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目的:探讨采用腺病毒做为载体介导基于RNA干涉的针对高HER2肿瘤的基因治疗的可能性。方法:构建HER2-绿色荧光蛋白融合蛋白表达质粒pHER2-GFP,并与9种针对HER2不同靶序列的siRNA表达质粒分别共转染CHO-K1细胞,根据荧光蛋白表达量从中筛选出沉默效率最高的质粒。将筛选出的质粒转染HER2高表达乳腺癌SKBR3细胞,检测它们对HER2表达的影响。随后,将siRNA转录单元克隆入腺病毒载体,成功包装病毒后感染SKBR3细胞,再次测定其下调HER2的效应及其对细胞生长的影响。结果:2种有效下调HER2表达的质粒被筛选出来。将此2种质粒所含siRNA转录单元包装入腺病毒后仍然保持了原有的基因沉默效应。HER2下调增加了SKBR3细胞中G1期细胞的比例,并且诱导部分细胞凋亡。MTT和细胞长期增殖抑制实验表明腺病毒介导的RNA干涉抑制了SKBR3细胞生长。结论:重组腺病毒介导的RNA干涉能够下调HER2的表达并且对高表达HER2的乳腺癌细胞有生长抑制作用。
AIM: To investigate the possibility of using adenovirus as a vector to mediate RNA interference-based gene therapy in high HER2 tumors. METHODS: The HER2-green fluorescent protein fusion protein expression plasmid pHER2-GFP was constructed and co-transfected into 9 kinds of siRNA expression plasmids targeting HER2 respectively. CHO-K1 cells were screened out from the expression levels of fluorescent proteins. Plasmid. The selected plasmids were transfected into HER2 overexpressing breast cancer SKBR3 cells and their effects on HER2 expression were examined. Subsequently, the siRNA transcription unit was cloned into an adenovirus vector and successfully infected with SKBR3 cells after packaging the virus. The effect of down-regulating HER2 and its effect on cell growth were determined again. RESULTS: Two plasmids that effectively down-regulated HER2 expression were screened out. The two kinds of plasmids containing siRNA transcribed unit into adenovirus still retains the original gene silencing effect. HER2 downregulation increased the proportion of G1 phase cells in SKBR3 cells and induced some apoptosis. MTT and long-term cell proliferation inhibition experiments showed that adenovirus-mediated RNA interference inhibited SKBR3 cell growth. Conclusion: Recombinant adenovirus-mediated RNA interference can down-regulate the expression of HER2 and inhibit the growth of breast cancer cells that express HER2.