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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2.9×10~7和2.8×10~7 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G_2/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G_2/M细胞比例上升。
Objective Glioma is the most common primary tumor of the central nervous system. The current malignant degree of glioblastoma multiforme has no significant improvement in the overall survival of patients with existing methods of operation and radiotherapy and chemotherapy. In this study, we observed the overexpression of CIB1 gene in the human glioma cell line U251 and its relationship with tumor proliferation and cycle in recombinant lentivirus Proliferation and cycle effects. Methods The CIB1 gene fragment was amplified by PCR. The lentiviral vector of CIB1 recombinant pLVX-IRES-CIB1-tdTomato was constructed and the recombinant lentiviral vector was transfected into 293T packaging cells. The virus was packaged and tested for viral titers. The infection of U251 glioma cells, the expression of pLVX-IRES-CIB1-tdTomato lentivirus was observed by fluorescence microscopy, the effect of CIB1 overexpression on the proliferation of human glioma cells was detected by CCK-8 assay, the effect of CIB1 overexpression on cell cycle Impact. Results The lentiviral vector pLVX-CIB1-IRES-tdTomato was successfully constructed and the recombinant lentiviral vector was constructed. The virus titers were 2.9 × 10-7 and 2.8 × 10-7 ifu / mL, respectively. Viral Infections U251 cells were infected with a multiple of 100. CCK-8 test showed that CIB1 infection significantly promoted cell proliferation compared with blank control group and blank control group (P <0.05), suggesting that CIB1 overexpression had a growth-promoting effect on U251 human glioma cells. Flow cytometry showed that the percentage of cells in G 2 / M phase of U251 cells was significantly increased and the percentage of cells in S phase was decreased in CIB1 infected cells. Conclusion Overexpression of CIB1 can promote the proliferation of U251 cells and increase the proportion of G 2 / M cells.