flow tube;

(4) the following procedure was carried out in the dark: respectively adding 5 μ l of fluorescence labeled antibody (CD29, CD33, CD34, CD45, CD73, CD90, CD105, CD133, SSEA-4, HLA-ABC, HLA-DR, PE-IgG isotype control, APC-IgG isotype control, PC7-IgG isotype control, FITC-IgG isotype control) into the cell suspension, and incubating at room temperature in a dark place for 30 min;

(5) the cells were washed 2 times with sterile PBS (1300rpm/min, 5 min) and then resuspended in 300. mu.l PBS and examined by flow cytometry.

1.2.5 detection of differentiation potential of amniotic fluid Stem cells

Induction of differentiation into adipocytes

(1) Preparing a lipid-induced differentiation medium A liquid and a lipid-induced differentiation medium B liquid according to the instruction of an OriCell Blb/c mouse bone marrow mesenchymal stem cell lipid-induced differentiation medium kit (Cyagen company, product number MUCMX-90031);

(2) taking the 5 th generation amniotic fluid stem cells with good growth state,when the cells fused to 80%, the cells were collected by trypsinization, and the concentration of the cell suspension was adjusted to 1X 10 ⁶ Uniformly inoculating the solution to a 6-pore plate in a concentration of ml;

(3) adding 2ml complete culture medium into each well of 6-well plate, placing 6-well plate at 37 deg.C and 5% CO ₂ Culturing in an incubator;

(4) changing the solution for 1 time in 3 days, when the cells in the holes grow to be 100% fused or over-fused, removing the supernatant, adding 2ml of solution A into the holes, sucking away the solution A after inducing for 3 days, adding 2ml of solution B, sucking away the solution B after 24 hours, adding the solution A again, and continuing to induce and culture;

(5) alternately culturing the solution A and the solution B for about 5 times, continuously maintaining the culture with the solution B for 4-7 days, changing fresh solution B every 2-3 days, observing under the mirror every day, and finishing the induction when the lipid droplets become large and round;

(6) the supernatant was discarded, washed gently twice with PBS solution, 1ml of oil red O dye solution was added to each well, after half an hour of dyeing, the dye solution was aspirated, washed 3 times with PBS solution, and observed under the mirror.

Induced differentiation into osteocytes

(1) Preparing a complete osteogenic differentiation medium according to the instruction of an osteogenic differentiation medium kit (Cyagen company, product number MUCMX-90021) of bone mesenchymal stem cells of OriCell Blb/c mice;

(2) collecting 5 th generation amniotic fluid stem cells with good growth state, collecting cells when the cells are fused to 80%, collecting cells by trypsinization, adjusting the concentration of cell suspension, and making into 1 × 10 ⁶ Uniformly inoculating the mixture in a concentration of/ml on a 6-well plate coated with 0.1% gelatin in advance;

(3) adding 2ml complete culture medium into each well of 6-well plate, placing cells at 37 deg.C and 5% CO ₂ Culturing in an incubator;

(4) changing the solution for 1 time in 3 days, removing the supernatant when the cells grow and fuse to about 70%, and adding 2ml of osteogenesis inducing and differentiating complete culture medium into the holes;

(5) changing the solution every 3 days for 1 time, and carrying out induction culture for about 20 days;

(6) observing the morphological change and the growth condition of the cells, discarding the supernatant, washing the cells for 2 times by using PBS (phosphate buffer solution), adding 2ml of 4% paraformaldehyde solution into each well, incubating the wells for 30min, sucking away the fixing solution, washing the wells for 2 times by using the PBS solution, adding 1ml of alizarin red staining solution into each well, staining the cells for 3-5min, discarding the supernatant, washing the cells for 2 times by using PBS, and observing the cells under a microscope.

1.3 results of the experiment

1.3.1 morphological Observation of amniotic fluid Stem cells

There are many different forms of primary amniotic fluid cells which have been isolated from amniotic fluid and inoculated onto a culture dish, including some impurities, including spindle, round, strip, irregular, etc. (as shown in fig. 1A), and when cultured for about 3 days, it can be seen that individual adherent cells are scattered at the bottom of the culture dish, and there are about 3-5 cells per culture dish on average (there are also no adherent cells in the culture dish) (as shown in fig. 1B). After scraping the epithelioid cell colony with a cell scraper about 3 times, more than 95% of the cells are fibroblast-like and have basically uniform morphology and are arranged in a vortex or radial shape in a culture dish and are tightly arranged when cultured about 15 days, so that the primarily purified cells are obtained (as shown in FIG. 1C). Although a small amount of epithelioid cells were mixed in the cells, they were substantially disappeared after 3 to 4 passages. The fibroblast-like cells after subculture grow very vigorously and have stable morphology, and the cell morphology does not change obviously after multiple passages. The results were preliminary to suggest that the isolated and cultured cells were amniotic fluid stem cells.

1.3.2 identification of amniotic fluid Stem cell surface markers

The result of detecting cell surface markers by flow cytometry shows that the cells cultured and separated by the invention express the surface markers CD29, CD44, CD73, CD90 and CD105 of mesenchymal stem cells, do not express the surface markers CD34, CD45 and CD133 of hematopoietic stem cells, express the pluripotent stem cell marker SSEA-4, express MHC class I molecule HLA-ABC but not express MHC class II molecule HLA-DR (figure 2), and the separated cells are proved to have low immunogenicity which indicates that the cells generally do not have serious rejection reaction when being transplanted and are more suitable for in vivo transplantation treatment of diseases. The flow cytometry results further demonstrate that the fibroblast-like cells isolated in this experiment are amniotic fluid stem cells.

1.3.3 characterization of differentiation potential of amniotic fluid Stem cells

The amniotic fluid stem cells of the 5 th generation are directionally induced and differentiated to the adipocytes, after the amniotic fluid stem cells are alternately induced by A, B liquid and maintained and cultured by B liquid, the large and round lipid droplets can be seen through microscopic observation, and the lipid droplets are dyed pink after oil red O dyeing (figure 3A), so that the induced differentiation of the amniotic fluid stem cells to the adipocytes is proved.

The 5 th generation amniotic fluid stem cells are directionally induced and differentiated to osteocytes, and after 20 days of induction culture by osteogenic induction liquid and staining by alizarin staining liquid, a large amount of orange mineralized nodules are formed (calcium salt deposition) (figure 3B), so that the amniotic fluid stem cells are induced and differentiated to the osteocytes.

The cells separated and cultured in the experiment can be determined to be the amniotic fluid stem cells by combining the cell morphological expression, the identification result of the cell surface marker and the measurement result of the cell induced differentiation capability, the cells are successfully separated and cultured, and the cells can be used for carrying out the next experiment.

Example 2 in vivo efficacy study of amniotic fluid Stem cells for treatment of lupus nephritis

2.1 Experimental materials

2.1.1 Experimental animals

30 mice (MRL/lpr) with 3-4 weeks of age were purchased from Shanghai Sprek laboratory animals, LLC. Feeding conditions are as follows: SPF grade, room temperature 26 ℃, relative humidity 50%, constant temperature and humidity, light and dark alternate once within 12 hours, mice freely eat water in the rearing cage, and padding is replaced every 3 days. The experiment was reviewed by the ethical committee of the hospital.

2.1.2 Experimental reagents

1% periodic acid, Schiff's solution, hematoxylin stain, ammonia water, xylene, alcohol, umbilical cord mesenchymal stem cells (Tianjin national stem cell center), a BCA protein concentration determination kit (Shanghai Binyan), and a GSM-CAA-4000 kit (the kit comprises the following contents in Table 1):

TABLE 1

2.2 Experimental methods

2.2.1 animal grouping, Stem cell therapy

(1)30 MRL/lpr mice were randomly divided into 3 groups of 10 mice each;

(2) the first group was injected with 5 th generation amniotic fluid stem cells 1X 10 per mouse at 16 and 18 weeks, respectively, through tail vein ⁶ 0.5ml, then called AF group (Amniotic fluid stem cell group, Amniotic) (n 10);

(3) the second group was injected with 1X 10 umbilical cord stem cells per mouse at 16 and 18 weeks, respectively, each time via tail vein ⁶ Each 0.5ml, and then called UC group (Umbilical cord stem cell group, Umbilical) (n is 10);

(4) the third group was given 0.5ml of saline per mouse at 16 and 18 weeks, each time via tail vein, and then named as CON group (Model group) (n 10).

2.2.2 Kidney Material selection, fixation, embedding and slicing

Kidney material selection and fixation: at 20 weeks, after anaesthetizing the mice, the mice were disinfected with 75% alcohol, then the abdominal skin was cut along the median line of the abdomen, the kidney tissues on both sides were separated, the capsule on the surface of the kidney was gently torn off with forceps, then the upper and lower poles of the kidney were cut off and fixed in 4% paraformaldehyde solution for 48 hours, and the whole procedure was performed on ice with attention paid to the rapidity of the action. After each kidney is cut off a kidney tissue, the kidney tissue is cut into small granules by using a tissue shear, the small granules are quickly frozen and compacted in liquid nitrogen, then the small granules are placed in a freezing storage tube for marking, and the small granules are placed in an ultra-low temperature refrigerator at minus 80 ℃ for long-term storage.

Dehydrating, embedding and slicing kidney tissues:

(1) kidney tissue was fixed in 4% paraformaldehyde solution for at least 48 hours;

(2) taking out kidney tissue, sequentially soaking in 75%, 85%, 95%, and 100% ethanol for dehydration, and gradient for 2 times, each for 30 min;

(3) then soaking the glass substrate in dimethylbenzene for transparency for 40 min;

(4) immersing in melted wax for 2 hr;

(5) taking out, ventilating and airing at room temperature for 24 hours;

(6) opening an embedding machine for preheating 2 hours in advance, and carrying out paraffin embedding;

(7) freezing the embedded paraffin block in a refrigerator at-20 deg.C for half an hour, and taking down;

(8) can be preserved for a long time at room temperature;

(9) the paraffin block is placed on a cold table for precooling and slicing, and can be stored for a long time.

2.2.3 PAS staining evaluation of pathological injury of mouse Kidney

(1) Sequentially immersing the paraffin sections into dimethylbenzene and gradient ethanol for dewaxing, wherein the sequence is 20 minutes for the first time of a dimethylbenzene solution, 20 minutes for the second time of the dimethylbenzene solution, 20 minutes for 100% ethanol, 20 minutes for 95% ethanol, 20 minutes for 85% ethanol, 20 minutes for 75% ethanol and 5 minutes for deionized water;

(2) immersing into 1% periodic acid solution for oxidizing for 25-30 minutes, and washing for several seconds;

(3) immersing into Schiff's solution for dyeing for 30 minutes;

(4) soaking in warm water for 30 minutes;

(5) dyeing with hematoxylin solution for 1-2 minutes, and washing with water for several seconds;

(6) differentiating in 1% ethanol hydrochloride solution for several seconds, and washing with water for several seconds;

(7) the ammonia water returns to blue for several seconds and then is washed by running water;

(8) observing the cell nucleus to be blue under a mirror, and dyeing the basement membrane to be pink;

(9) the stained section is sequentially immersed into gradient ethanol and xylene solution for dehydration and transparency, wherein the sequence is 75% ethanol for 10 minutes, 85% ethanol for 10 minutes, 95% ethanol for 10 minutes, 100% ethanol for 10 minutes, xylene solution for the first time for 10 minutes and xylene solution for the second time for 10 minutes;

(10) after the neutral gum is encapsulated, the gel is observed under a mirror.

2.2.4 detection of mouse urine protein by ELISA

(1) Collecting samples: collecting urine of the mice for 24 hours by using a metabolism cage in 16 th, 18 th and 20 th weeks respectively, centrifuging for 40 minutes at 1500rpm/min at 4 ℃, taking supernatant, recording urine volume, subpackaging in an EP tube at-80 ℃ and ultralow temperature refrigerator for storage, and paying attention to no repeated freeze thawing;

(2) preparing a protein standard substance according to the kit instruction;

(3) preparing a BCA working solution, wherein the BCA working solution can be stably stored for 24 hours at the temperature of 4 ℃;

(4) diluting a sample to be detected by 300 times;

(5) adding BCA working solution into a 96-well plate, wherein each well is 200 mu l;

(6) respectively adding a protein standard substance and a sample to be detected into corresponding holes, wherein each sample is provided with two multiple holes;

(7) then incubating the 96-well plate at 37 ℃ for 30 minutes;

(8) and detecting the absorbance of each hole at the wavelength of 570nm by using a multifunctional microplate reader, fitting a standard curve, and calculating the protein concentration of the sample to be detected.

2.2.5 protein chip technology for detecting inflammatory factors of mouse kidney tissues

Preparing a sample to be tested:

tissue lysis:

(1) diluting 2X Cell Lysis Buffer by 2 times with double distilled water for later use;

(2) preparing a protease inhibitor: centrifuging the tube filled with protease inhibitor, adding 60 μ L diluted 1X Cell Lysis Buffer into the tube, and mixing to dissolve protease inhibitor;

(3) preparing lysate according to the proportion of 10 mu L of protease inhibitor and 990 mu L of 1X Cell Lysis Buffer;

(4) adding the above lysis solution to each tissue;

(5) cracking the mixture on ice for 30min, and shaking the mixture once every 5 minutes;

(6) centrifuging: 13000rpm/min, 20 minutes, and the supernatant was collected.

Protein concentration determination:

(1) preparing BCA standard solution

9 clean centrifuge tubes, labeled A, B, C, D, E, F, G, H, I, were added to B, C, D, E, F, G, H, I in 125. mu.L, 325. mu.L, 175. mu.L, 325. mu.L, 400. mu.L of PBS solution at pH 8.0. Taking BSA standard substance storage liquid in the kit, respectively adding 300 mu L, 375 mu L and 325 mu L into a A, B, C tube, and uniformly mixing to obtain standard substances with the concentrations of 2000 mu g/mL, 1500 mu g/mL and 1000 mu g/mL. Extracting 175 mu L of the standard substance in the tube B, adding the standard substance into the tube D, uniformly mixing to obtain 750 mu G/mL of standard substance D, extracting 325 mu L of the standard substance in the tube C, adding the standard substance into the tube E, uniformly mixing to obtain 500 mu G/mL of standard substance E, extracting 325 mu L of the standard substance in the tube E, adding the standard substance into the tube F, uniformly mixing to obtain 250 mu G/mL of standard substance F, extracting 325 mu L of the standard substance in the tube F, adding the standard substance into the tube G, uniformly mixing to obtain 125 mu G/mL of standard substance G, extracting 100 mu L of the standard substance in the tube G, adding the standard substance H into the tube H, uniformly mixing to obtain 25 mu G/mL of standard substance H, and enabling the protein concentration of the tube I to be 0.

The standard line dilution is shown in table 2 below:

TABLE 2

(2) Preparing a BCA working solution

The required total BCA working solution volume was first calculated:

total BCA working solution volume (standard + sample to be tested) × repeat well number × 200 μ L

Preparing a BCA working solution: to 50 volumes of BCA reagent a, 1 volume of BCA reagent B (a: B50: 1) was added and mixed well. Note that: BCA reagent B, when added to BCA reagent a, is rapidly cloudy and becomes clear immediately upon mixing.

(3) Detection on machine

Respectively taking 10 mu L of standard substance or sample to be detected and adding the standard substance or the sample to be detected into a micropore plate:

a. add 200. mu.L BCA working solution to each well, mix well by gentle shaking, and stand at 37 ℃ for 30 min.

b. Cooling to room temperature, and detecting absorbance at a wavelength range of 540-595 nm on an enzyme-labeling instrument, wherein the wavelength of 562nm is the best.

c. A standard curve was drawn based on the absorbance of the BSA standard. The protein concentration of the sample was calculated from the standard curve and the dilution factor of the sample.

The measurement results are shown in the following table 3:

TABLE 3

Sample ID	ug/ml
		1	37026.67
2	23210
		3	27193.33
4	28893.33
		5	25326.67
6	29543.33
		7	32493.33
8	17560

The sample was loaded at a final concentration of 500. mu.g/ml

Complete drying of the slide chip: taking out the slide chip from the box, balancing at room temperature for 20-30min, opening the packaging bag, uncovering the sealing strip, and then placing the chip in a vacuum drier or drying at room temperature for 1-2 hours.

Chip operation:

(1) adding 100 mul of sample diluent (the final concentration of the sample protein is 500 mug/ml) into each well, incubating for 1h on a shaking table at room temperature, and sealing the quantitative antibody chip;

(2) remove the buffer from each well, add 100. mu.l of sample to the well, incubate overnight at 4 ℃ on a shaker;

(3) cleaning: the glass slide is cleaned by a Wellwash Versa chip plate cleaning machine in two steps, firstly, 1 Xwashing liquid I is used for cleaning, 250 microliter of 1 Xwashing liquid I is used for cleaning 10 times in each hole, each time the washing is carried out for 10s, the oscillation intensity is selected to be high, and 20 Xwashing liquid I is diluted by deionized water. Then, cleaning by using a 1 Xwashing liquid II channel, wherein 250 mu L of 1 Xwashing liquid II is used for each hole, cleaning is carried out for 6 times, each time of oscillation is carried out for 10s, the oscillation intensity is selected to be high, and 20 Xwashing liquid II is diluted by deionized water;

(4) incubation of the detection antibody mixture: the test antibody mixture vials were centrifuged and then 1.4ml of sample diluent was added, mixed well and then centrifuged quickly again. Add 80. mu.l of detection antibody to each well and incubate for 2 hours on a shaker at room temperature;

(5) cleaning, the step is the same as (3);

(6) incubation of Cy 3-streptavidin: the Cy 3-streptavidin vial was centrifuged, then 1.4ml of sample diluent was added, mixed well and centrifuged quickly again. Adding 80. mu.l of Cy 3-streptavidin to each well, wrapping the slide with aluminum foil paper, incubating in the dark, and incubating for 1 hour on a shaker at room temperature;

(7) cleaning, the step is the same as (3);

(8) fluorescence detection: the slide frame was first removed, carefully not touching the antibody-printed side of the slide by hand, and the signal was scanned using an InnoScan 300 laser scanner, using either Cy3 or a green channel (excitation frequency 532nm), using the data analysis software of GSM-CAA-4000 for data analysis.

2.2.6 statistical analysis

Statistical analysis and mapping of data using Graphpad Prism 8.0, differential analysis between multiple sets of data and multiple comparisons Single factor analysis of variance (ANOVA) were considered statistically significant when P <0.05, and denoted as "+" when P < 0.01. ltoreq. P <0.05, and denoted as "+" when P < 0.01.

2.3 results of the experiment

2.3.1 morphological Change in Kidney tissue in MRL/lpr mice after Stem cell therapy

The morphological expression of PAS stained kidney tissue was observed under a light microscope, and CON group mainly showed diffuse mesangial hyperplasia, basement membrane thickening accompanied by glomerular sclerosis, and severe infiltration of renal interstitial inflammation and fibrosis (FIG. 4A). Compared with CON group, pathological results of AF group and UC group showed decreased proliferation of mesangial, and a lesser degree of interstitial inflammatory cell infiltration and fibrosis (fig. 4B, 4C).

2.3.2 changes in urinary protein levels in MRL/lpr mice after Stem cell therapy

The 24-hour urinary protein levels at 3 time points (weeks 16, 18 and 20) were measured and showed no significant difference in the urinary protein levels (P >0.05) in mice in the three groups before treatment (week 16), but significant differences in both AF and UC groups at weeks 18 and 20 (P <0.05) compared to CON group, elevated urinary protein levels at 24 hours in CON group, and decreased urinary protein levels after stem cell treatment. The results show that both amniotic fluid stem cell and umbilical cord stem cell treatment can reduce the urinary protein level of mice with lupus nephritis (figure 5).

2.3.3 Change in inflammatory factor levels in Kidney tissue of MRL/lpr mice after Stem cell therapy

The differential expression of 200 cytokines was measured between the AF group and the CON group at week 20 using the GSM-CAA-4000 cytokine protein chip, and the AF group was defined as group A and the CON group as group B.

Differentially expressed proteins: proteins with P <0.05, fold difference (fold change) > 1.2 or < 0.83 were defined as difference proteins (FIG. 6), for a total of 22, see FIG. 7 and Table 4. The results show that: 22 proteins with different expression are respectively LOX-1, Fas L, Nope, BLC, MIG, MMP-2, IL-1ra, CD30L, KC, LIX, TCA-3, TACI, Epigen, EGF, ALK-1, IL-12p40, IL-17, ADAMTS1, MIP-1b, IGFBP-6, Pentraxin 3 and SCF.

The 22 differentially expressed proteins are primarily involved in inflammatory factors and immune regulation related cytokines, with the interleukins: IL-1ra, IL-12p40, IL-17; metalloprotease: MMP-2; growth factor: EGF, IGFBP-6; tumor necrosis factor receptor super receptor: TACI; chemokines: BLC, MIP-1 b.

Up-regulated protein after amniotic fluid stem cell therapy: LOX-1, Fas L, BLC, MIG, MMP-2, IL-1ra, KC, TACI, Epigen, ALK-1, ADAMTS1, MIP-1b, Pentraxin 3, SCF, and 14 in total. Wherein, the expression of the anti-inflammatory factor IL-1ra (interleukin-1 receptor antagonist) is obviously up-regulated after the stem cell treatment.

Protein down-regulated following amniotic fluid stem cell therapy: nope, CD30L, LIX, TCA-3, EGF, IL-12p40, IL-17, IGFBP-6, 8 in total. Wherein, the expression level of proinflammatory factors IL-17 and IL-12p40 is obviously reduced after stem cell treatment.

TABLE 4A, B proteins with differential expression between the two groups

Protein name	Group A	Group B	P value	Multiple of difference	Change after treatment
						LOX-1	11.92708	10.73743	0.000845	2.280979645	Up regulation
Fas L	11.85661	9.207617	0.002907	6.272293022	Is adjusted upwards
						Nope	11.05583	11.58727	0.003711	0.691860724	Down-regulation of
BLC	15.13278	14.41592	0.00938	1.643599586	Up regulation
						MIG	15.46676	14.91895	0.007363	1.461867684	Up regulation
MMP-2	13.11321	12.21812	0.008554	1.859724263	Up regulation
						IL-1ra	12.92793	11.87011	0.017221	2.081786311	Is adjusted upwards
CD30L	12.69193	13.14146	0.011292	0.732282187	Down-regulation of
						KC	13.03979	12.17368	0.014437	1.822736574	Up regulation
LIX	13.79605	14.21718	0.020389	0.74683897	Down-regulation of
						TCA-3	14.11132	14.52687	0.01335	0.749732261	Down-regulation of
TACI	13.4177	12.71262	0.019678	1.630230357	Up regulation
						Epigen	15.09863	14.80404	0.01872	1.226535954	Is adjusted upwards
EGF	15.32317	15.76714	0.023804	0.735108386	Down-regulation of
						ALK-1	12.51808	12.15998	0.024086	1.281736475	Is adjusted upwards
IL-12p40	12.79769	13.09734	0.026653	0.812449149	Down-regulation of
						IL-17	12.84524	13.22226	0.031287	0.770025761	Down-regulation of
ADAMTS1	11.63876	11.28076	0.03458	1.281644176	Up regulation
						MIP-1b	14.76906	12.80431	0.038954	3.903460524	Is adjusted upwards
IGFBP-6	14.615	15.36213	0.044529	0.595786915	Down-regulation of
						Pentraxin 3	12.77267	12.07166	0.043371	1.625646959	Is adjusted upwards
SCF	10.98159	10.00251	0.049508	1.971206791	Is adjusted upwards

The invention directly reflects the renal function of the mouse by detecting urine protein of the mouse, and the result shows that the renal function of the MRL/lpr mouse can be improved by both the amniotic fluid stem cell transplantation and the umbilical cord mesenchymal stem cell transplantation: at week 16, there was no significant difference in urinary protein levels in the three groups of mice, whereas at weeks 18 and 20, the urinary protein levels in the AF and UC groups were significantly lower than in the CON group. The results demonstrate that amniotic fluid stem cell therapy is equally effective for lupus mice as umbilical cord mesenchymal stem cells.

According to the invention, kidney PAS staining is adopted, pathological changes of the kidney of lupus mice before and after treatment are observed, and the results show that CON group mainly shows diffuse mesangial hyperplasia, basement membrane thickening and glomerular sclerosis accompanied, and renal interstitial inflammation infiltration and fibrosis degrees are heavier. The results show that the amniotic fluid stem cells can improve the kidney structure to a certain extent when used for treating lupus mice, as with umbilical cord mesenchymal stem cells.

The invention utilizes protein chip technology to detect the cytokine expression difference of AF group and CON group (because the umbilical cord mesenchymal stem cell group is only used as a reference for researching the effectiveness of the amniotic fluid stem cell for treating lupus nephritis in the invention, so that the discussion is omitted), 200 kinds of cytokines are detected in total, and the protein with P <0.05 and fold difference (fold change) > 1.2 or < 0.83 is defined as the difference protein, and the result shows that 22 kinds of difference proteins are totally and respectively LOX-1, Fas L, Nope, BLC, MIG, MMP-2, IL-1ra, CD30L, KC, LIX, TCA-3, TACI, Epigen, EGF, ALK-1, IL-12P40, IL-17, ADAMTS1, MIP-1b, IGFBP-6, Pentraxin 3 and SCF. 14 of them were upregulated (LOX-1, Fas L, BLC, MIG, MMP-2, IL-1ra, KC, TACI, Epigen, ALK-1, ADAMTS1, MIP-1b, Pentraxin 3, SCF), and 8 were downregulated (Nope, CD30L, LIX, TCA-3, EGF, IL-12p40, IL-17, IGFBP-6). Interleukin IL-1 family members are central mediators of innate immunity and inflammation, playing a key role in a variety of immunological diseases. Most IL-1 family cytokines have pro-inflammatory effects (e.g., IL-1. alpha., IL-1. beta., etc.), but IL-1ra has anti-inflammatory effects, and the researchers reported a decrease in IL-1ra in serum from SLE patients, and the present invention demonstrated the efficacy of treatment by detecting an increase in IL-1ra after treatment of lupus mice with amniotic fluid stem cells.

In conclusion, the analysis of the results shows that the amniotic fluid stem cells are effective in treating lupus nephritis mice.

Example 3 immune regulation and mechanism of amniotic fluid stem cells in lupus nephritis

3.1 Experimental materials

3.1.1 Experimental specimens

Each sample was prepared by mixing 2 identically treated MRL/lpr mouse kidneys, and the experiment was divided into 3 groups of 3 samples each, for a total of 9 samples. Wherein: CON group 3, AF group 3, UC group 3.

3.1.2 laboratory instruments and apparatus

Helios Mass Spectrometry flow cytometer (FLUIDIGM, USA)

3.1.3 Experimental reagents

42 Mass Spectrometry Metal isotope-coupled antibodies (see Table 2 for labeling different cell subsets (T cells, B cells, NK cells, granulocytes, myeloid cells, etc.) (U.S. FLUIDIGM)

194Pt (U.S. FLUIDIGM), Cisplatin stain (U.S. FLUIDIGM), FACS Buffer (U.S. FLUIDIGM), Block Solution reagent (U.S. FLUIDIGM), Intercalator-Ir stain (U.S. FLUIDIGM), Fix and Perm Buffer reagent (U.S. FLUIDIGM), 10 × Permeabilization Buffer reagent (U.S. FLUIDIGM), fire/Permeabilization reagent (U.S. FLUIDIGM), and fire/Permeabilization Concentrate reagent (U.S. FLUIDIGM).

3.2 Experimental methods

3.2.1 Kidney tissue digestion

(1) Taking out the kidney tissue from the tissue protection solution, and putting the kidney tissue into a culture dish;

(2) adding 1640 basic culture medium to wash the tissue for 3 times;

(3) weighing the tissues;

(4) then, cutting the kidney tissue into pieces by using tissue scissors, wherein the weight of each part is 0.1-1 g;

(5) the minced tissue was placed in 5ml EP tubes, and 177. mu.l (1mg/ml) of DMEM solution and 1250. mu.l (1mg/ml) of Liberase TM were added to each tube, followed by addition of DMEM solution to 5ml for digestion;

(6) digestion conditions are as follows: digesting for 30min at 37 ℃ and 250rpm/min until no residue is left on the tissue;

(7) the digested liquid was filtered once using a 70 μm sieve, the sieve and the digestion tube were washed with FACS buffer, and then centrifuged: rotating at 400g, centrifuging for 10min, discarding supernatant, and loosening cells;

(8) lysis with erythrocyte lysate for 1 min, centrifugation: 400g, 5 minutes, observing that red blood cells need to be lysed again if the red blood cells are not lysed completely;

(9) the cells were resuspended in FACS buffer, 10. mu.L counted for 2 counts, and viable and dead cells were counted separately.

3.2.2 Mass Spectrometry flow cytostaining

(1) Cell death and viability staining: the 194Pt dead and live staining solution was prepared with PBS solution, the cells were resuspended, stained on ice for 5min, and then washed twice with staining buffer.

(2) Non-specific blocking: the Fc receptor blocking solution was prepared using staining buffer, the cells were resuspended, and blocked on ice for 20 min.

(3) Extracellular staining: preparing extracellular staining antibody mixed liquor, adding the extracellular staining antibody mixed liquor into a cell sample containing Fc receptor confining liquid, uniformly mixing, staining for 30min on ice, then resuspending cells by using a staining buffer solution, centrifuging, discarding supernatant, and repeating the washing operation once.

(4) DNA staining: the DNA stain was prepared with fixed rupture buffer, cells were resuspended, and incubated overnight at 4 ℃.

(5) Intracellular staining: and (3) adding a membrane breaking buffer solution into the sample to clean the cells, centrifuging, then discarding the supernatant, adding a fixing solution, fixing at room temperature for 30 minutes, then cleaning the cells by using the membrane breaking buffer solution, resuspending the cells by using a pre-prepared intracellular staining antibody mixed solution, and placing on ice for staining for 30 minutes.

(6) Sample coding marking: the cells were washed with staining buffer, centrifuged, the supernatant discarded, the cells resuspended using a preconfigured coded labeling reagent (Barcode), and labeled on ice for 20 min.

(7) Cleaning cells before loading the machine: the cells were washed with staining buffer, centrifuged and the supernatant discarded, and the cells were resuspended in deionized water and filtered into a flow tube for cell counting.

3.2.3 mass cytometry detection on machine

(1) And opening the Helios mass spectrometer, preheating for 30 minutes, and then entering a starting program.

(2) And starting to debug the instrument and control the quality standard.

(3) After passing the quality control, the same cell number (1X 10) was taken from each sample ⁶ ) Mix and add the calibration pellet to the sample to be tested.

(4) Naming each detection channel by antibody name and coupled metal isotope name, naming data according to different groups, setting collection speed (300- ⁵ One), after all the settings are finished, the computer collects the original data, and then the data is further analyzed.

3.2.4 bioinformatics analysis method of raw data

(1) Preprocessing raw data and controlling quality: and (3) carrying out standardized pretreatment on the original data by adopting a standardized method to eliminate batch effect, thereby obtaining the data of the single surviving immune cell for further analysis.

(2) Clustering analysis: and selecting a signal mark (marker) appointed in the computer scheme (panel), analyzing data by adopting an Xshift clustering algorithm, and if the data amount is overlarge, processing the data by a random down-sampling method, so that real and effective data can still be obtained. Clustering analysis was then performed in a data-driven manner, resulting in a classification of immune cell subpopulations (clusters) of the sample.

(3) Statistical analysis and visual display of clustering results: and (3) performing dimensionality reduction visualization on the clustering result by adopting a visualization method based on t-distributed stochastic domain embedding (t-SNE). And finally, drawing a corresponding visual storage network embedding (viSNE) and heat map (heatmap) according to the obtained clustering subset and the t-SNE coordinate. The samples were then analyzed for differences in the percentage (frequency) of each subpopulation, and differences in the expression of each subpopulation of immune cells between different groups were analyzed using the two-sided t-test method.

3.3 results of the experiment

3.3.1 construction of immune cell subpopulation profiles of MRL/lpr mouse Kidney Using Mass Spectrometry flow cytometry

An immune cell subset map of the kidney of the MRL/lpr mouse is established by using a mass spectrometry flow cytometry technology, and immune cells in the kidney of the MRL/lpr mouse are clustered and divided into 22 subsets (figure 8A-figure 8C and figure 9A-figure 9C) by using 42 immune cell markers (table 5), wherein the specific definitions of the subsets are shown in table 6.

42 immune cell markers selected in Table 5

Serial number	Marker	Serial number	Marker
					1	CD45	22	CD11c
2	CD19	23	MHC II
				3	CD138	24	CD317
4	B220	25	CD11b
				5	IgD	26	FcεR1
6	IgM	27	CD117
				7	CD3	28	Ly6G
8	CD4	29	Siglec F
				9	CD183	30	F4/80
10	IL-33R	31	Ly6C
				11	CD196	32	CD64
12	CD185	33	CD68
				13	CD25	34	CD115
14	Foxp3	35	CD163
				15	CD8	36	CD206
16	CD197	37	CX3CR1
				17	KLRG1	38	CD80
18	CD279	39	CD86
				19	CD278	40	CD49b
20	CD28	41	gdTCR
				21	CD62L	42	Ki67

TABLE 6 subpopulations of immune cells in kidney tissue of MRL/lpr mice and definitions

	Cell type	Subgroup definition
			1	B	Ki67+MHCll+Ly6C+CD19+CD317+CD80
2	Mast	Ki67+CD117+CD11c+CD317+CD68+CD11b
			3	Mono/Macrophage	Ki67+MHCll+CX3CR1+CD11c+F4_80+CD64+CD86+CD317+CD80+CD68+IgM+CD11b
4	Mono/Macrophage	CX3CR1+CD11c+F4_80+CD64+CD86+CD317+CD80+CD196+CD68+IgM+CD11b
			5	DC	Ki67+CD11C+CD68+CD11b
6	DC	CX3CR1+CD11c+F4_80+CD80+CD68+CD11b
			7	MDSC	Ki67+Ly6G+Ly6C+CD11b
8	Mono/Macrophage	Ki67+Ly6C+F4_80+CD64+CD86+CD317+CD80+CD68+CD11b
			9	Eosinophils	Ki67+Ly6C+Siglec_F+F4_80+CD11b
10	DN T	CD3+Ly6C+CD8
			11	CD8+T	CD3+Ly6C+CD86+CD8+
12	CD8+T	CD3+CD11c+CD8+
			13	CD8+T	CD3+CD8+
14	CD8+T	CD3+PD_1+CD86+CD8+
			15	CD8+T	CD3+PD_1+CD8+
16	CD4+T	CD3+Ly6C+ICOS+CD4+
			17	CD4+T	CD3+PD_1+CD86+ICOS+CD4+
18	CD4+T	CD3+PD_1+ICOS+CD4+
			19	CD4+T	CD3+ICOS+CD4+
20	CD4+T	CD3+CD4+
			21	CD4+T	CD3+PD_1+CD4+
22	CD4+T	CD3+CD4+

3.3.2 percentage of 22 subpopulations of immune cells changed after Stem cell therapy

Comparing the ratio change of 22 immune cell subsets after stem cell treatment (fig. 10), 7 significantly different subsets were found between the amniotic fluid stem cell treatment group and the model control group (fig. 11), i.e.

subsets

1, 3, 4, 5, 6, 9, 16. These 7 subpopulations were defined and analyzed separately according to the present invention based on their surface markers (table 7). Comparing the changes in three major immune cell subsets following stem cell therapy, CD4 was found following stem cell therapy ⁺ T cell, CD8 ⁺ The proportion of T cells is increased, and the proportion of dendritic cells, mononuclear macrophages, MDSC and eosinophil cells is reduced. Of these variations, CD4 ⁺ T cells were significantly elevated in the amniotic fluid stem cell group compared to the model control group, with the remaining changes being insignificant (fig. 12A-12D).

TABLE 7 subgroups differing between amniotic fluid stem cell group and model control group

The invention utilizes 42 markers to immunize the kidney of the MRL mouseThe cells were divided into 22 subsets; from the change of 22 immune cell subsets, 7 subsets with significant difference between the amniotic fluid stem cell group and the model control group are provided. Compared with the model control group, the proportion of proinflammatory M1 type macrophages (subgroup 3) in the amniotic fluid stem cell group is reduced, the proportion of dendritic cells (subgroup 6) playing a role in antigen presentation is reduced, the proportion of eosinophils (subgroup 9) reflecting an inflammatory state is reduced, and the proportion of inflammatory-inhibiting Th2 cells (subgroup 16) is increased. The result shows that the inflammatory state of the kidney of the lupus mouse is improved after the amniotic fluid stem cell treatment; from the change in the proportion of major immune cell types, CD4 following stem cell therapy ⁺ T cells, CD8 ⁺ There was an increase in T cells and a decrease in cells of each myeloid lineage (including DCs, monocytes macrophages, MDSCs and eosinophils). Of these variations, CD4 ⁺ T cells are obviously increased in the amniotic fluid stem cell group compared with the model control group, and the rest changes are not significant.

Claims

1. The application of the amniotic fluid stem cells in preparing the medicine for treating lupus nephritis, wherein the expression rate of CD34 of the amniotic fluid stem cells is below 0.5%; the expression rate of CD45 is below 2.5%; the expression rate of CD133 is below 0.5%; the expression rate of CD29 is more than 95%; the expression rate of CD44 is more than 99%; the expression rate of CD73 is more than 99%; the expression rate of CD90 is more than 99%; the expression rate of CD105 is more than 95%; the expression rate of HLA-ABC is more than 95 percent; the HLA-DR expression rate is below 0.5%; the expression rate of SSEA-4 is more than 80%;

wherein the amniotic fluid stem cells are prepared according to the following method: filtering amniotic fluid in middle stage of pregnancy with 200 mesh filter to remove tissue debris, centrifuging to collect cell precipitate, suspending the cells with complete culture medium (DMEM/F12 medium containing 20% FBS, 1% streptomycin and 10ng/ml bFGF) at saturated humidity, 37 deg.C and 5% CO ₂ Culturing in an incubator until scattered adherent cells appear, changing a fresh complete culture medium, continuously culturing the adherent cells until the adherent cells grow like colonies, scraping the epithelioid cell colonies, and culturing fibroblast-like cells;

the medicine is used for reducing the urinary protein level of a lupus nephritis individual, down-regulating the expression of proinflammatory factors IL-17 and IL-12p40 of the lupus nephritis individual and up-regulating the expression of an inflammation inhibiting factor IL-1 ra.

2. The use of claim 1, wherein the amniotic fluid stem cells have the ability to differentiate into adipocytes and/or osteocytes.

3. The use of claim 1, wherein the medicament is for down-regulating the proportion of pro-inflammatory M1-type macrophages, dendritic cells and eosinophils in kidney immune cells in an individual with lupus nephritis; and/or the ratio of anti-inflammatory Th2 cells in kidney immune cells for up-regulating lupus nephritis individuals.