Application of amniotic fluid stem cells in preparation of medicine for treating lupus nephritis
阅读说明：本技术 羊水干细胞在制备治疗狼疮肾炎的药物中的应用 (Application of amniotic fluid stem cells in preparation of medicine for treating lupus nephritis ) 是由 陈香美 白雪源 张俊铭 于 2020-07-14 设计创作，主要内容包括：本发明提供了羊水干细胞在制备治疗狼疮肾炎的药物中的应用。所述羊水干细胞能够下调促炎因子IL-17、IL-12p40的表达、上调抑炎因子IL-1ra的表达,同时,能够下调促炎性的M1型巨噬细胞、树突状细胞和嗜酸性粒细胞、上调抑炎性的Th2细胞从而发挥治疗效果。所述羊水细胞具有易获取、无伦理纠纷；培养简单；分化能力强以及免疫原性低等特点。本发明为系统性红斑狼疮及其并发症狼疮肾炎的治疗提供了新的策略。(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.)
1. Application of amniotic fluid stem cells in preparing medicine for treating lupus nephritis is provided.
2. Application of amniotic fluid stem cells in preparation of medicines for treating Systemic Lupus Erythematosus (SLE).
3. The use of claim 1 or 2, wherein the amniotic fluid stem cells have the ability to differentiate into adipocytes and/or osteocytes.
4. The use of claim 1 or 2, wherein the amniotic fluid stem cells are HLA-ABC positive for MHC class i molecules and HLA-DR negative for MHC class ii molecules; express CD29, CD44, CD73, CD90, CD105 and SSEA-4, and do not express CD34, CD45, CD 133.
5. The use of claim 1 or 2, wherein the medicament is for reducing urinary protein levels in a lupus subject and ameliorating renal pathological damage in a lupus subject.
6. The use according to claim 1 or 2, wherein the medicament is for down-regulating the expression of the pro-inflammatory factors IL-17, IL-12p40 in an individual; up-regulating the expression of the inflammation-inhibiting factor IL-1 ra.
7. The use according to claim 1 or 2, wherein the medicament is for down-regulating pro-inflammatory macrophages of type M1, dendritic cells and eosinophils; and/or for up-regulating inflammatory Th2 cells.
8. Use according to claim 1 or 2, wherein the amniotic fluid stem cells are derived from amniotic fluid in the middle of pregnancy in humans.
9. The use of claim 1 or 2, wherein the amniotic fluid stem cells are isolated by differential adherence and mechanical separation.
10. An amniotic fluid stem cell preparation, 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; preferably, the amniotic fluid stem cell product is obtained by differential adherence and mechanical separation.
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 immune complexes are deposited on kidney nephrons, 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 in some refractory lupus nephritis patients and 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 application 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 such as 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 The transplantation of amniotic fluid stem cells could improve The self-repair ability of The kidney after acute kidney injury [ Hauser P V, De Fazio R, Bruno S, et al.Stem cell derived from human immunological fluid recovery [ J ]. The American journel of Pathology,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 amniotic fluid at the middle pregnancy stage of a 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 of the lupus mice, reducing the production of proinflammatory factors (IL-17 and IL-12p40) and increasing the production of inflammation-inhibiting factors (IL-1ra), 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 specific embodiment of the invention, the amniotic fluid stem cells of the invention are derived from amniotic fluid in the middle of pregnancy in humans.
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-
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, the 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 was observed following stem cell therapy+T cell, 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 remarkably increased in the amniotic fluid stem cell group compared with the model control groupThe remaining changes were 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 obtained 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.C2Culturing 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 fine particles with substantially uniform cell colony morphologyThe cells 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 regulating down the inflammatory M1 type macrophages, dendritic cells and eosinophils and regulating up 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 capacity: 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 drawing of 2 nd generation amniotic fluid stem cells; FIG. 1C is a diagram of
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 an AF group; fig. 4C shows UC panels.
FIG. 5 is a graph showing 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 heat map 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.