南方医科大学学报

南方医科大学学报 ›› 2015, Vol. 35 ›› Issue (03): 348-.

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VHL慢病毒表达和干扰载体的构建及对肾癌细胞增殖和凋亡的影响

沈东来，马鑫，张瑜，高宇，李新涛，顾良友，巩会杰，牛少曦，张旭   

  • 出版日期:2015-03-20 发布日期:2015-03-20

Construction of a recombinant lentiviral vector for VHL and VHL shRNA and its effect
on proliferation and apoptosis of renal cell carcinoma cells

  • Online:2015-03-20 Published:2015-03-20

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摘要： 目的构建人VHL重组慢病毒表达载体及干扰载体，建立稳定转染细胞株，观察VHL对肾癌细胞株增殖和凋亡的影响。
方法构建pZsGreen1-VHL及pLL3.7-shVHL重组慢病毒载体，与3质粒包装系统用脂质体法共同转染293T细胞，包装成病毒
颗粒，分别感染A498、Caki-1细胞，并进行RT-PCR和Western blot检验细胞中VHL的表达。用MTS法和流式细胞仪检测VHL
对肾癌细胞增殖和凋亡效应的影响。结果重组慢病毒载体及稳定转染细胞株构建成功，转染后VHL在细胞中表达明显变化；
VHL过表达细胞的增殖速度明显低于各对照组，而凋亡率明显高于各对照组；VHL干扰细胞的增殖速度明显高于各对照组，而
凋亡率明显低于各对照组（P<0.05）。结论VHL对肾癌细胞具有抑制增殖和诱导凋亡的作用。

Abstract: Objective To construct a lentiviral expression vector for human VHL and its shRNA vector, and study the effect of
VHL on proliferation and apoptosis of renal cell carcinoma cell lines. Methods Lentiviral vectors pZsGreen1-VHL and
pLL3.7-shVHL were constructed and transfected into 293T cells with 3 packaging plasmids by LipofectamineTM 2000 reagent.
The supernatant was collected to infect A498 and Caki-1 cells, respectively. VHL mRNA and protein levels were detected by
RT-PCR and Western blotting, respectively. The effect of VHL on the proliferation, cell cycle and cell apoptosis were analyzed
by MTS and flow cytometry. Results The recombinant lentiviral vectors were successfully constructed. The proliferation of
A498 cells with reconstituted wild-type VHL was significantly inhibited, while the proliferation of Caki-1 cells with VHL
knockdown was significantly enhanced as compared with the control cells (P<0.05). VHL induced G0/G1-S cell cycle arrest. The
apoptosis rate of A498 cells with reconstituted wild-type VHL was significantly increased while that of Caki-1 cells with VHL
knockdown was significantly lowered compared with the control cells (P<0.05). Conclusion VHL can inhibit the proliferation
and induce apoptosis of renal cell carcinoma cells.