第10号染色体上缺失与张力蛋白同源的磷酸酯酶基因人脑胶质瘤SHG44细胞凋亡及B细胞淋巴瘤/白血病-2蛋白下调表达

摘要

目的 观察第10号染色体上缺失与张力蛋白同源的磷酸酯酶基因(PTEN)对人脑胶质瘤SHG44细胞凋亡及凋亡相关基因B细胞淋巴瘤/白血病-2(bcl-2)表达的影响,探讨PTEN基因抑制肿瘤细胞增殖的机制.方法 将SHG44细胞常规条件下培养于含10％小牛血清的RPMI 1640培养基中.脂质体介导法将pBP-PTEN和pBP载体分别转染对数生长期的SHG44细胞,筛选阳性细胞克隆,将分别成功转染pBP-PTEN和pBP载体的SHG44细胞设为SHG44-pBP-PTEN和SHG44-pBP细胞.利用原位杂交方法分别检测SHG44-pBP-PTEN、SHG44-pBP和SHC-44细胞.通过免疫组织化学方法检测PTEN基因在3种细胞中的表达,采用透射电镜、流式细胞仪和核DNA琼脂糖凝胶电泳检测细胞的凋亡情况以免疫荧光法检测bcl-2基因的表达.结果 PTEN基因转染的SHG44细胞有PTEN基因和蛋白的表达.透射电镜下可见SHC-44-pBP-PTEN细胞核染色质浓缩、边集,细胞核碎裂,胞质浓缩;并检测到明显的凋亡小体,为典型的肿瘤细胞凋亡表现.SHG44和SHG44-pBP细胞超微结构未见异常.流式细胞仪示细胞周期从G1期到S期发生抑制,并且在G1期峰前出现明显的凋亡峰,SHG44细胞G1期、S期、G2期和凋亡(AP)期所占比例分别为61.2％、28.2％、8.3％和2.3％,SHG44-pBP细胞分别为64.1％、24.6％、8.9％和2.4％,SHG44-pBP-PTEN细胞分别为75.3％、9.6％、2.2％和12.9％.SHG44-pBP-PTEN细胞的生长速度较另两组细胞明显降低.细胞核DNA琼脂糖凝胶电泳呈现凋亡细胞特有的梯状条带;bcl-2的表达也显著下调.SHG44-pBP细胞的平均荧光强度(MIF)为对照细胞的100.4％(P＞0.05),而SHG44-pBP-PTEN细胞的MIF为对照细胞的40.2％,两组比较差异有统计学意义(P＜0.01).结论 PTEN基因可诱导SHG44细胞凋亡,并下调bcl-2蛋白的表达,这可能是PTEN基因抑制肿瘤细胞增殖的分子机制之一.%Objective To investigate the effect of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene on the apoptosis and expression of apoptosis related gene B cell lymphoma/leukemia-2 (bcl-2) in human glioma SHG44 cells,and to elucidate the mechanism of PTEN gene to inhibit tumor cell proliferation.Methods The SHG44 cells were cultured in RPMI 1640 medium supplemented with 10％ calf serum.pBP-PTEN and vector pBP were transfected into SHG44 cells in logarithmic phase by Lipofectamine.Positive cell clones were screened,and SHG44 cells successfully transfected with pBP-PTEN and vector pBP served as SHG44-pBP-PTEN cells and SHG44-pBP cells respectively.The SHG44-pBP-PTEN,SHG44-pBP and SHC44 cells were detected by in situ hybridization.The immunohistochemistry was used to detect the expression of PTEN gene in three kinds of cells.Transmission electron microscopy,flow cytometry and nuclear DNA agarose gel electrophoresis were applied to examine the apoptosis,and immunofluorescence was used to detect the expression of B cell lymphoma/leukemia-2 (bcl-2) gene.Results Expression of PTEN gene and protein in SHG44 cells transfected by PTEN gene.Under the transmission electron microscope,SHC44-pBP-PTEN nuclear chromatin condensation,edge set,nuclear fragmentation,cytoplasmic condensation,and obvious apoptotic bodies were detected,which showed the typical apoptosis of tumor cells.The structure of SHG44 and SHG44-pBP cells was not found abnormal.Flow cytometry showed that cell cycle from G1 phase to S phase inhibition,and apoptosis peak appeared before G1 peak,SHG44 cell G1 phase,S phase,G2 phase and apoptotic peak (AP) phase proportion was 61.2％,28.2％,8.3％ and 2.3％,SHG44-pBP cells were 64.1％ 24.6％,8.9％ and 2.4％,SHG44-pBP-PTEN cells were 75.3％,9.6％,2.2％ and 12.9％.The growth rate of SHG44-pBP-PTEN cells was significantly lower than that of the other two cells.Nuclear DNA agarose gel electrophoresis showed a typical apoptotic cell ladder;bcl-2 expression was also significantly reduced.The mean intensity of fluroscence (MIF) of SHG44-pBP cells was 100.4％ (P ＞0.05) of control cell,while the MIF of SHG44-pBP-PTEN cells was 40.2％,and the difference between the 2 groups was statistically significant (P ＜ 0.01).Conclusion PTEN gene can induce apoptosis of SHG44 cells and down regulate the expression of bcl-2 protein,which may be one of the molecular mechanisms of PTEN gene inhibiting the proliferation of tumor cells.