大鼠增殖抑制基因抑制大鼠胶质瘤细胞的增殖及其机制

Expression and mechanism of rat hyperplasia suppressor gene suppressing growth of rat C6 glioma cells

目的观察腺病毒介导的大鼠增殖抑制基因(rat hyperplasia suppressor gene,rHSG)在大鼠胶质瘤细胞(C6)中的表达及其对大鼠胶质瘤细胞增殖的影响,并探讨其作用的机制。方法用含有rHSG基因的重组腺病毒载体(Adv-rHSG-GFP)转染C6细胞,采用Western blot和免疫细胞化学法检测rHSG蛋白的表达;使用流式细胞仪分析细胞周期,并结合MTT、细胞计数法和平板克隆形成实验观察rHSG对细胞增殖的影响;应用Western blot检测增殖细胞核抗原(PCNA)和细胞周期调控蛋白p27Kip1、p21Cip1的蛋白表达变化。结果 Adv-rHSG-GFP转染组C6细胞中:rHSG蛋白的相对表达量均明显高于未转染组和Adv-GFP转染组(P<0.01);流式细胞仪分析结果表明细胞周期被阻滞在G0/G1期,且MTT、细胞计数和平板克隆形成实验显示大鼠胶质瘤细胞增殖受到明显抑制(P<0.01);与未转染组和Adv-GFP转染组相比,周期调控蛋白p27Kip1、p21Cip1表达升高,PCNA表达降低(P<0.05,P<0.01)。结论 rHSG基因过表达能够抑制大鼠胶质瘤细胞增殖,其机制可能是通过参与细胞周期调节发挥作用。

Objective To investigate the expression of rat hyperplasia suppressor gene （rHSG） in rat glioma cell as well as the effect of rHSG on rat glioma cell proliferation, and to explore the mechanism.MethodsThe rat glioma cells （C6 cells） were transfected with recombinant adenovirus vector containing rHSG （Adv-rHSG-GFP）. Western blotting and immunohistochemical assay were applied for determining the expression of rHSG protein. The effect of rHSG on cell cycle was analyzed by flow cytometry. Together with MTT assay, cytometry and plate cloning formation method were used for identifying the effect of rHSG on cell proliferation. The expression of proliferating cell nuclear antigen （PCNA） and cell cycle regulatory proteins p27Kip1 and p21Cip1 were determined by Western blotting.ResultsIt was observed that in C6 cells transfected with Adv-rHSG-GFP, the protein expression level of rHSG was higher than that in untransfected cells and cells transfected with Adv-GFP （P〈0.01）. The results from flow cytometry indicated that the cell cycle of C6 cells transfected with Adv-rHSG-GFP was arrested in G0/G1 phase. MTT assay, cytometry and plate cloning formation results showed that the proliferation of C6 cells transfected with Adv-rHSG-GFP was markedly inhibited （P〈0.01）. The expression levels of p27Kip1 and p21Cip1 were increased while that of PCNA was decreased in C6 cells transfected with Adv-rHSG-GFP, as compared to untransfected cells and cells transfected with Adv-GFP （P〈0.05, P〈0.01）.ConclusionThe over-expression of rHSG can suppress the proliferation of rat glioma cells, with the possible mechanism of cell cycle regulation.