大鼠骨髓间充质干细胞促进RSC96细胞凋亡并抑制其增殖和迁移

Rat bone marrow mesenchymal stem cells promote apoptosis and inhibit proliferation and migration of RSC96 cells

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 effect of rat bone marrow mesenchymal stem cells（ BMSCs） on the proliferation and migration of rat RSC96 Schwann cells in vitro. Methods BMSCs of Sprague Dawley（ SD） rats were isolated by bone marrow cell adherent method. The morphology of the third generation of BMSCs was observed by inverted microscopy. The cell surface antigen makers CD19,CD34,CD73,CD105 were detected by flow cytometry. The third-passage BMSCs differentiated into osteoblasts and adipocytes after exposed to bone-inducing and lipid-inducing media for 3 and 2 weeks,respectively,and then were identified by oil red O and alkaline phosphatase staining. The third generation of BMSCs was co-cultured with RSC96 cells for 24 hours in 24 mm co-culture plate. The cell proliferation and colony formation of RSC96 cells were determined by MTT assay and plate cloning assay. The migration of RSC96 cells was detected by scratch and TranswellTMchamber assay. Bax and Bcl-2 expression levels of RSC96 cells were detect by Western blot analysis. Results The third generation of BMSCs showed fusiform,spiral arrangement with similar cell size under an inverted microscope. The CD73 and CD105 expression levels of BMSCs were high,however,the CD34 and CD19 were low. BMSCs had large lipid droplets after adipogenic induction for 3 weeks; and the alkaline phosphatase staining of BMSCs was positive after osteogenic induction for 2 weeks. Compared to the control group,co-culture of RSC96 cells and BMSCs induced a significant decrease in the proliferation,colony formation and migration of RSC96 cells. Meanwhile,Bax protein expression increased,and Bcl-2protein expression decreases in RSC96 cells co-cultured with BMSCs. Conclusion BMSCs could promote the apoptosis and inhibit the proliferation and migration of RSC96 cells.