慢病毒介导的CIB1过表达对人脑胶质瘤细胞增殖和周期影响

目的 神经胶质瘤是中枢神经系统最常见的原发性肿瘤,其中恶性度最高的多形性胶质母细胞瘤现有的手术及放化疗等治疗方式对患者总体生存率无显著改善.本研究观察重组慢病毒中与肿瘤增殖、周期相关的钙整合素结合蛋白1(calcium-and integrin-binding protein 1,CIB1)基因,在人胶质瘤细胞系U251中的过表达情况及对其增殖和周期的影响.方法 PCR扩增CIB1目的片段,构建CIB1重组pLVX-IRES-CIB1-tdTomato慢病毒载体,将重组慢病毒表达载体感染293T包装细胞,进行病毒的包装并检测病毒滴度,筛选最佳病毒感染复数并感染U251胶质瘤细胞,荧光显微镜观察pLVX-IRES-CIB1-tdTomato慢病毒表达,CCK-8法检测CIB1过表达对人胶质瘤细胞增殖的影响,流式细胞仪检测CIB1过表达对细胞周期的影响.结果 成功构建慢病毒表达载体pLVX-CIB1-IRES-tdTomato,制备了重组CIB1慢病毒和空载体慢病毒,病毒滴度分别为2.9×107和2.8×107 ifu/mL;用最佳病毒感染复数100感染U251细胞.CCK-8检测显示,CIB1感染组与空载体对照组及空白组相比明显促进细胞增殖(P＜0.05),提示CIB1过表达对人脑胶质瘤细胞U251有生长促进效应.流式细胞检测显示,CIB1感染组U251细胞G2/M期细胞百分比明显增加,S期细胞比例下降.结论 CIB1过表达可以促进U251细胞增殖,并使细胞G2/M细胞比例上升.

Effects of lentivirus-mediated CIB1 overexpression on proliferation and cell cycle of human glioma cells

OBJECTIVE Glioma is the most common primary tumor of the central nervous system,in which the highest degree of malignant pleomorphic glioblastoma existing surgery and radiotherapy and chemotherapy treatment of patients with no significant improvement in overall survival.This study was designed to investigate the effect of lentiviral vector-mediated CIB1gene expression on the proliferation and cell cycle of human glioma U251 cells.METHODS The ORF of CIB1 gene was amplified by PCR and pLVX-IRES-td Tomato lentiviral vector were constructed.The recombinant plasmid was transfected into 293T packaging cells.The transfection efficiency was observed by fluorescence microscopy.CCK-8 assay was used to observe the survival ratio of U251 cells growth.FCM analysis was used to detect the change of cell cycle of U251 cells.RESULTS The lentiviral expression vector pLVX-CIB1-IRES-tdTomato was successfully constructed.The titers of recombinant lentivirus and empty vector lentiviruses were 2.9 × 107 ifu/mL and 2.8 × 107 ifu/mL respectively.CCK-8 results showed that it could increase the growth of U251 cells (P＜0.05).FCM results showed that CIB1 significantly increased the proportion of G2/M phase U251 cells.CONCLUSION Lentiviral vector-mediated CIB1 could induce cell proliferation and increase the proportion of G2/M cells in U251 cells.