Application of umbilical cord mesenchymal stem cells in treatment and / or prevention of Alport syndrome renal injury

Abstract

The invention provides application of umbilical cord mesenchymal stem cells in treatment and / or prevention of Alport syndrome renal injury, and relates to the technical field of biomedicine.Experiments prove that hUC-MSCs can effectively relieve proteinuria degree of mice with Alport syndrome, relieve renal function injury progress, improve glomerular sclerosis and podocyte loss proportion, improve renal function loss rate and improve renal function loss rate. In-vitro experiments show that the function protection of the MSC on the podocyte is one of mechanism targets of the MSC for relieving the renal injury. The invention proves that the umbilical cord mesenchymal stem cells can relieve, treat and / or prevent the Alport syndrome renal injury and can prolong the lifetime. The invention discloses the treatment potential of the human umbilical cord mesenchymal stem cells in the Alport syndrome, and expands the treatment means of the disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an application of umbilical cord mesenchymal stem cells in treating and / or preventing Alport syndrome renal injury. Background Art

[0002] Alport syndrome (AS) is a rare disease that often develops in childhood. Alport syndrome patients mainly show renal involvement, including hematuria, proteinuria, and progressive decline in renal function. Patients also often have sensorineural hearing loss and eye abnormalities. Col4a5 gene mutation is the most common pathogenic gene for Alport syndrome (80% to 85%). Col4a5 gene mutation is an X-linked dominant inheritance. Male patients tend to progress faster than female patients, and more than 90% of male patients enter end-stage renal disease before the age of 40. There is currently a lack of effective methods for the treatment of Alport syndrome. The existing clinical treatment strategies are mainly based on non-specific drug intervention and symptomatic supportive treatment, and the treatment effect is very limited.

[0003] Studies have found that bone marrow mesenchymal stem cells can significantly inhibit the infiltration of macrophages in the kidneys of rats with diabetic nephropathy, reduce the secretion of inflammatory cytokines, slow down glomerular sclerosis, and improve renal function. MSCs injection can significantly reduce the level of autoantibodies in LN model mice, such as anti-dsDNA antibodies, reduce the deposition of C3 and IgG in the glomeruli, and alleviate renal damage. hUC-MSCs have been widely used in a variety of renal diseases, including diabetic kidney disease (DKD), lupus nephritis (LN), acute kidney injury (AKI), and management after renal transplantation. However, whether hUC-MSC can be applied to the degree of renal injury in Alport syndrome mice has not been reported, and its possible mechanism is still lacking.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide an application of umbilical cord mesenchymal stem cells in the preparation of a product for treating and / or preventing Alport syndrome renal injury, so as to solve the technical problem of the lack of an effective method for treating Alport syndrome in the prior art.

[0006] A second object of the present invention is to provide an application of umbilical cord mesenchymal stem cells in the preparation of a product for treating and / or preventing Col4a5 gene mutation-induced kidney damage.

[0007] The third object of the present invention is to provide an application of umbilical cord mesenchymal stem cells in the preparation of a product for repairing glomerular basal cell damage.

[0008] A fourth object of the present invention is to provide an application of umbilical cord mesenchymal stem cells in the preparation of a product for repairing glomerular podocyte damage.

[0009] A fifth object of the present invention is to provide an application of umbilical cord mesenchymal stem cells in the preparation of a product for improving renal ischemia.

[0010] The sixth object of the present invention is to provide an application of umbilical cord mesenchymal stem cells in the preparation of a product for treating and / or preventing renal sclerosis.

[0011] A seventh object of the present invention is to provide a product for the above applications.

[0012] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0013] In a first aspect, the present invention provides the use of umbilical cord mesenchymal stem cells in the preparation of a product for treating and / or preventing Alport syndrome renal injury.

[0014] Furthermore, the Alport syndrome is X-linked Alport syndrome.

[0015] In a second aspect, the present invention provides the use of umbilical cord mesenchymal stem cells in the preparation of a product for treating and / or preventing Col4a5 gene mutation-induced kidney damage.

[0016] In a third aspect, the present invention provides the use of umbilical cord mesenchymal stem cells in the preparation of a product for repairing glomerular basal cell damage.

[0017] In a fourth aspect, the present invention provides the use of umbilical cord mesenchymal stem cells in the preparation of a product for repairing glomerular podocyte damage.

[0018] In a fifth aspect, the present invention provides the use of umbilical cord mesenchymal stem cells in the preparation of products for improving renal ischemia.

[0019] In a sixth aspect, the present invention provides the use of umbilical cord mesenchymal stem cells in the preparation of a product for treating and / or preventing renal sclerosis.

[0020] Furthermore, the umbilical cord mesenchymal stem cells are P2 to P4 generation umbilical cord mesenchymal stem cells.

[0021] Furthermore, the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0022] In a seventh aspect, the present invention provides a product having any of the following functions, comprising umbilical cord mesenchymal stem cells:

[0023] A1. Treat and / or prevent Col4a5 gene mutation-induced renal injury;

[0024] A2. Treatment and / or prevention of X-linked Alport syndrome;

[0025] A3, repair glomerular basement damage;

[0026] A4, repair glomerular podocyte damage;

[0027] A5. Improve renal ischemia;

[0028] A6. Treat and / or prevent glomerulosclerosis.

[0029] The application of umbilical cord mesenchymal stem cells provided by the present invention in the preparation of products for treating and / or preventing Alport syndrome renal injury has been experimentally proven to be able to effectively alleviate the degree of proteinuria in Alport syndrome mice, alleviate the progression of renal function damage, and improve the proportion of glomerular sclerosis and podocyte loss. In vitro experiments have shown that the functional protection of MSC on podocytes is one of its mechanism targets for alleviating renal injury. The present invention confirms that umbilical cord mesenchymal stem cells can alleviate, treat and / or prevent Alport syndrome renal injury and can prolong survival. The therapeutic potential of human umbilical cord mesenchymal stem cells in Alport syndrome has been revealed, expanding the means of treating the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 A comparison chart of the survival rate and body weight of KO mice and WT mice at different culture times provided in Example 1 of the present invention;

[0032] Figure 2 A comparison of the kidney appearance and weight of KO mice and WT mice at 28 weeks of age provided in Example 1 of the present invention;

[0033] Figure 3 A comparison chart of urine albumin / urine creatinine between KO mice and WT mice at different culture times provided in Example 1 of the present invention;

[0034] Figure 4 A comparison chart of serum renal function indicators of KO mice and WT mice at different culture times provided in Example 1 of the present invention;

[0035] Figure 5A comparison of kidney histological observations of KO mice and WT mice at different culture times provided in Example 1 of the present invention;

[0036] Figure 6 A comparison chart of kidney histological analysis data of KO mice and WT mice at different culture times provided in Example 1 of the present invention;

[0037] Figure 7 This is a morphological observation diagram of hUC-MSCs of generations P2 to P4 provided in Example 2 of the present invention;

[0038] Figure 8 The FACS test results of hUC-MSCs provided in Example 2 of the present invention;

[0039] Fig. 9 The hUC-MSCs differentiation function test results provided in Example 2 of the present invention;

[0040] Fig.10 Observation diagram of hUC-MSCs labeled with DiR after tail vein injection at different times provided in Example 2 of the present invention;

[0041] Fig.11 This is a quantitative analysis diagram of the residence of DiR-labeled hUC-MSCs in the kidney at different injection times provided in Example 2 of the present invention;

[0042] Fig.12 A flow chart of tail vein administration and sampling of human umbilical cord mesenchymal stem cells for improving renal injury in Alport syndrome mice provided in Example 4 of the present invention;

[0043] Fig.13 A comparison chart of the survival rate and body weight of KO mice under different treatments provided in Example 4 of the present invention;

[0044] Fig.14 Comparison diagram of kidney appearance and urine of KO mice under different treatments provided in Example 4 of the present invention;

[0045] Fig.15 A comparative diagram of quantitative analysis of urine protein and urine red blood cells of KO mice at different culture times under different treatments provided in Example 4 of the present invention;

[0046] Fig.16 A comparison chart of urine albumin / urine creatinine of KO mice at different culture times under different treatments provided in Example 4 of the present invention;

[0047] Fig.17 A comparison chart of serum renal function indicators of KO mice at different culture times under different treatments provided in Example 4 of the present invention;

[0048] Fig.18This is a comparison diagram of kidney histological observation of KO mice under different treatments provided in Example 4 of the present invention;

[0049] Fig.19 This is a comparison chart of kidney histological analysis data of KO mice under different treatments provided in Example 4 of the present invention;

[0050] Fig. 20 Electron microscope observation images of the glomerular basement membrane of KO mice under different treatments provided in Example 4 of the present invention;

[0051] Fig.21 This is a comparison diagram of immunohistochemical staining of KO mouse kidneys under different treatments provided in Example 4 of the present invention;

[0052] Fig. 22 A flow chart of the co-culture analysis of MSC and iPSC-derived podocytes provided in Example 5 of the present invention;

[0053] Fig.23 A comparison of the renal podocyte morphology of AS patients and CTRL at different cell differentiation culture times provided in Example 5 of the present invention;

[0054] Fig.24 A comparison chart of the expression levels of the skeleton protein F-actin in in vitro differentiated renal podocytes from AS patients and CTRL provided in Example 5 of the present invention;

[0055] Fig.25 A comparison diagram of renal podocyte immunofluorescence detection of AS patients and CTRL provided in Example 5 of the present invention;

[0056] Fig.26 A diagram showing the classification of the degree of compliance of the podocyte skeleton provided in Example 5 of the present invention;

[0057] Fig. 27 A comparison chart of the expression levels of the renal podocyte cytoskeleton protein F-actin under different in vitro cell co-culture treatments provided in Example 5 of the present invention;

[0058] Fig.28 A statistical graph of the proportion of renal podocytes under different in vitro cell co-culture treatments provided in Example 5 of the present invention;

[0059] Fig.29 This is an observation diagram of renal podocyte adhesion under different in vitro cell co-culture treatments provided in Example 5 of the present invention;

[0060] Fig.30 The flow cytometry apoptosis detection results of renal podocytes under different in vitro cell co-culture treatments provided in Example 5 of the present invention;

[0061] Fig.31This is a comparison chart of the early apoptosis rates of renal podocytes under different in vitro cell co-culture treatments provided in Example 5 of the present invention. DETAILED DESCRIPTION

[0062] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0063] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0064] In one aspect, the present invention provides the use of umbilical cord mesenchymal stem cells in preparing a product for treating and / or preventing Alport syndrome renal injury.

[0065] The present invention experiments prove that hUC-MSCs can effectively alleviate the degree of proteinuria in Alport syndrome mice, alleviate the progression of renal function damage, and improve the proportion of glomerular sclerosis and podocyte loss. In vitro experiments show that the functional protection of MSC on podocytes is one of its mechanism targets for alleviating renal damage. The present invention confirms that umbilical cord mesenchymal stem cells can alleviate, treat and / or prevent Alport syndrome renal damage and prolong survival. It reveals the therapeutic potential of human umbilical cord mesenchymal stem cells in Alport syndrome and expands the treatment methods for this disease.

[0066] In some specific embodiments, the Alport syndrome is X-linked Alport syndrome.

[0067] The present invention discloses the phenotypic changes of XLAS Alport syndrome mice caused by Col4a5 gene mutation during the AS pathogenesis. It was found that mice with Col4a5 gene knockout began to lose weight and showed abnormal serum renal function indicators at 14 weeks of age. At 28 weeks of age, the indicators significantly deteriorated and the kidneys atrophied and hardened. +(80cell / μl) began to appear in Col4a5 knockout mice at 14 weeks and continued throughout the disease process, which is consistent with XLAS models in dogs, rats and other mice. Ultrastructural observation of kidneys of different ages revealed that the glomerular basement membrane was the initial site of injury, followed by extensive fusion of podocyte foot processes, podocyte loss, and glomerular sclerosis. In the end stage of the disease, tubular injury and renal interstitial fibrosis were manifested, which is consistent with the pathological characteristics of human XLAS kidneys.

[0068] According to another aspect of the present invention, there is also provided the use of umbilical cord mesenchymal stem cells in the preparation of a product for treating and / or preventing Col4a5 gene mutation-induced kidney damage.

[0069] The present invention first confirmed that intravenous injection of hUC-MSCs can prolong the survival time of Col4a5 knockout mice, delay the deterioration of renal function, and improve the degree of partial pathological damage in the kidney. The injection time of the tail vein is from 7 weeks of age, and the second injection is injected at an interval of 2 weeks. This administration mode is observed to improve the renal damage of model mice.

[0070] According to another aspect of the present invention, there is also provided the use of umbilical cord mesenchymal stem cells in preparing a product for repairing glomerular basal cell damage.

[0071] The experiment of the present invention proves that the texture and thickness uniformity of the basement membrane of the glomerulus are improved after the administration of hUC-MSCs. The foot process morphology is also improved. The extensive fusion degree of the foot processes of podocytes is reduced. The secondary cell bodies of adjacent podocytes are staggered and restored to regularity. The role of umbilical cord mesenchymal stem cells in local repair of glomerular basement damage.

[0072] According to another aspect of the present invention, there is also provided the use of umbilical cord mesenchymal stem cells in preparing a product for repairing glomerular podocyte damage.

[0073] The experiment of the present invention proved that the administration of hUC-MSCs reduced the number of podocytes lost at week 28, and the recovery of podocyte cell body morphology was also observed under an electron microscope. This in vivo administration improved the function of podocytes. Podocytes differentiated from iPSCs of AS patients with Col4a5 gene mutations were further co-cultured with hUC-MSCs to observe the functional changes of podocytes. The results showed that the co-culture of hUC-MSCs with podocytes from AS patients could improve the proportion of PAN-induced cytoskeleton disorder and reduce cell apoptosis. This direct protective effect is related to the stimulation of podocytes by exosomes produced by hUC-MSCs. Umbilical cord mesenchymal stem cells have the function of repairing glomerular podocyte damage.

[0074] The present invention compared the appearance of the kidneys of 28-week-old mice and found that, unlike the obvious atrophy and sclerosis of the kidneys of the KO+Saline group of mice, the renal ischemia and sclerosis of the KO+huMSC group of mice were improved. Umbilical cord mesenchymal stem cells can improve renal ischemia and renal sclerosis.

[0075] According to another aspect of the present invention, there is also provided the use of umbilical cord mesenchymal stem cells in preparing a product for improving renal ischemia.

[0076] According to another aspect of the present invention, there is also provided the use of umbilical cord mesenchymal stem cells in preparing a product for treating and / or preventing renal sclerosis.

[0077] In some specific embodiments, the umbilical cord mesenchymal stem cells are P2 to P4 umbilical cord mesenchymal stem cells.

[0078] In some specific embodiments, the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0079] According to another aspect of the present invention, there is also provided a product having any of the following functions, comprising umbilical cord mesenchymal stem cells:

[0080] A1. Treat and / or prevent Col4a5 gene mutation-induced renal injury;

[0081] A2. Treatment and / or prevention of X-linked Alport syndrome;

[0082] A3, repair glomerular basement damage;

[0083] A4, repair glomerular podocyte damage;

[0084] A5. Improve renal ischemia;

[0085] A6. Treat and / or prevent glomerulosclerosis.

[0086] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0087] Experimental Materials

[0088] Col4a5 knockout mice: purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., culture conditions include raising them at suitable temperature and humidity until 7 weeks of age, with free access to water and food under a 12-h light / dark cycle.

[0089] hUC-MSCs: provided by Guanhao Bio GMP Laboratory, hUC-MSCs were obtained from the umbilical cords of healthy pregnant women (test results for HIV, syphilis antibodies, 5 hepatitis markers, 5 TORCH items, etc. were all negative), and all participants have signed informed consent.

[0090] Human induced pluripotent stem cells (hiPSCs): Peripheral venous blood was collected from healthy subjects and AS patients, and mononuclear cells were isolated and cultured using the following protocol:

[0091] Culture medium and culture protocol for inducing human induced pluripotent stem cells (hiPSCs) into renal podocytes

[0092] D-4_D0: ipsc culture medium (manufacturer STEMCELL TM No. 05876),

[0093] D0_D2: Differentiation medium 1: DMEM / F-12: Manufacturer Gibco TM , Cat. No. 11320033; plus 1% MEM NEAA; 0.5% GlutaMAX; 0.1mM β-mercaptoethanol; 6uM CHIR99021; 100U / ml penicillin and 0.1mg / ml streptomycin;

[0094] D2-D16: Differentiation medium 2: hESFM: Manufacturer Gibco TM , Product No. 11111044; plus 2% B27; 100U / ml penicillin and 0.1mg / ml streptomycin;

[0095] D16-: (Maintenance medium) DMEM / Ham F-12: Manufacturer Gibco TM , Catalog No. 11320033; 10% FBS; 100U / ml penicillin and 0.1mg / ml streptomycin;

[0096] After differentiation, the cells were seeded onto Matrigel-embedded slides, fixed with 4% paraformaldehyde, and hiPSCs were identified using Podocin, WT-1, Synaptopodin (podocyte marker), and FITC-phalloidin.

[0097] Experimental methods

[0098] Histological analysis: Tissues were fixed in 4% paraformaldehyde for at least 24 h, embedded in paraffin and sectioned for histological examination. H&E staining, Periodic Acid-Schiff (PAS) staining, and Massonˊstrichrome staining were performed according to the manufacturer's instructions (Servicebio, Wuhan, China). Case Viewer was used for image acquisition. Collagen content after Masson red staining was quantified using ImageJ software.

[0099] Immunofluorescence staining: The kidney tissue was immersed in 4% paraformaldehyde for 15 minutes and permeabilized with 0.2% Triton X-100 for 15 minutes. Then, the slides were immunostained with the primary antibody. Fluorescently labeled secondary antibodies were used. The samples were stained with DAPI and observed. Photos were taken using a Leica SP8 confocal scanning microscope. Image J was used for quantitative immunofluorescence analysis.

[0100] Transmission electron microscopy (TEM): The cortical area of ​​renal tissue was 1 mm 3 Small pieces were placed in electron microscopy fixative, embedded in tissue after gradient dehydration, prepared into 40-50 nm electron microscopic sections and stained, and observed and imaged using a transmission electron microscope.

[0101] In vivo tracing: DiR fluorescent dye was used to prepare 1-5 μM working solution, and hUC-MSCs were added to the resuspended dye solution to make the density of 1x10 6 / mL. After incubating hUC-MSCs at 37°C for 20 minutes, the cells were resuspended, and hUC-MSCs labeled with DiR fluorescent dye (experimental group) and PBS (negative control group) were injected into the tail vein of mice. Different observation times were set, and the mice were tracked in vivo using the small animal in vivo imaging system (IVIS).

[0102] Cytoskeleton staining: Fix the cell samples on the cell slide, permeabilize the cell samples with 0.5% Triton X-100, wash them, add TRITC-labeled phalloidin working solution and incubate them at room temperature in the dark for 30 minutes, counterstain the cell nuclei with DAPI solution and seal the slides, select TRITC excitation / emission filter (Ex / Em=545 / 570nm) and DAPI excitation / emission filter (Ex / Em=364 / 454nm). Quantitative calculation of actin stress fibers: A: More than 90% of the cell area is filled with thick stress fibers; B: At least 2 thick stress fibers run under the cell nucleus, and the rest of the cell area is filled with thin stress fibers; C: There are no thick stress fibers, but some stress fibers exist; D: Stress fibers are not seen in the central area of ​​the cell. Randomly select 4 fields of view, calculate all cells in the field of view, and count the percentage of each type.

[0103] Apoptosis detection: AnnexinV / 7AAD apoptosis detection was performed according to the manufacturer's instructions. Briefly, hiPS-podocytes were seeded in six-well plates. After different treatments, cells were digested with 0.25% trypsin without EDTA and washed with PBS. Centrifuged at 1500 rpm for 5 minutes, resuspended with 500 μl staining buffer and stained with 5 μl AnnexinV at room temperature for 15 minutes in the dark. Flow cytometric analysis (CytoFLEX, BD, USA) was performed 5 minutes after adding 5 μl 7AAD reagent.

[0104] RNA sequencing (RNA-seq): After extracting the total RNA from each group of cell samples, mRNA is separated, fragmented, and cDNA chains are synthesized, double-stranded cDNA is purified, products are purified, PCR amplified and purified, and the library is sequenced after quality inspection.

[0105] Statistical analysis: Data processing was performed. For quantitative data, the t-test was used for comparison between the two groups, and one-way analyses of variance (ANOVA) was used for comparison between multiple groups. Image J 1.45 software was used for quantitative analysis of renal pathological staining, and the data were expressed as the ratio of positive staining area to the total analysis area. All statistical processes were completed by the statistical software GraphPad Prism9. When P < 0.05, the difference was considered statistically significant.

[0106] The relevant indicators of the following embodiments were detected according to the above experimental methods. All animal protocols in the following embodiments were approved by the Animal Ethics Committee of Guangzhou Women and Children's Medical Center.

[0107] Example 1 Renal phenotype characteristics of Col4a5 gene knockout Alport syndrome mouse model

[0108] In this example, mice with the Col4a5 gene knocked out (KO mice) were used to observe the renal phenotype characteristics. The results showed that the KO mice showed renal atrophy and obvious fibrosis as they aged. Figure 1 As shown, a is the survival rate, b is the body weight, and the survival time of KO mice was significantly shorter than that of WT mice; at 7 and 14 weeks of age, there was no significant difference in weight, morphology, and behavioral habits between KO mice and wild-type mice in the same litter control group. At 21 and 28 weeks of age, KO mice were smaller than WT mice, had less activity, more white secretions from the eyeballs, and some of their whiskers and body hair fell off, and their weight did not increase; Figure 2The figure c shows the kidney comparison, and the figure d shows the kidney weight. The kidneys of KO mice at 28 weeks of age were dissected, and it can be seen that the kidneys were reduced in size, the tissues were severely hypocongested, the kidney surface was uneven, the nephron structure was absent, the kidney tissue was hard, and the kidney tissue had granular changes. The kidney mass analysis showed that the kidney mass of KO mice at 28 weeks of age was significantly reduced compared with that of WT mice.

[0109] Urine protein results such as Figure 3 As shown in the figure, compared with the WT mice, the KO mice had increased urine albumin / urine creatinine from 7 weeks of age and there was a difference (P=0.0220). As the age increased, the urine protein of the KO mice gradually worsened until the difference in urine albumin / urine creatinine was significant at 28 weeks of age (P=0.0009).

[0110] Serum renal function test results Figure 4 As shown in the figure, g is serum creatinine, h is urea nitrogen (BUN), and i is cystatin C (Cys-C). There were no significant differences in serum creatinine, urea nitrogen and cystatin C between the KO group and the WT group at 7 weeks of age. At 14 weeks of age, serum creatinine (P=0.0330) and urea nitrogen (P=0.0361) in the KO group were significantly increased compared with those in the WT group. At 21 weeks of age, cystatin C in the KO group was significantly increased compared with that in the WT group (P=0.0019), indicating that the renal function of the KO mice further deteriorated. At the 28th week of age, all serum renal function tests of the mice were higher than those of the control group, indicating that the renal function of the KO mice further deteriorated and showed signs of renal decompensation.

[0111] like Figure 5 and Figure 6 As shown, Figure 5 These are the observation pictures of PAS staining, Masson staining and HE staining. Figure 6 l is the glomerular sclerosis score, m is the Masson negative area, and n is the tubular injury score. PAS staining of renal pathology confirmed that with the increase of age, the glomerular capsule wall of KO mice was thickened, the glomerular mesangial cells and matrix were slightly proliferated, the mesangial basement membrane was thickened, and the glomeruli were sclerotic. Masson staining showed glomerular pericapsular fibrosis, tubular dilatation, and renal interstitial fibrosis. HE staining showed different degrees of tubular dilatation and atrophy, and the tubular brush border fell off.

[0112] Example 2 Cultivation and identification of human umbilical cord mesenchymal stem cells (hUC-MSCs)

[0113] 1. hUC-MSCs culture

[0114] hUC-MSCs were cultured in DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin and streptomycin; then, they were incubated at 37°C and 5% CO 2The cells were routinely cultured and subcultured in an incubator until P4; the cells obtained by subculture were digested with 0.25% trypsin, and the stably proliferating cells were collected.

[0115] 2. Identification of hUC-MSCs

[0116] 1) The morphology of hUC-MSCs from P2 to P4 was observed to be long spindle-shaped, similar to fibroblasts, with monolayer adherent growth and cell aggregation in a vortex-like pattern, such as Figure 7 shown.

[0117] 2) Perform FACS detection, the results are as follows Figure 8 As shown, hUC-MSCs strongly expressed MSC markers CD73, CD90, and CD105, with a positive rate greater than 99%, but did not express CD11b, CD19, CD34, CD45, and HLA-DR, with a positive rate less than 1%, indicating that the hUC-MSCs cultured in this example had a high purity.

[0118] 3) Explore the differentiation function of hUC-MSCs, such as Fig. 9 As shown (scale bar is 100 μm), a is alizarin red staining to detect osteogenic differentiation of cells, b is alizarin blue staining to detect chondrogenic differentiation, and c is oil red staining to detect adipogenic differentiation of cells. As a result, after appropriate induction, hUC-MSCs showed osteocyte characteristics, alizarin red staining showed calcium deposition in bone matrix, and hUC-MSCs also showed adipocyte characteristics after induction, with the appearance of oil red-positive lipid droplets, and after alizarin blue staining, the acidic mucopolysaccharide in the cartilage tissue can be stained blue-green by alizarin blue. This shows that hUC-MSCs have good osteogenic, adipogenic and chondrogenic differentiation abilities, and have good mesenchymal stem cell characterization.

[0119] 3. In vivo tracing of hUC-MSCs

[0120] hUC-MSCs were labeled with DiR (Fluorescence Biotechnology) and in vivo imaging was performed 1, 4, 7, 10, 24, 48, 72, and 120 hours after tail vein injection. Fig.10 and Fig.11 As shown, hUC-MSCs were injected into mice via the tail vein 24 hours after the kidney fluorescence signal intensity reached the strongest, and continued until 120 hours after the fluorescence imaging was performed again, indicating that hUC-MSCs were still resident in the kidney.

[0121] Example 3 hUC-MSCs tail vein administration regimen

[0122] This example explores the following two aspects of hUC-MSCs injection dosage and administration time:

[0123] ① hUC-MSCs injection dose and frequency: First, three dosage methods were designed, namely 200,000 cells / mouse, once a week for four times, 500,000 cells / mouse, once a week for four times, and 1 million cells / mouse for four times, and urine and kidneys of mice were collected regularly. The results showed that a dose of 1 million cells / mouse and a total of 4 injections would increase the number of deaths from pulmonary embolism in mice. Based on this, this study finally chose 200,000 cells / mouse, once a week for two times, with an interval of 2 weeks.

[0124] ② hUC-MSCs administration time: In order to evaluate the time point of stem cell intervention in KO mice, the preliminary experiment was designed with three groups of administration times of 9 weeks, 14 weeks, and 21 weeks respectively.

[0125] Example 4 Human umbilical cord mesenchymal stem cells improve renal injury in Alport syndrome mice

[0126] In this example, the experimental animals were divided into the following three groups (5 mice / group): ① Control group: WT group, which was the wild type of the same littermate of Col4a5 knockout mice, without intervention; ② Disease group: KO+Saline, Col4a5 knockout mice were treated with PBS; ③ Experimental group: KO+huMSC, Col4a5 knockout mice were treated with hUC-MSCs; ④ Comparative group: KO+ACEI, Col4a5 knockout mice were given angiotensin converting enzyme inhibitor (ACEI) as a positive control. Col4a5 knockout Alport syndrome model mice were adapted for 2 weeks at the beginning of the disease (7th week), and they were fed and watered by themselves, maintained at a breeding temperature of 21-26°C, 50-60% suitable humidity, and provided with an appropriate (12h light, 12h dark) day and night light change cycle. Starting from 9 weeks of age, hUC-MSCs (2×10 5 cells / cells) treatment, the specific tail vein administration and sampling scheme are as follows Fig.12 shown.

[0127] 1. Analysis of biochemical parameters of Alport syndrome mice improved by human umbilical cord mesenchymal stem cells

[0128] like Fig.13 As shown, b is the survival rate, c is the body weight,

[0129] The results showed that the survival rate of WT mice on day 196 was 100%, the survival rate of KO+Saline was 57.1%, the survival rate of KO+ACEI was 40%, and the survival rate of KO+huMSC was 75%. The mice in the KO+Saline group and the KO+ACEI group began to lose weight at the 14th week of age, while the weight of the mice in the KO+huMSC group gradually increased. hUC-MSCs treatment improved the growth of KO mice. Comparison of the kidney appearance of 28-week-old mice showed that Fig.14 As shown, d is the kidney comparison, and e is the urine comparison. Unlike the obvious atrophy and sclerosis of the kidneys in the KO+Saline group of mice, the renal ischemia and sclerosis of the KO+huMSC group of mice were improved. The appearance of urine in each group of mice was observed. The urine volume of the WT group of mice was 2.8-3.0 ml / day, and the urine was light yellow, clear, transparent, and had no obvious odor. The urine volume of the KO+Saline mice was 1.3-1.7 ml / day, and the urine was dark yellow, turbid, white or red flocculent, with a lot of foam on the upper layer of urine and a clear smell of mouse urine. After ACEI and hUC-MSCs treatment, the KO mice had a urine volume of about 1.9-2.5 ml / day, and part of the urine turned light yellow, the white flocculent was improved, and the foam on the upper layer of urine was reduced. Among them, the improvement of the appearance of urine in KO+huMSC was more obvious than that in KO+ACEI.

[0130] In this study, random urine samples of mice in different age groups were semi-quantitatively tested for urine protein and urine red blood cells. The results were as follows: Fig.15 Compared with the KO+Saline group, the degree of proteinuria in the KO+ACEI group was alleviated, but there was no statistical difference; the difference in proteinuria between the hUC-MSCs group and the KO group was significant, and the proportion of urine red blood cells 3+ in the hUC-MSCs treatment group was significantly decreased (≥200 cells / μl).

[0131] This study also collected 24-hour urine samples from mice at different observation time points and found that Fig.16 As shown in the figure, the urinary albumin / urinary creatinine in the KO+Saline group gradually increased with the increase of age until it reached 25.72±0.96μg / ml at 28 weeks of age. The KO+ACEI group had no significant improvement in urinary albumin / urinary creatinine, while the KO+huMSC group delayed urinary albumin / urinary creatinine by 21 weeks (P=0.0399) and 28 weeks (P=0.0110). Fig.17As shown, i is creatinine, j is urea nitrogen (BUN), and k is cystatin C (Cys-C). After two doses of administration, the decrease in creatinine and urea nitrogen (BUN) in the hUC-MSCs treatment group was significantly alleviated. Compared with the KO+Saline group, there were significant differences in creatinine (P=0.0302) and urea nitrogen (P=0.0078) at week 14, and serum cystatin C (P=0.0344) was also significantly improved at week 28. In comparison, the therapeutic effects of the KO+ACEI treatment group on serum creatinine, urea nitrogen, and serum cystatin C were similar to those of the normal saline group, and there was only a statistical difference in the reduction of serum creatinine at week 28 (P=0.0112).

[0132] 2. Analysis of human umbilical cord mesenchymal stem cells improving renal pathology in Alport syndrome mice

[0133] To investigate the protective effect of hUC-MSCs on the renal pathological structure of Alport syndrome model mice, kidney tissues of mice in each group at 28 weeks of age were collected. Fig.18 As shown in Figure 2, PAS staining showed that compared with the KO+Saline group, the proportion of glomerular sclerosis in the KO+huMSC group was reduced, the reduction in the number of glomerular podocytes was alleviated, and the thickening of the glomerular capsule wall was significantly improved (e.g. Fig.19 b); Masson staining showed that the degree of fibrosis around the glomerular capillary loops in the KO+huMSC group was significantly reduced compared with that in the KO+Saline group, and the tubular and interstitial fibrosis was significantly reduced (e.g. Fig.19 c); HE staining showed that the morphology of renal tubular epithelial cells in the KO+huMSC group improved, including epithelial cell lumen expansion, cell flattening, and vacuolar degeneration. In comparison, the KO+ACEI treatment group could improve the renal pathological damage of KO mice to a certain extent, but there was no statistical difference compared with the KO+Saline group (e.g. Fig.19 d).

[0134] Electron microscopy analysis Fig. 20 As shown in the figure, the glomerular basement membrane of mice in the KO+Saline and KO+ACEI treatment groups showed different degrees of uneven basement membrane thickness and extensive fusion of foot processes. Compared with the KO+Saline group, the texture and thickness uniformity of the glomerular basement membrane in the KO+huMSC group were improved. The foot process morphology was also improved. The extensive fusion of the foot processes of podocytes was reduced. The secondary cell bodies of adjacent podocytes were staggered and restored to regularity. Podocyte loss is an important pathological manifestation of the progression of Alport syndrome. Immunohistochemical staining in this study is shown in Figure 2. Fig.21 As shown, the number of renal podocytes (WT1 positive staining) in the KO+Saline group of mice at 28 weeks of age was significantly reduced compared with that in the WT group, and the proportion of glomerular podocyte loss in the KO+huMSC group was significantly decreased.

[0135] Example 5 Construction of human induced pluripotent stem cells (hiPSC) differentiated renal podocytes and hUC-MSCs to improve podocyte injury in vitro

[0136] In order to explore the possible effects of hUC-MSCs on renal podocyte function, iPSCs from healthy individuals and patients with Alport syndrome were reprogrammed and induced to differentiate into podocytes. Fig. 22 As shown in the figure, MSCs were co-cultured with iPSC-derived podocytes to evaluate whether MSCs could directly improve podocyte function. In clinic, AS patients often experience worsening proteinuria under the stimulation of pathological factors such as infection. In this study, PAN was used to treat podocytes in each group to induce pathological damage.

[0137] In vitro differentiation experiments showed that the nuclear-cytoplasmic ratio of cells increased on the 6th day of cell differentiation culture, and the cells were flattened into clusters with clear boundaries. On the 16th day, the cell bodies of renal podocytes could be clearly observed to have protrusions (such as Fig.23 Compared with healthy human renal podocytes, AS patient-derived renal podocytes have reduced cell polarity and significantly reduced expression of the cytoskeleton protein F-actin (as shown in Figure 2A). Fig.24 As shown). Immunofluorescence detection of iPSC-induced differentiated cells in this study showed positive staining of podocyte markers WT1 and Podocin. (As shown Fig.25 shown).

[0138] The cell experiment groups are as follows: ① Control group: CRTL, healthy human peripheral blood induced differentiation of renal podocytes; ② Disease group: AS+DMEM, Alport syndrome patient peripheral blood induced differentiation of renal podocytes (AS-hiPS-podocytes); ③ Intervention group: AS+PAN, the attached AS-hiPS-podocytes were pretreated with puromycin (PAN) 0.5μg / mL for 24 hours; ④ Treatment group: AS+PAN+huMSC, AS-hiPS-podocytes were stimulated with PAN for 24 hours and then co-cultured with hUC-MSCs for 48 hours. The cell concentration of hiPS-podocytes was adjusted to 2×10 5 cell / ml, inoculated in the lower chamber of Transwell, the concentration of hUC-MSCs was 2×10 5 cell / ml, inoculated in the upper chamber of Transwell, and set up 3 sub-wells for each group. After 48 hours of co-culture, the lower chamber of Transwell was taken out, the culture medium was discarded, a part was counted, and a part was fixed with PFA for subsequent experiments.

[0139] Comparison of the skeleton rearrangement of podocytes in each group: According to the number, morphology and distribution of actin staining in cells, e.g. Fig.26As shown, the degree of podocyte skeleton compliance is divided into four types from high to low: A, B, C, and D. Compared with podocytes from healthy individuals, podocytes in the AS+DMEM group had skeleton rearrangement, and the proportion of podocytes of type C (P=0.0070) and type D (P=0.0200) increased. After puromycin treatment, the phenomenon of loss of polarity of skeleton stress fiber arrangement in the AS+PAN group was more common, that is, the proportion of type D podocytes (P=0.0008) was significantly higher than that in the AS+DMEM group.

[0140] Analysis of the improvement effect of hUC-MSCs on the skeletal rearrangement of AS-derived podocytes: Fig. 27 and Fig.28 As shown in the figure, compared with the healthy control group, the proportion of podocytes of type A in the AS group decreased (P=0.0125), while the proportion of type D increased (P=0.0031). When the podocytes of the AS group were treated with PAN, the proportion of type D podocytes (P=0.0008) was significantly higher than that of the AS+DMEM group. Compared with the AS+PAN group, the proportion of type A podocytes in the AS+PAN+huMSC group increased significantly (P<0.0001), while the proportion of type D podocytes (P<0.0001) decreased. The loss of podocyte adhesion was analyzed, as shown in the figure. Fig.29 As shown, the AS+PAN+huMSC group had significantly fewer floating cells than the AS+PAN group, and a higher proportion of podocytes growing in cord-like monolayers.

[0141] Comparison of podocyte apoptosis in each group. Fig.30 and Fig.31 As shown, the apoptosis rate of AS+DMEM group (10.59%: Q2+Q3) and AS+PAN group increased (21.35%: Q2+Q3). ​​Compared with the AS+PAN group, the apoptosis rate of AS+PAN+huMSC cells was significantly reduced (12.19%: Q2+Q3). ​​It is worth noting that hUC-MSCs significantly reduced the occurrence of early cell apoptosis.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of umbilical cord mesenchymal stem cells in the preparation of products for the treatment and / or prevention of Alport syndrome renal injury.

2. The use according to claim 1, characterized in that: The Alport syndrome is X-linked Alport syndrome.

3. Application of umbilical cord mesenchymal stem cells in the preparation of products for the treatment and / or prevention of Col4a5 gene mutation-induced kidney damage.

4. Application of umbilical cord mesenchymal stem cells in the preparation of products for repairing glomerular basal cell damage.

5. Application of umbilical cord mesenchymal stem cells in the preparation of products for repairing glomerular podocyte damage.

6. Application of umbilical cord mesenchymal stem cells in the preparation of products to improve renal ischemia.

7. Use of umbilical cord mesenchymal stem cells in the preparation of products for the treatment and / or prevention of renal sclerosis.

8. The use according to any one of claims 1 to 7, characterized in that: The umbilical cord mesenchymal stem cells are P2 to P4 generation umbilical cord mesenchymal stem cells.

9. The use according to claim 8, characterized in that: The umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

10. A product having any of the following functions, comprising umbilical cord mesenchymal stem cells: A1. Treat and / or prevent Col4a5 gene mutation-induced renal injury; A2. Treatment and / or prevention of Alport syndrome; A3, repair glomerular basement damage; A4, repair glomerular podocyte damage; A5. Improve renal ischemia; A6. Treat and / or prevent glomerulosclerosis.

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