Application of mesenchymal stem cells in preparation of medicine for treating polycystic ovarian syndrome

Abstract

The invention discloses application of mesenchymal stem cells in preparation of a medicine for treating polycystic ovarian syndrome, and belongs to the technical field of biological medicine. Research finds that injection of MSCs cells into PCOS mouse uterine cavities can effectively reverse the core effect of lumen epithelial cell-macrophage-matrix fibroblast axis, reduce chemotaxis from macrophages to lumen epithelial cells and differentiation from fibroblasts to myofibroblasts, relieve PCOS mouse uterine fibrosis, and improve PCOS mouse uterine fibrosis resistance. The invention provides a novel therapeutic drug for treating uterine fibrosis and endometrial receptivity decline which may occur in PCOS diseases, and is expected to solve the bad reproduction phenomenon caused by PCOS.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of mesenchymal stem cells in the preparation of drugs for treating polycystic ovary syndrome. Background Art

[0002] Polycystic ovary syndrome (PCOS) is the most common reproductive endocrine disease in women of childbearing age, with an incidence of about 8%-13%, causing 80% of ovulatory infertility. At present, the pathogenesis of PCOS is still unclear, and there is a lack of effective treatment methods, which has become a bottleneck problem that needs to be solved in reproductive medicine. Successful pregnancy depends on good embryos, endometrium, and synchronous development between mother and fetus. PCOS patients are infertile due to anovulation and poor endometrial receptivity, and usually use assisted reproductive technology in vitro fertilization-embryo transfer to help pregnancy. At present, consensus guidelines and clinical programs for the treatment of PCOS infertility mainly focus on improving ovulatory function and treating infertility related to oligoovulation or anovulation. However, a large amount of data shows that even after the transplantation of euploid embryos confirmed by preimplantation genetic diagnosis, more than 40% still fail to implant, which seriously affects the outcome of assisted reproductive technology pregnancy. Among clinical research data, most studies have shown that women with PCOS have a higher miscarriage rate and worse pregnancy outcomes, proving that the endometrium of PCOS is poorly receptive. Studies have reported that PCOS is a chronic, low-grade inflammatory disease with immune disorders in the uterus. However, the mechanism of how immune disorders lead to poor endometrial receptivity is unclear.

[0003] Mesenchymal stem cells (MSCs) are a type of adult stem cells derived from the early mesoderm of embryonic development. They have the potential for self-renewal and multidirectional differentiation. They are widely available and are the preferred seed cells for cell therapy and tissue engineering due to their immunomodulatory, homing migration, paracrine, and multidirectional differentiation abilities. They have broad clinical application prospects. Currently, the application of MSCs in infertility has been proven to be effective and safe. However, whether MSCs can regulate the endometrium immunomodulation in the PCOS microenvironment and thus achieve the regulation of endometrial receptivity has not yet been reported. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide the use of mesenchymal stem cells in the preparation of drugs for treating polycystic ovary syndrome, so as to solve the problem of the lack of drugs for treating poor endometrial receptivity and uterine fibrosis in PCOS patients.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] Application of mesenchymal stem cells in the preparation of drugs for the treatment of polycystic ovary syndrome.

[0007] The beneficial effects of the present invention are as follows: Through experiments, it is found that luminal epithelial cells (LE) can promote macrophage polarization, highly express fibrosis marker genes, and become pro-fibrotic macrophages. Cell communication occurs between macrophages and myofibroblasts, promoting the activation of myofibroblasts and inducing uterine fibrosis. Intrauterine injection of MSCs can reduce the chemotaxis of luminal epithelial cells to macrophages, reverse the differentiation of receptive fibroblasts into myofibroblasts, and reverse uterine fibrosis in PCOS mice, thereby improving endometrial receptivity.

[0008] Furthermore, polycystic ovary syndrome includes uterine fibrosis caused by polycystic ovary syndrome and reduced endometrial receptivity.

[0009] A drug for treating polycystic ovary syndrome, the active ingredient of the drug includes mesenchymal stem cells.

[0010] The present invention has the following beneficial effects:

[0011] The present invention combines single-cell transcriptome sequencing and Xenium in situ spatial analysis technology to map the single-cell atlas of the uterus of PCOS mice. It is found through analysis that due to the chemotaxis of luminal epithelial cells to macrophages, the number of macrophages in the uterus increases, and pro-fibrotic macrophages drive the differentiation of receptive fibroblasts into myofibroblasts, promoting the fibrosis of the endometrium in PCOS mice. Furthermore, through research, it is found that intrauterine injection of MSCs can effectively weaken the chemotaxis of luminal epithelial cells to macrophages, reduce the differentiation of fibroblasts into myofibroblasts, alleviate uterine fibrosis in PCOS mice, and improve endometrial receptivity, providing a new therapeutic drug for treating uterine fibrosis and decreased endometrial receptivity caused by PCOS, and is expected to solve the adverse reproductive phenomena caused by PCOS. Description of the Drawings

[0012] Figure 1 For the detection results of the fibrosis phenomenon and the expression level of fibrosis marker proteins in the endometrium of PCOS patients in Example 1, where A is the HE staining result, B is the MASSON staining result, and C is the volume fraction of collagen fibers;

[0013] Figure 2Detection results of endometrial fibrosis and decreased receptivity in PCOS patients in Example 1. Among them, A is the qRT-PCR quantitative analysis results of the mRNA contents of Hoxa10, Prl, and Igfbp1 genes in the endometrium of PCOS patients, B is the number of cystic glands in the endometrium of PCOS patients, C is the diameter of cystic glands in the endometrium of PCOS patients, D is the qRT-PCR quantitative analysis results of the mRNA contents of Timp2, Mmp2, and Fn genes in the endometrium of PCOS patients, and E is the WB detection results of the contents of ColⅢ, TGF-β, and α-SMA proteins in the endometrium of PCOS patients;

[0014] Figure 3 Construction results and analysis of PCOS model mice in Example 1. Among them, A is the gross view and HE staining of the ovaries of PCOS model mice, B is the number of cystic follicles in PCOS model mice, C is the diameter of cystic follicles in PCOS model mice, D is the testosterone level of PCOS model mice, E is the litter size after co-housing of PCOS model mice, F is the body weight change trend of PCOS model mice, and G is the estrous cycle of PCOS model mice and the control group;

[0015] Figure 4 Uterine analysis results of PCOS model mice in Example 1. Among them, A is the HE staining results of the uterus of PCOS model mice, B is the endometrial thickness of PCOS model mice, C is the myometrial thickness of PCOS model mice, and D-F are the mRNA contents of Hoxa10, Hand2, and Esr1 genes in the uterine tissue of PCOS model mice in sequence;

[0016] Figure 5 Analysis of uterine fibrosis and impaired endometrial receptivity and related protein and mRNA contents in PCOS model mice in Example 1. Among them, A is the MASSON staining results of the uterus of PCOS model mice, B is the statistical results of MASSON staining, C is the relative contents of uterine fibrosis marker proteins (Col III, TGF-β, and α-SMA proteins) detected by WB in PCOS model mice, D-F are the statistical charts of the relative contents of uterine fibrosis marker proteins, and G-I are the relative mRNA contents of Bmp2, Timp2, and Pdgf genes in sequence;

[0017] Figure 6Results of the comparison of macrophages in the endometrial tissues of PCOS patients and the uteri of PCOS model mice in Example 1. Among them, A is the statistical chart of the number of macrophages in the endometrial tissues of PCOS patients analyzed by flow cytometry, B is the statistical chart of the number of macrophages in the uterine tissues of PCOS mouse models analyzed by flow cytometry, and C-D are the qRT-PCR quantitative analyses of the expressions of macrophage chemokines Ccl2 and M-csf in the uteri of PCOS mice in sequence;

[0018] Figure 7 For the construction of Lyz2 in Example 1 Cre Td-tomato flox / flox PCR results of the genotypes of the mice used in the construction of the PCOS model in female mice. Among them, A is the identification result of the Lyz2 Cre genotype, and B is the identification result of the Td-tomato flox / flox genotype;

[0019] Figure 8 For the analysis of the differentiation of Col1a1 + macrophages driving Hoxa11 + fibroblasts (F0 and F1) into myofibroblasts (F2) in the PCOS uterus in Example 2. Among them, A and B are the schematic diagrams of the differentiation of Hoxa11 + fibroblasts into myofibroblasts in the control group and the PCOS group respectively, C is the in-situ expression result of collagen in PCOS uterine macrophages shown by Xenium, D is the expression amount of collagen in each macrophage on average, E is the number of myofibroblasts around each macrophage on average, F is the percentage of F0, F1, and F2 subsets of cells, G is the change of receptive marker genes in fibroblasts, and H is the change of fibrotic marker genes in myofibroblasts;

[0020] Figure 9 For the morphology, distribution, cell number, and percentage of LE in each group shown by the Xenium in-situ spatial map in Example 2. Among them, A is the morphology and distribution of LE in each group, B is the cell number of LE in each group, and C is the percentage of LE in each group;

[0021] Figure 10 For the expression of cell proliferation-related genes in LE shown by Xenium in-situ and statistical analysis in Example 2. Among them, A is the Xenium in-situ display map, B is the statistical analysis map. In Figure A, purple represents LE, blue represents the cell nuclei located based on DAPI, the scale bar length is 50 μm, * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001;

[0022] Figure 11In-situ expression, statistical analysis, and immunohistofluorescence images of key genes in the LAMININ signaling pathway in uterine LE and macrophages in Example 2. Among them, A shows the in-situ expression results, B shows the statistical analysis results, and C shows F4 / 80 + Immunohistofluorescence image of macrophages;

[0023] Figure 12 Analysis results of gene expression characteristics of LAMININ ligand-receptor pairs in LE and macrophages in PCOS model mice in Example 2. Among them, A shows the expression of laminin ligand produced by LE, and B shows the inter-group differential expression of integrin receptor produced by macrophages;

[0024] Figure 13 Experimental results of MSCs improving receptivity by reversing uterine fibrosis in PCOS mice in Example 3. Among them, A shows the morphological structure diagram of the uterus in the MSCs treatment group by HE staining, B shows the endometrial thickness, C shows the number of cystic glands in the uterus, D-F successively show the expression of endometrial receptivity markers Esr1, Hand2, and Hoxa10 at the mRNA level, G shows the percentage of F0-F2 subgroup cells, and H shows Hoxa11 + Changes in receptivity marker genes in fibroblasts, and I shows changes in fibrosis marker genes in myofibroblasts;

[0025] Figure 14 Masson staining and statistical results of MSCs treatment inhibiting uterine fibrosis in PCOS mice in Example 3. Among them, A shows the Masson staining results of collagen deposition in the mouse uterus, B shows the local enlarged view, and C shows the statistical chart.

[0026] Figure 15 WB analysis of the relative contents of ColⅢ, TGF-β, and α-SMA proteins and the relative expression changes of protein gray scale analysis in the endometrium of mice in the MSCs treatment group in Example 3. Among them, A shows the WB detection results, and B-D successively show the relative expression change diagrams of ColⅢ, TGF-β, and α-SMA proteins;

[0027] Figure 16 Experimental results of MSCs treatment inhibiting the chemotaxis of luminal epithelial cells to macrophages in Example 3. Among them, A shows the LAMININ pathway ligand-receptor pair of LE chemotaxing macrophages shown by Xenium in-situ, and B shows the distribution map of macrophages before and after MSCs treatment by immunohistofluorescence. The position circled by the red dotted line is LE;

[0028] Figure 17For the in-situ expression of collagen in uterine macrophages in the MSCs treatment group in Example 3, the expression level of collagen in each macrophage, and the number of surrounding myofibroblasts, where A is the in-situ expression map of collagen in uterine macrophages, B is the expression level of collagen in each macrophage on average, C is the number of myofibroblasts around each macrophage on average, and D is the Xenium in-situ distribution map of myofibroblasts. Detailed implementation mode

[0029] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0030] Example 1: Construction of PCOS model mice

[0031] (1) Samples of the endometrium of PCOS patients and the control group were taken for characterization. HE staining and MASSON staining were performed on the endometrial tissue to analyze the volume fraction of collagen fibers; the mRNA contents of genes such as Hoxa10, Prl, Igfbp1, Timp2, Mmp2, and Fn in the endometrium of PCOS patients were quantitatively analyzed by qRT-PCR; the number and diameter of cystic glands in the endometrium of PCOS patients were detected, and the characterization results are as Figure 1 - Figure 2 shown.

[0032] The results of HE staining and MASSON staining showed that compared with the control group, the gland density of the endometrial stromal cells in PCOS patients decreased, the glandular epithelial cells were low columnar or flat, and the glandular lumen expanded into a cystic shape. The qRT-PCR results showed that the endometrium of PCOS patients showed a lower receptive gene expression level (as shown in Figure A in Figure 2 ), Figure 2 Figures B and C in Figure 1 showed an increase in the number and diameter of cystic glands in the endometrium of PCOS patients. The results indicated that the endometrial receptivity of PCOS women decreased. According to the analysis results of the volume fraction of collagen fibers in Figure C in Figure 2 , it was known that fibrosis occurred in the endometrium of PCOS patients. At the same time, the qRT-PCR results showed an increase in the mRNA contents of genes related to uterine fibrosis, Timp2, Mmp2, and Fn, in PCOS patients (as shown in Figure D in Figure 2 ). At the same time, the WB results in Figure E in

[0033] (2) Establish PCOS model mice. All mice were raised in the SPF-level environment of the animal center. After weaning, the mice were adaptively fed for 1 week and then randomly assigned to each group according to their body weight. The PCOS model mouse group was treated with intragastric administration of letrozole at a dose of 1 mg / kg and fed with normal diet for 5 weeks. The uterine tissues of the PCOS model mice were obtained for characterization, and the control group was intragastrically administered with an equal volume of normal saline. HE staining of the ovaries and uteri of the mice was performed respectively, the number and diameter of cystic follicles were detected, the testosterone level was detected, the pregnancy rate after co-housing the mice was detected, Masson staining analysis of the endometrium was performed, the body weight and estrous cycle of the mice, the thickness of the endometrium and myometrium of the mice, as well as qRT-PCR and WB analysis were carried out. The experimental results are as Figure 3 - Figure 5 shown.

[0034] Compared with the control group, the body weight and serum testosterone level of the PCOS model mice were significantly increased, the estrous cycle was disordered and always in the diestrus phase, and HE staining showed that multiple cystic follicles were visible in the ovaries. The density of uterine stromal cells and the number of normal glands in PCOS mice decreased, the thickness of the endometrial layer and myometrium became thinner, the number of cystic glands increased, and the glandular epithelial cells were low columnar or flattened. After co-housing the PCOS model mice with male mice, the pregnancy rate of the PCOS model mice was significantly reduced, and the expression levels of Hoxa10, Hand2, and Esr1 genes in the uterus were significantly decreased. The results of Masson staining showed that the collagen content in the endometrium of the PCOS model mice was significantly increased, and the results of WB and qRT-PCR showed that the expression of fibrosis marker genes increased.

[0035] In summary, the PCOS model mice were successfully constructed. The endometrium of PCOS mice developed fibrosis and the receptivity decreased, which was consistent with that of PCOS patients.

[0036] (3) Analyze the number and classification of macrophages in the uteri of PCOS patients and PCOS model mice. The results of immunohistofluorescence and flow cytometry are as Figure 6 shown. The results showed that compared with the control group, the number and classification of macrophages in the uteri of PCOS patients and PCOS model mice were abnormal, and the qRT-PCR results showed that the macrophage-related chemokines were abnormal.

[0037] Construct Lyz2 Cre Td-tomato flox / flox female mouse model. Specifically label bone marrow-derived macrophages The mice were female mice obtained by self-crossing of Td-tomato constructed by Shanghai Model Organisms Center, Inc. using CRISPR / Cas9 gene editing technology and male mice of Lyz2 purchased from Cyagen Biosciences (Suzhou) Inc. After mating, the resulting female and male mice were mated to obtain Lyz2 flox / + from the self-crossing of female mice and male mice of Lyz2 Cre obtained from the mating of female and male mice, and then mated to obtain Lyz2 CreTd-tomato flox / flox Female mice ( Figure 7 ), using the same method as wild mice to construct a PCOS model, and obtaining Lyz2 Cre Td-tomato flox / flox Female mouse PCOS model, which is consistent with the phenotype of the uterus of the PCOS model mice constructed above.

[0038] Example 2: Principle of uterine fibrosis and decreased receptivity in PCOS mice

[0039] (1) Macrophages in the PCOS uterus drive the differentiation of fibroblasts into myofibroblasts

[0040] The cytological basis for the fibrosis of tissues and organs is the activation of fibroblasts into myofibroblasts. Through research, it is found that matrix fibroblasts account for 50.55% in the control group and 30.99% in the PCOS model mouse group. Single-cell sequencing divides fibroblasts into 3 subpopulations, F0, F1, and F2. F2 is myofibroblasts, and F1 and F0 are Hoxa11 + fibroblasts. As shown in Figure A in Figure 8 , the three subpopulations of cells form a continuous differentiation trajectory ( Figure 8 Figure A in), which is F1→F0→F2. F1 is the origin of differentiation, and F2 is the end point of differentiation. Perform Xenium detection of the in-situ expression of collagen in uterine macrophages of PCOS mice. Compared with the control group, macrophages in PCOS mice drive the differentiation of Hoxa11 Figure 8 fibroblasts into myofibroblasts by highly expressing collagen signals ( + Figures C and D in), and the number of myofibroblasts increases significantly ( Figure 8 Figure B in). The result is that compared with the control group, the proportion of cells in the myofibroblast (F2) subpopulation increases in the PCOS model mouse group, and at the same time, the expression of fibrosis genes increases, the proportion of Hoxa11 Figure 8 fibroblasts decreases, and the expression of receptivity genes decreases ( + Figures F - H in). Figure 8 Figure F - H in

[0041] (2) Luminal epithelial cells (LE) in the uterus of PCOS mice regulate the chemotaxis of macrophages and promote their polarization into pro-fibrotic macrophages

[0042] Analyze the morphology, distribution, cell number and percentage of LE in each group by Xenium in situ, show the expression of cell proliferation-related genes (Cdk1, Cdk2, Cdk4, Cdk5, Cdk6, Cdk7, Cdk8, Cdk9, Mki67 and Pcna) in LE and perform statistical analysis; analyze the in situ expression and statistical analysis of key genes in the LAMININ signaling pathway in uterine LE and macrophages; perform immunohistofluorescence experiments on PCOS mice to show the distribution relationship between LE and macrophages; perform statistical analysis on the expression of Lama3, Lama5, Lamb2, Lamb3 and Lamc1 in the Lam-αβγ ligands produced by LE and the expression of Itg-αβγ receptors produced by macrophages.

[0043] The experimental results are as Figure 9 - Figure 12 shown. The results show that the cell number and percentage content of epithelial cells in the uterus of PCOS mice are significantly increased. According to the statistical analysis results of in situ expression in uterine LE and macrophages, the expression levels of ligand genes produced by LE and receptor genes produced by macrophages increase synchronously. And according to the immunohistofluorescence results, PCOS uterine macrophages tend to distribute towards LE.

[0044] In summary, in the uterus of PCOS model mice, LE can promote macrophage polarization, highly express fibrosis marker genes, and become pro-fibrotic macrophages. Macrophages communicate through multiple pathways to promote myofibroblast activation and induce uterine fibrosis.

[0045] Example 3: MSCs are used to treat PCOS to improve PCOS uterine fibrosis and endometrial receptivity

[0046] Construct PCOS model mice and Lyz2 according to Example 1 Cre Td-tomato flox / flox Use female PCOS model mice as the model, and inject 10 6 MSCs cells into the uterine cavity of the mice as the treatment group (inject 500,000 MSCs cells into each uterine horn. Each mouse has two uteri on both sides. Therefore, each mouse is injected with a total of 1 million cells). Two weeks after injection, collect uterine tissues for experiments, which are respectively used for single-cell transcriptome sequencing, prepare paraffin sections for HE staining, Xenium in situ verification, Masson staining to determine collagen in the mouse uterus, WB analysis of the relative contents of ColⅢ, TGF-β and α-SMA proteins in the mouse endometrium and protein gray-scale analysis of the relative expression changes of proteins, qRT-PCR analysis of the mRNA content of fibrosis marker genes, in situ expression of collagen in uterine macrophages and the expression amount of collagen in each macrophage on average and the number of surrounding myofibroblasts, immunohistofluorescence analysis of macrophage localization, and flow cytometry analysis of the proportion of macrophages.

[0047] The experimental results are as Figure 13 - Figure 17 shown.

[0048] The results showed that the endometrial receptivity of PCOS mice was significantly improved after injecting MSCs (as Figure 13 shown), the endometrial thickness of the mice increased (as shown in Figures A and B in Figure 13 ), the number of cystic glands decreased (as shown in Figure C in Figure 13 ), the expression levels of the endometrial receptivity marker genes Esr1, Hand2, and Hoxa10 were significantly increased ( Figure 13 Figures D and F in Figure 13 ), the number of receptive fibroblasts increased ( Figure 13 Figure G in

[0049] ), and the expression of the receptive marker genes was upregulated ( Figure 13 Figure H in

[0049] ), indicating that the endometrial receptivity of PCOS mice was effectively restored.

[0049] The results showed that uterine fibrosis in PCOS mice was improved after injecting MSCs (as Figure 13 - 17 ). As can be seen from the Masson staining results in Figure A and the statistical results in Figure B in Figure 14 , injecting MSCs could effectively improve collagen deposition in the uterus of PCOS mice, while reducing the number of myofibroblasts in the uterus. At the same time, the number of myofibroblasts decreased ( Figure 13 Figure G in Figure 13 ), and the expression of fibrosis marker genes was downregulated ( Figure 13 Figure I in

[0050] ), effectively inhibiting the uterine fibrosis phenomenon in PCOS mice.

[0050] According to Figure 15 the WB results and the statistical results of the relative protein expression levels in

[0051] ), injecting MSCs could effectively reduce the expression levels of the fibrosis-related marker proteins in the uterus, further demonstrating the effect of MSCs in treating uterine fibrosis caused by PCOS.

[0051] As Figure 16 shown by the Xenium in situ verification results in Figure A and the immunofluorescence results in Figure B in

[0052] According to Figure 17From the results of in-situ expression detection of collagen in macrophages and the statistical results of collagen and surrounding myofibroblasts, it can be seen that injecting MSCs can effectively reduce the expression of collagen in macrophages. By inhibiting the cell communication between macrophages and fibroblasts, the transformation of fibroblasts into myofibroblasts is inhibited, and the number of myofibroblasts in the endometrium is reduced, thereby effectively reducing the fibrosis of the endometrium caused by PCOS and improving the receptivity of the endometrium.

[0053] In summary, injecting MSCs can reduce the cell communication between luminal epithelial cells and macrophages, and between macrophages and myofibroblasts, alleviate the fibrosis of the uterus in PCOS mice, and improve the receptivity of the endometrium.

[0054] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of mesenchymal stem cells in the preparation of a drug for treating polycystic ovary syndrome.

2. Use of the mesenchymal stem cells according to claim 1 in the preparation of a medicament for treating polycystic ovary syndrome, characterized in that, Polycystic ovary syndrome includes uterine fibrosis and reduced endometrial receptivity caused by polycystic ovary syndrome.

3. A drug for treating polycystic ovary syndrome, characterized in that, The active ingredient of the drug includes the mesenchymal stem cells described in claim 1 or 2.

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