| Objectives:1. to observe the biological characteristic and multipotentail differentiation of bone marrow mesenchymal stem cells(BMSCs) in vitro, to establish a stable and lasting culture system and provide a source of cells therapy for stress urinary incontinence(SUI).2. to investigate the effects of 17 beta-estradiol(17β-E2) on BMSCs myogenic differentiation and proliferation, so that to enhance skeletal differentiation ratio of BMSCs by 5-aza cytidine(5-aza) induction, exploring possible biological mechanisms on estrogen treatment for SUI at cellular level, and providing enough cells for treating SUI.Methods:1. BMSCs were isolation from rats by plastic adherent method. The cell surface antigens were detected by flow cytometry for identification of BMSCs.2. The growth curve, adhesion rate was investigated to illuminate the proliferative potential. Multilineage differentiation capacity were observed through inducing BMSCs to osteogenic,adipogenic,chondrogenic differentiation.3. The passage 3 BMSCs were induced by 5-aza for 48h, then cells were contiguously cultured with non-5-aza medium. Immunofluorescence staining (IF) detected the markers of skeletal muscle differentiation on day 7,14 and 21, respectively, including myogenin,desmin,myosin heavy chain(MHC).4. MTT method was used to investigate the proliferation effects of 17beta-estradiol on BMSCs, to determine the optinal concentration of 17beta-estradiol.5. Hoechst33258 staining was used to measure the effect of 17β-E2l on BMSCs DNA content, to explore the proliferative potential. of 17β-E2 on BMSCs.6. The effects of 17β-E2 on BMSCs 5-aza-induced skeletal muscle differentiation were determined by IF and terminal deoxynucleotidyl transferase-mediated fluorescein-conjugated deoxyuridine triphosphate nick end-labeling(TUNEL) assay, to detect expression level of skeletal muscle differentiation markers and 5-aza-induced BMSCs apoptosis respectively.Results:1. High purity of BMSCs were obtained by plastic adherent method, and cells vitality was high. The primary BMSCs grew and adhered slow, culturing 24 hours latter, a few cells began to adherent to plastic plates and showed short spindle shape. After 3 days, adherent cells markedly increased, grew like colony, and got confluence in 10 to 14 days. Passage cells grew and adhered faster, 90 percent cells adhered to plastic plates after culturing 10 hours. The time of cells double populations was nearly 30 hours.2. Flow cytometry showed BMSCs CD34-/CD29+, Von Kossa,oil red O,toluidine blueour staining indicated BMSCs differentiating into osteogenic,adipogenic,chondrogenic cells. 3. IF showed green or red immunofluorescence in cytoplasm, indicated BMSCs positive for myogenin,desmin,MHC after 5-aza induction and skeletal muscle differentiation. Furthermore, the expression level of skeletal muscle differentiation markers was relative with culture time. The expression of myogenin declined along with culture time prolongation, however, the expression of desimin and MHC was opposite.4. MTT methd showed 17β-E2 concentation-dependently promoted BMSCs proliferation. The optimal concentration of 17β-E2 was 1nmol/L5. Hoechst33258 staining suggested 17β-E2 increase BMSCs DNA content with culture time dependency. On 48 hours, the effect was slight; on 7 days,14 days, the effect was significant compared with control group.6. 17β-E2 could increase the expression of skeletal muscle differentiation markers.7. TUNEL assay suggested 5-aza induced BMSCs apoptosis. Furthermore, TUNEL positive cells ratio increased by time dependency. 17β-E2 could reduce TUNEL positive cells, the difference was significant compared with single 5-aza induction group.Conclusion:1. We can establish a kind of simple and stable BMSCs lasting culture system by plastic adherent method. Furthermore isolated BMSCs were high vital, possessed multipotent differentiation, such as skeletal muscle cells differentiation.2. 17β-E2 could promote BMSCs proliferation, enhance skeletal muscle differentiation of BMSCs induced by 5-aza, and reduce BMSCs apoptosis by 5-aza indction.
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