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人骨髓间充质干细胞通过YAP影响人脂肪肉瘤SW872细胞的生物学行为
陈华,沙娜,刘宁,李阳,胡海军
PDF(1444 KB)
PDF(1444 KB)
人骨髓间充质干细胞通过YAP影响人脂肪肉瘤SW872细胞的生物学行为
)Effect of human bone marrow mesenchymal stem cells on biological behavior of human liposarcoma SW872 cells through YAP
)目的 观察人骨髓间充质干细胞（hMSCs）条件培养基（CM）与人脂肪肉瘤SW872细胞共培养后对肿瘤细胞增殖和迁移能力的影响，探讨hMSCs CM对脂肪肉瘤细胞的作用及可能的作用机制。 方法 体外培养hMSCs，采用慢病毒方法分别转染慢病毒空载体shNS（对照组）和慢病毒shRNA Yes相关蛋白（YAP）（shYAP-hMSCs组），采用实时荧光定量PCR （RT-qPCR）法和Western blotting 法检测各组hMSCs中YAP mRNA和蛋白表达水平，提取CM。体外培养SW872细胞，分为对照组（正常培养）、hMSCs CM组和shYAP-hMSCs CM组。采用CCK-8法检测各组细胞增殖活性，流式细胞术检测各组细胞凋亡率，细胞划痕实验检测各组细胞划痕愈合率，Western blotting法检测各组细胞中YAP、基质金属蛋白酶9（MMP-9）和细胞周期蛋白D1（cyclin D1）蛋白表达水平。 结果 与对照组比较，shYAP-hMSCs组hMSCs中YAP mRNA和蛋白表达水平降低（P<0.01），表明成功构建了shYAP-hMSCs稳定转染细胞株。CCK-8法，与对照组比较，hMSCs CM组SW872细胞增殖活性升高（P<0.05），shYAP- hMSCs CM组SW872细胞增殖活性降低（P<0.01）；流式细胞术，与对照组比较，hMSCs CM组SW872细胞凋亡率无明显变化（P>0.05），shYAP-hMSCs CM组SW872细胞凋亡率升高（P<0.01）；细胞划痕实验，与对照组比较，hMSCs CM组SW872细胞划痕愈合率升高（P<0.05），shYAP-hMSCs CM组SW872细胞划痕愈合率降低（P<0.01）；Western blotting法，与对照组比较，hMSCs CM组SW872细胞中YAP、MMP-9和cyclin D1蛋白表达水平差异无统计学意义（P>0.05），shYAP-hMSCs组SW872细胞中YAP、MMP-9和cyclin D1蛋白表达水平降低（P<0.05或P<0.01）。 结论 hMSCs参与调控人脂肪肉瘤SW872细胞增殖和迁移，其机制可能与YAP表达有关。
Objective To observe the effect of human mesenchymal stem cells （hMSCs） conditioned medium （CM） co-cultured with the human liposarcoma SW872 cells on the proliferation and migration of the tumor cells， and to discuss the effect of hMSCs CM on the liposarcoma cells and the possible mechanism. Methods The hMSCs were cultured in vitro and transfected with either lentiviral vector control shNS （control group） or lentiviral shRNA targeting Yes-associated protein （YAP） （shYAP-hMSCs group） by lentiviral methods. The expression levels of YAP mRNA and protein in the hMSCs in various groups were detected by real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods. The CM was then harvested. The SW872 cells were cultured in vitro and divided into control group （normal culture）， hMSCs CM group， and shYAP-hMSCs CM group. The proliferation activities of the cells in various groups were detected by CCK-8 assay； the apoptotic rates of the cells in various groups were detected by flow cytometry； the scratch healing rates of the cells in various groups were detected by cell scratch assay； the expression levels of YAP， matrix metallopeptidase-9 （MMP-9）， and cyclin D1 proteins in the cells in various groups were detected by Western blotting method. Results Compared with control group， the expression levels of YAP mRNA and protein in the cells in shYAP-hMSCs group were decreased （P<0.01）， indicating the successful establishment of a stable transfected cell line. The CCK-8 assay results showed that compared with control group， the proliferation activity of the cells in hMSCs CM group was increased （P<0.05）， and the proliferation activity of the cells in shYAP-hMSCs CM group was decreased （P<0.01）. The flow cytometry results showed that compared with control group， there was no significant change in the apoptotic rate of the cells in hMSCs CM group （P>0.05）， while the apoptotic rate of the cells in shYAP-hMSCs CM group was increased （P<0.01）. The cell scratch assay results showed that compared with control group，the scratch healing rate of the cells in hMSCs CM group was increased （P<0.05）， and the scratch healing rate of the cells in shYAP-hMSCs CM group was decreased （P<0.01）. The Western blotting results showed that compared with control group， there were no significant differences in the expression levels of YAP， MMP-9， and cyclin D1 proteins in the cells in hMSCs CM group （P>0.05）， while the expression levels of YAP， MMP-9， and cyclin D1 proteins in the cells in shYAP-hMSCs group were decreased （P<0.05 or P<0.01）. Conclusion The hMSCs regulate the proliferation and migration of the human liposarcoma SW872 cells， and its mechanism may be related to the expression of YAP.
骨髓间充质干细胞 / 条件培养基 / 脂肪肉瘤 / 细胞增殖 / 细胞迁移 / Yes相关蛋白
Human bone marrow mesenchymal stem cell / Conditioned medium / Liposarcoma / Cell proliferation / Cell migration / Yes-associated protein
R73-3
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