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目的探讨大鼠骨髓间充质干细胞(BMSC)对大鼠RSC96施万细胞增殖及迁移的影响。方法全骨髓细胞贴壁法分离并提取SD大鼠BMSC,倒置显微镜观察第3代BMSC形态;流式细胞术检测细胞表面CD19、CD34、CD73、CD105;第3代BMSC经成脂成骨诱导液分别诱导3周和2周后,分别应用油红O染色和碱性磷酸酶检测BMSC的多向分化能力。应用24 mm共培养板,将第3代BMSC与RSC96细胞共培养24 h,采用MTT法和平板克隆形成实验检测RSC96细胞增殖及克隆形成能力;划痕实验及TranswellTM小室实验检测RSC96细胞的迁移能力;Western blot法检测RSC96细胞Bax和Bcl-2蛋白表达水平。结果SD大鼠第3代BMSC显微镜下呈梭形、旋涡状排列,形态大小相似;BMSC表面标志CD73、CD105高表达,造血干细胞表面标志CD34和淋巴细胞表面标志CD19低表达;BMSC经成脂诱导培养3周后,细胞有大量脂滴;经成骨诱导培养2周后,碱性磷酸酶染色呈阳性;BMSC与RSC96细胞共培养后,与对照组相比,RSC96细胞的增殖活性显著降低,克隆形成能力显著减弱、细胞迁移能力降低;共培养后RSC96细胞Bax蛋白水平升高,Bcl-2蛋白表达降低。结论 BMSC能促进RSC96细胞凋亡,抑制其增殖、迁移。
Objective To investigate the effects of bone marrow mesenchymal stem cells (BMSCs) on proliferation and migration of rat RSC96 Schwann cells. Methods BMSC was isolated and extracted from bone marrow of SD rats by whole bone marrow adherent method. The morphology of BMSCs of the third passage was observed under inverted microscope. The surface of cells were examined by flow cytometry for CD19, CD34, CD73 and CD105. After induction for 3 weeks and 2 weeks respectively, the oil red O staining and alkaline phosphatase were used to detect the multi-directional differentiation ability of BMSCs. The third passage of BMSCs and RSC96 cells were co-cultured with 24 mm co-culture plates for 24 h. The proliferation and colony formation ability of RSC96 cells were detected by MTT assay and plate clone formation assay. Scratch assay and TranswellTM chamber assay were used to detect the migration ability of RSC96 cells Western blot was used to detect the protein expression of Bax and Bcl-2 in RSC96 cells. Results The 3rd generation BMSCs of SD rats were spindle-shaped and arranged in a swirling pattern with the same size and shape. The expression of CD73 and CD105 on the surface of BMSCs was high, and the expression of CD34 on the surface of hematopoietic stem cells and CD19 on the surface of lymphocytes were low. After cultured for 3 weeks, the cells had a large number of lipid droplets. After two weeks of osteogenic induction culture, alkaline phosphatase staining was positive. After co-cultured with BMSC and RSC96 cells, the proliferation activity of RSC96 cells was significantly decreased, The colony formation ability was significantly weakened and the cell migration ability was decreased. The Bax protein level of RSC96 cells increased and the expression of Bcl-2 protein decreased after co-culture. Conclusion BMSC can promote the apoptosis of RSC96 cells and inhibit its proliferation and migration.