[胰岛素样生长因子-II P4启动子驱动的胸苷激酶表达载体的构建]
[The construction of IGF-II P4 promoter-driven tk expression vector].
作者信息
Zhou Hong-Ke, Yang Dong-Hua, Tang Shao-Hui, Huang Wei
机构信息
Department of Gastroenterology, First Affiliated Hospital, Jinan University, Guangzhou 510630, China.
出版信息
Zhonghua Gan Zang Bing Za Zhi. 2011 Jun;19(6):460-3.
OBJECTIVE
To investigate the selective killing effect of herpes simplex virus-thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene system controlled by human IGF-II P4 promoter on HCC cells in vitro.
METHODS
Recombinant shuttle plasmid vectors driven by IGF-II P4 promoter and driven by CMV promoter were constructed by techniques of gene recombination. The recombinant shuttle plasmids were then transfected into HepG2 and HeLa cells by techniques of lipidosome transfection. EGFP expression was detected by fluoroscopy. Tk and EGFP mRNA expression were detected by RT-PCR. The selective killing effect after GCV application was determined with MTT method. Statistical analysis was performed with ANOVA analysis.
RESULTS
Identification of pDC316-tkEGFP-P4 by enzyme digestion and sequencing analysis showed that the construction of the recombinant shuttle plasmid was correct. It was found that green fluorescence protein could only be seen in HepG2 cells, not in HeLa cells. The results of RT-PCR showed only two bands could be seen in the samples of pDC316-tkEGFP-P4 transfected HepG2 cells. The growth of HepG2 cells transfected with pDC316-tkEGFP-CMV and pDC316-tkEGFP-P4 were inhibited remarkably, the growth inhibition rates were 6.95% +/- 0.67%, 24.99% +/- 1.53%, 49.68% +/- 1.68%, 71.85% +/- 3.28% and 4.83% +/- 0.35% vs 17.34% +/- 1.15%, 30.17% +/- 1.30%, 40.39% +/- 0.82% (F = 24.055, P < 0.05), respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-CMV were also inhibited, the growth inhibition rates were 6.36% +/- 0.83%, 23.95% +/- 1.72%, 45.13% +/- 1.64% and 69.38% +/- 3.17%, respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-P4 was not inhibited. The growth inhibition rates were 0.91 +/- 0.04, 1.18 +/- 1.32, 1.19 +/- 0.10 and 1.32 +/- 0.05 (F = 26.469, P < 0.01) , respectively.
CONCLUSION
The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-II P4 promoter has been constructed successfully and its specific expression in HepG2 cells provided the basis for targeted gene therapy for HCC.
目的
探讨人胰岛素样生长因子-II P4启动子调控的单纯疱疹病毒胸苷激酶/更昔洛韦（HSV-tk/GCV）自杀基因系统对肝癌细胞的体外选择性杀伤作用。
方法
采用基因重组技术构建由IGF-II P4启动子驱动及由CMV启动子驱动的重组穿梭质粒载体。然后通过脂质体转染技术将重组穿梭质粒转染至HepG2和HeLa细胞。通过荧光显微镜检测绿色荧光蛋白（EGFP）表达。采用逆转录-聚合酶链反应（RT-PCR）检测Tk和EGFP mRNA表达。应用MTT法测定加入更昔洛韦后的选择性杀伤作用。采用方差分析进行统计学分析。
结果
经酶切及测序分析鉴定pDC316-tkEGFP-P4，表明重组穿梭质粒构建正确。发现绿色荧光蛋白仅在HepG2细胞中可见，而在HeLa细胞中不可见。RT-PCR结果显示，在转染pDC316-tkEGFP-P4的HepG2细胞样本中仅可见两条条带。转染pDC316-tkEGFP-CMV和pDC316-tkEGFP-P4的HepG2细胞生长受到显著抑制，生长抑制率分别为6.95%±0.67%、24.99%±1.53%、49.68%±1.68%、71.85%±3.28%和4.83%±0.35%，而对照组为17.34%±1.15%、30.17%±1.30%、40.39%±0.82%（F = 24.055，P < 0.05）。转染pDC316-tkEGFP-CMV的HeLa细胞生长也受到抑制，生长抑制率分别为6.36%±0.83%、23.95%±1.72%、45.13%±1.64%和69.38%±3.17%。转染pDC316-tkEGFP-P4的HeLa细胞生长未受抑制，生长抑制率分别为0.91±0.04、1.18±1.32、1.19±0.10和1.32±0.05（F = 26.469，P < 0.01）。
结论
成功构建了由IGF-II P4启动子驱动携带tkEGFP融合蛋白基因的穿梭质粒载体，其在HepG2细胞中的特异性表达为肝癌的靶向基因治疗提供了依据。
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