The Effects Of Adipose Tissue-Derived Mesenchymal Stem Cell Transplantation Therapy In Mice With Systemic Lupus Erythematosus

BackgroundSystemic lupus erythematosus (SLE) is a destructive autoimmune disease, which can damage multiple organ systems, and are characterized by autoantibodies, immune complex formation, chronic inflammation, and end-organ damage. Antigen-antibody complexes can be deposited in the small blood vessels, skin basement membrane, kidney and other organ systems after formed. The clinical manifestations of the disease is mainly concentrated in the skin, kidney, skeletal muscle and blood system, but it also can affect the lungs, and central nervous system, serous and almost all of the other organ systems.The epidemiological survey of SLE shows that SLE occurs worldwide, but there are obvious differences between different countries and regions. The prevalence of SLE is higher in our country, and has a tendency to rise year by year. At present, the pathogenesis of SLE is not studied very clearly. It is generally recognised the that SLE is influenced by many factors, including genetic gene, the environment of growth, the estrogen and gender, the epigenetic regulation of gene expression, the immune cells and cytokines. Persistent immune cell activation due to inadequate immune surveillance or suppression is associated with the development of SLE. The abnormal regulation of a variety of cells in the body dysregulation makes the treatment of SLE become very difficult, because a wide range of immunosuppression will bring many side effects.At present, the treatment of SLE mainly use the glucocorticoid combined with immune inhibitors. This therapy is effective in most cases, but there is poor efficacy and adverse reactioms in the treatment of some intractable, severe lupus erythematosus.To cure autoimmune disease, we urgently need to find a new treatment which can inhibit immune cells, and do not affect the body’s other immune system work. With the deep understanding of the pathogenesis of autoimmune diseases, more and more scholars began to switch their attention to the stem cell therapy.Stem cells is a kind of very different cell groups. Despite the different sources, stem cells have two characteristic properties. First of all, under certain conditions, stem cells have continued, unlimited self-renewal ability. Second, the stem cells retain the potentiality that differentiate into many other special type of cell. Hematopoietic stem cell (HSC) transplantation is one kind of the most widely used stem cell therapy. As to the shortcomings of allogeneic hematopoietic stem cell,such as, the big risk of use, the matching donor is difficult to search, immune rejection after transplantation,etc. In order to solve these problems, the researchers began to look for other stem cells. Mesenchymal stem cells (MSC) is a kind of pluripotent stem cells which can differentiate into osteoblasts, chondrocytes, fat cells.They have the function of the induction of immune tolerance, immunosuppression, and low immunogenicity, and can avoid allograft rejection. So the transplantation can be done without genetic matches, and it is widely used in autoimmune diseases, including systemic lupus erythematosus, having a very broad application prospects in treatment. The earliest known MSC is derived from bone marrow, with the name of bone marrow mesenchymal stem cell (BMSC).AS it needs a lot of bone marrow mesenchymal stem cells to get the BMSC.bone it brought misery to the donor and the risk of infection, which limits the application of BMSC. The researchers around the world began to look for a new source of mesenchymal stem cells, including dental pulp tissue, cord blood, placenta, fat, etc., the fat source is the centre of attraction. Compared with the mesenchymal stem cells getted from bone marrow, the adipose-derived stem cell (ADSC) has many advantages such as abundant source, easy to collection, more content, donor suffered less pain and less ethical limits, and it becomes the main content of ectomesenchymal stem cell research in recent years. ADSC has the characteristic of MSC, which includes dividing into osteogenesis, cardiac muscle, smooth muscle, fat, cartilage and nerves and endothelial cells, and it also has similar BMSC immunity activity. There have been some studies which confirmed the ADSC’s effect in neurological damage repair, myocardial damage repair, autoimmune disease treatment,etc,mostly using human mesenchymal stem cells to do the experiment. For follow-up experiments need lupus rat animal model of mice, so this experiment adopted the same extracted allogeneic mouse as the source of adposie mesenchymal stem cells. Although mesenchymal stem cell transplantation therapy has been used clinically, but the exact mechanism of MSC therapy has not been clearly expounded.The mechanism reported in the literature includes:MSC immunosuppressive effects that are mediated by activation and differentiation Treg cells to produce; By secreting an enormous amount of IL-10 or TGF-beta 1 cytokines, which can make antigen presenting cells of mature restrained, MSC can induce T cells not to produce an immune response; MSC can be activated T cells apoptosis, and prevent proliferation of T cells at the same time, in order to give play to the role of immunosuppression, etc.Autoantibodies plays an important role in SLE patients’ multiple organ dysfunction, the rise of anti-dsDNA antibodies is one of the criteria for determining that the SLE patients’autoimmune is active or not.By observing whether anti-dsDNA antibodies reduce or not,this experiment evaluate that ADSC treatment is effective against lupus mice or not. With the deepening of the research, people realize that Th17 cells characteristic and its secretion of cytokines IL-17 in the pathogenesis of autoimmune diseases such as SLE has played a key role. IL-17 missed mice cannot induce the lupus erythematosus with the expression of increased Lupus antibody expression and lupus nephritis. So could ADSC therapy reduce lupus mice of the expression of IL-17 or not? RORyt is the key factor that regulate the Th17 cells differentiation and the expression of the main effect factors of IL-17. RORyt can be adjusted by IL-17 to disease affected by Th17 cells play a role. Whether ADSC adjust the generation of IL-17 and release by RORyt?There are many kinds of animal models used in autoimmune disease research, and B6.MRL Faslpr/J mice is one of mostly used animal models for the study of systemic lupus erythematosus. The systemic autoimmune reaction appears at about 30 weeks, characterized by swollen lymph nodes, glomerular kidney disease.By doing the the same allogeneic mesenchymal stem cell transplantation therapy for the rat model of systemic lupus erythematosus, this experiments observe its efficacy and safety, so as to provide laboratory evidence for clinical application. And to explore its possible mechanism based on the research of the related transcription factors and cytokines.ObjectiveTo study influence that the same allogeneic mesenchymal stem cell transplantation give on the the main indicators of systemic lupus erythematosus between fat rat body weight, urine protein, anti-dsDNA antibodies and renal pathological changes, as well as ROR gamma t to the kidney, IL-17 mrna expression quantity, And explore the transplantation’s possible mechanisms in the body.MethodsUse the collagenase digestion method to separate the mesenchymal stem cells between fat of mice’s fat and make the cell’s subculture in vitro. Observe the cell’s morphology,and take the third-generation cell which grows well by flow cytometry instrument to identify the cell phenotype.Divide the female B6. MRL Faslpr/J lupus mice into 2 groups, each group of five mices (the 2 groups animals have on difference in group weight and urine protein concentration); Five C57BL/6 mices of the same age are the normal control group; ADSC treatment group:inject the 1-3 generation of ADSC into the tail vein (every 10 g weight 0.1 X106 cells), once every two weeks,3 times; As the lupus mice and normal control group, use the same amount of saline instead of ADSC to do the tail vein infusion.The main indexes detection:the coomassie brilliant blue method (Brandford law) for the detection of urine protein concentration; Method of enzyme-linked immunosorbent (ELISA) to detect serum anti-dsDNA; Conventional histopathological H.E staining for observing the pathological changes of kidney in mice; Use the Immunofluorescence in mice kidney pathological to observe the IgG deposition; Real-time fluorescent PCR for the detection of the expression of RORγt、 IL-17mRNA in kidney tissue.StatisticsStatistical software SPSS13.0 was used to conduct statistical analysis for all the experimental data. The data were interpreted by错误！未找到引用源。±SD and analyzed by single factor analysis of variance (One-Way ANOVA). Multiple comparisons were addressed by Bonferroni test. When P<0.05, there was significant difference.ResultsThe mesenchymal stem cells of mice’s fat have a adherent growth, with a shape of long fusiform or polygon, arranged in a beam mixed or into spiral shape. The third-generation ADSC expression of cell surface CD29 was 99.66%, the expression of CD34 is 1.41%, CD45 expression was 0.3%, the expression of CD44 was 72.58%, CD90 expressed as 90.23%.(1) the experiment lasts eight weeks, no death is occurred in both groups of mice, eating, mental health, weight has no obvious difference. (2) there is no statistical difference (P> 0.05) in urine protein concentration between the two groups.; the urine protein concentration of Lupus mice group was obviously higher than the normal control group’s, while the urine protein concentration of the ADSC treatment group was significantly lower than the lupus mice group’s (P< 0.05). (3) lupus mice group’s serum anti-dsDNA antibody concentration was significantly higher than the normal control group’s, while the ADSC treatment group’s was significantly lower than lupus mice group’s (P< 0.05). (4) the kidney pathological H.E staining observation:the mice’s glomerular of lupus mice group has more immune complex deposition,while the ADSC treatment group has less immune complex deposition than the lupus mice group.The kidney expression amount of RORγt、IL-17mRNA of the lupus mice group was higher than the normal control group, while the ADSC treatment group’s was lower than the lupus mice group’s.Conclusion1. The ADSC of mice can be cultured in vitro, positive expression of CD29, CD44 and CD90, no expression of CD34 and CD45 hematopoietic stem cell surface marker.2. Adipose Mesenchymal stem cell transplantation can reduce the concentration of B6 MRL-Faslpr/J lupus mice’s serum anti-DNA antibody, and also reduce proteinuria and IgG immune complex deposition in the kidney caused by the renal injury, which has a therapeutic effect on systemic lupus erythematosus.3. By cutting the expression of transcription factors of RORyt to inhibit the expression and release of IL-17, the ADSC might play a role in the treatment of SLE.