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目的构建下调及过表达SWAP-70的慢病毒载体,并检测其在神经胶质瘤细胞中的表达效果。方法采用PCR扩增目的基因片段,构建SWAP-70慢病毒表达载体,与慢病毒转染系统共转染至293T细胞。收集病毒颗粒上清液,感染神经胶质瘤细胞。荧光显微镜下观察细胞中绿色荧光蛋白的表达情况,并采用Western blot法鉴定SWAP-70蛋白表达水平。结果成功构建下调及过表达SWAP-70的慢病毒载体,感染神经胶质瘤细胞的效率达90%左右。Western blot结果显示,下调SWAP-70的神经胶质瘤细胞中SWAP-70蛋白表达降低(P<0.01),而过表达SWAP-70的神经胶质瘤细胞中内源性及外源性SWAP-70蛋白均有表达。结论成功建立下调及过表达SWAP-70的慢病毒载体,并有效转染神经胶质瘤细胞,为胶质瘤的基因治疗研究提供参考。
Objective To construct a lentiviral vector that down - regulates and overexpresses SWAP - 70 and test its expression in glioma cells. Methods The target gene fragment was amplified by PCR. The SWAP-70 lentiviral expression vector was constructed and transfected into 293T cells with lentiviral transfection system. The virus particle supernatant was collected and infected with glioma cells. Fluorescence microscopy was used to observe the expression of green fluorescent protein (EGFP) in cells. The expression of SWAP-70 protein was identified by Western blot. Results The lentiviral vector with down-regulated and overexpressed SWAP-70 was successfully constructed and the efficiency of inoculating glioma cells was about 90%. The result of Western blot showed that SWAP-70 expression was down-regulated in SWAP-70-treated glioma cells (P <0.01), while in SWAP-70-overexpressing SW90-SW70 cells, 70 protein are expressed. Conclusion The successful construction of lentiviral vector down-regulated and overexpressed SWAP-70 and its efficient transfection into glioma cells provide a reference for gene therapy of glioma.