Preliminary Study On Murine Bone Marrow Mesenchymal Stem Cells Repairing Endometrial Damage

Intrauterine adhesions(IUA) is a common gynecological secondary disease, whose incidence rate was increasing in recent years. It is usually caused by improper operation of the uterine cavity and occured after abortion or endometrial resection. The pathological mechanism of IUA is due to the fibrosis of endometrial epithelial and mesenchymal caused by trauma and inflammation. The lack of endometrium covering lead up to anterior and posterior wall of uterine adhere with each other. At present, most studies have shown that the incidence of IUA is due to the destruction of intrauterine environment,also the scope and depth of damage. Adhesion separation hysteroscopic surgery combined with postoperative artificial cycle is used to restore the shape of the uterine cavity and stimulate the regeneration of endometrium. The current clinical data confirm that this approach is universally effective treatment for mild patients, but for severe patients is limited. symptoms often relapse after surgery.In recent years, endometrial mesenchymal stem cell hypothesis has been confirmed by scholars at home and abroad.They pointed out that the regeneration of endometrial functional layer shedding in the menstrual cycle may be a result of proliferation and differentiation of stem cell in stroma. This theory give a new ideas to study the mechanism of endometrial damage and repair barriers.In this study, we choose bone marrow mesenchymal stem cells (BMSCs) as seed cells, transplanted into murine model with endometrial damage over the cross-transplantation of male and female. Preliminary study with the cell therapy of endometrial repair barriers by Situ hybridizationby to locate the cell with SRY (Sex-determining region Y) gene fragment and pathological examination of endometrial tissue in transplanted receptors. Objectivepreliminary study of the mechanism and effect of bone marrow mesenchymal stem cells cultured and amplified in vitro in the endometrial damage repairement.Materials and methods1. Isolation and Identification of murine bone marrow mesenchymal stem cells Mouse bone marrow mesenchymal stem cells were isolated and cultured with adherent screening. Purified and amplified the cell by passaged several times. Identified cultivated mesenchymal stem cells by test cell surface antigens with flow cytometry, and induced cell differentiation into osteoblasts and adipocytes with conditioned medium, as well as the morphological characteristics.2. Murine endometrial damage model:A duct with hot water at a tempretrue of100℃in which is used to injury the model’s endometrium in the cornual of "Y" shaped uterine, tissue was harvest at different time points as6h,48h,7d and14d to observe the progress of the damage repair process in mouse endometrium.3. Located SRY-positive cells and pathological analysis of models’ endometrium: Murine model were divided into experimental and control groups randomly, both of two group have15models,the untreated side of experimental group as a self-control group. Bone marrow mesenchymal stem cells were transplanted into experimental group mice by intrauterine and intravenous injection. The control group was injected with the same amount of PBS. After transplantation, the mice were killed according to the established time. HE staining to observe endometrial repairment of two groups, the expression of SRY-positive cells in the tissue was observed by situ hybridization.4. Statistical analysis Statistical analysis was performed with SPSS16.0software. the positive rate is expressed as a percentage. The comparision between experimental and control groups was checked by the χ2test. The testing standard is set at α=0.05.Result 1. Isolation and Identification of murine bone marrow mesenchymal stem cells Bone marrow-derived mesenchymal stem cells isolated by adherence to screening have the ability of differentiating into osteoblast and adipocyte. Identification of bone marrow mesenchymal stem cell surface antigen with flow cytometry shown that CD90(88.3%) and CD44(87.9%)positive expression, hematopoietic stem cell surface antigen CD34(3.8%) and monocyte surface antigen CD45(2.3%) Were negative.2. Murine endometrial damage model:Hot catheter can injury endometrium and destroy the normal structure of the endometrial epithelium and stroma,then lead to the phenomenon of fibrosis.3. Located SRY-positive cells and pathological analysis of model’s endometrium:Situ hybridization showed that experimental group appeared colonization of the SRY-positive cells, especially the site of injury and repair, there is a certain degree of aggregation.Untreated side appeared scattered SRY-positive cells occasionally. The control group did not show the SRY-positive cells.HE staining of endometrial tissue with local signs of hyperplasia, and rare fibrosis repairment in experimental group, as well as the control group mainly shown the fibrosis repairment.4. Statistical analysis:The positive rate of SRY expression in the endometrial tissue in the experimental group (93.3%) is higher than their control group (73.3%) and PBS control group(15.6%). Compare the SRY positive rate of endometrial tissue in the experimental group with the other two groups,the differences was statistically significant(P<0.05);the positive-rate of experimental group and PBS control group significant difference(P<0.01).Conclusion1. Bone marrow mesenchymal stem cells with proliferative and differentiation capacity can be isolated from murine bone marrow with method of adherence screening. 2. Transplantation of bone marrow mesenchymal stem cells can promote the repairment of the endometrial injury, and the cells can located both in the damage and normal endometrial tissue.