南方医科大学学报

南方医科大学学报 ›› 2017, Vol. 37 ›› Issue (12): 1603-.

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shRNA-PAX6慢病毒载体构建及其对胶质瘤U251细胞增殖的影响

廖晓红，尹蔚兰，王芳，邬力祥，黄柏胜   

  • 出版日期:2017-12-20 发布日期:2017-12-20

Construction of a lentiviral vector carrying short-hairpin RNA targeting PAX6 and its effect on proliferation of glioma U251 cells in vitro

  • Online:2017-12-20 Published:2017-12-20

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摘要： 目的构建shRNA-PAX6慢病毒载体并观察其对胶质瘤U251细胞增殖的影响。方法根据文献报道的PAX6靶点序列， 设计两条PAX6基因的siRNA靶点干扰序列，引物退火形成带粘性末端的双链，与经BamH I、EcoR I双酶切后线性化的慢病毒 载体进行连接、转化，构建shRNA-PAX6慢病毒重组载体，再经菌落PCR及测序鉴定重组载体；在293T细胞中包装病毒，将包装 后的shRNA-PAX6慢病毒重组载体感染U251细胞。Real-time PCR检测PAX6 mRNA的表达水平，Western blot检测PAX6蛋 白的表达，MTT法检测U251细胞增殖。结果慢病毒载体pLKD-CMV-G&NR-U6-shRNA经双酶切后可见线性化的8208 bp大 片段，菌落PCR及测序鉴定证实shRNA-PAX6慢病毒重组载体构建成功，构建的shRNA-PAX6慢病毒载体在293T细胞完成包 装，测得慢病毒滴度为6.73×108 TU/mL；Real-time PCR结果显示，沉默PAX6的表达可见U251细胞PAX6 mRNA表达水平明显 低于正常对照组及慢病毒空载体组（P<0.05）；Western blot结果显示，PAX6蛋白表达亦明显低于正常对照组及慢病毒空载体组 （P<0.05）；MTT结果显示，与正常对照组及慢病毒空载体组细胞比较，感染shRNA-PAX6慢病毒重组载体组的细胞，细胞增殖 能力明显增强（P<0.05）。结论成功构建人PAX6基因的shRNA-PAX6慢病毒重组载体，沉默PAX6基因的表达，U251细胞的 增殖能力增强。

Abstract: Objective To construct a lentiviral vector for delivering short hairpin RNA (shRNA) targeting PAX6 and investigate its effect on the proliferation of glioma U251 cells in vitro. Methods Two small interfering RNA sequences targeting PAX6 gene were designed based on the reported sequence of PAX6 and annealed to form a double-stranded chain, which was inserted into a lentiviral vector to construct the recombinant lentiviral vector shRNA-PAX6. The recombinant vector was infected into U251 cells, and the expression of PAX6 mRNA and protein in the cells was detected by real-time PCR and Western blotting, respectively. The changes in the proliferation of U251 cells after the infection was assessed using MTT assay. Results Double enzyme digestion of the lentiviral vector pLKD-CMV-G&NR-U6-shRNA yielded an 8208-bp fragment, and colony PCR and sequencing analysis confirmed successful construction of the lentiviral vector shRNA-PAX6. Infection of the cells with shRNA-PAX6 caused a significant reduction of the expressions of PAX6 mRNA and protein (P<0.05) and resulted in obviously increased proliferation of U251 cells (P<0.05). Conclusion We successfully constructed the recombinant vector shRNA-PAX6 for silencing PAX6 gene. PAX6 gene silencing results in increased proliferation of U251 cells in vitro.