Stat3-siRNA表达质粒在AFP阳性肿瘤基因治疗中的应用

The value of stst3-siRNA plasmid on AFP-positive tumor gene therapy

目的设计和构建针对stat3基因的siRNA表达框架,并在肝癌细胞中观察其干扰效果。方法 stat3合成并修饰靶序列后PCR扩增siRNA表达框架,通过脂质体介导,直接将扩增产物转染至肝癌细胞HepG2中,48 h后提取细胞总RNA进行RT-PCR和Western blot检测干扰效果。结果 RT-PCR结果显示HepG2细胞内靶基因sTAT3 mRNA水平比空白组和阴性对照组明显下降(P<0.01)。流式细胞仪结果显示,转染stat3-siRNA的细胞与阴性对照组、空白对照组比较在G1期出现明显阻滞,在G1期出现的阻滞为(78.17±21.73)%,凋亡率达(21.39±9.47)%,与空白组和阴性对照组比较有明显差异(P<0.01)。结论本实验成功设计并构建的siRNA表达框架能干扰人肝癌HepG2细胞内sTAT3基因的表达。

Objective To construct a siRNA expression cassette targeted to gene stat3 and observe the efficiency to RNA interference in AFP-positive tumor gene.Methods After synthesizing and modifying the target sequence,the siRNA expression cassette was amplified by polymerase chain reaction（PCR）.Mediated by liposome,the amplified product was transfected into AFP-positive tumor cells HepG2.After 48 hours,the total RNA was extracted for reverse transcriptional-polymerase chain reaction（RT-PCR） to detect the efficiency of RNA interference and apoptosis through Western blot.Results The STAT3 gene expression was decreased at the mRNA level in comparison with the blank group and the control-negative group by RT-PCR detection.Through flow cytometry,the control-positive group demonstrated G1 phase significantly block in comparison with the blank group and the control-negative group by Western blot detection.The G1 phase block was（78.17±21.73）% and the rate of apoptosis was（21.39±9.47）%.There was a significant defference between the control-positive group and the other two groups（P〈0.01）.Conclusion We successfully contruct a siRNA expression cassette targeted to gene stat3 and it can interfere the expression of stat3 gene in AFP-positive tumor cells.