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目的:观察大鼠骨髓间充质干细胞对HepG2肝癌细胞增殖的影响,并初步探讨其机制。方法:全骨髓贴壁法分离培养大鼠骨髓间充质干细胞,流式细胞术鉴定其分子表面标志,体外扩增培养并收集培养上清。MTT法、平板克隆实验及软琼脂克隆实验检测大鼠间充质干细胞对人肝癌细胞株HepG2增殖的影响,流式细胞术检测肝癌细胞周期的改变,Western blot法检测细胞周期相关蛋白Cyclin D1表达情况。结果:骨髓间充质干细胞CD105和CD90表达阳性,CD34和CD45表达阴性。在骨髓间充质干细胞培养上清作用下,MTT法显示实验组肝癌细胞光密度值增加(P<0.05),平板克隆法显示实验组肝癌细胞克隆形成率比对照组明显升高(P<0.05),软琼脂克隆法显示实验组肝癌细胞空间克隆形成率比对照组明显升高(P<0.05),流式细胞术显示实验组细胞周期G1期比例降低,S期、G2期比例增加。Western blot显示实验组细胞Cyclin D1表达量增加(P<0.05)。结论:骨髓间充质干细胞可促进HepG2肝癌细胞的增殖及空间克隆形成能力,其促进肝癌细胞体外增殖可能与Cyclin D1表达上调有关。
OBJECTIVE: To observe the effect of rat bone marrow mesenchymal stem cells on the proliferation of HepG2 hepatocarcinoma cells and to explore its mechanism. Methods: Bone marrow mesenchymal stem cells were isolated and cultured by whole bone marrow adherent method. The molecular surface markers were identified by flow cytometry. The supernatants were expanded and cultured in vitro. MTT assay, plate clone assay and soft agar cloning assay were used to detect the effects of MSCs on the proliferation of human hepatocellular carcinoma cell line HepG2. Flow cytometry was used to detect the cell cycle of hepatocellular carcinoma cells. Western blot was used to detect the expression of Cyclin D1 Happening. Results: The expression of CD105 and CD90 in bone marrow mesenchymal stem cells was negative and the expressions of CD34 and CD45 were negative. MTT assay showed that the optical density of hepatocellular carcinoma cells in experimental group increased (P <0.05), and the colony formation rate of hepatocellular carcinoma cells in experimental group was significantly higher than that in control group (P <0.05) by using the supernatant of bone marrow mesenchymal stem cells ), Soft agar cloning method showed that the rate of clone formation in hepatocellular carcinoma cells in experimental group was significantly higher than that in control group (P <0.05). Flow cytometry showed that the proportion of cell cycle G1 phase decreased and the proportion of S phase and G2 phase increased. Western blot showed that the expression of Cyclin D1 in experimental group increased (P <0.05). Conclusion: Bone marrow mesenchymal stem cells can promote the proliferation and space clone formation of HepG2 hepatoma cells, which may be related to the up-regulation of Cyclin D1 expression.