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目的构建含木薯linamarase(lis)基因的稳定转染肝癌细胞系,并对其生物学特性进行研究。方法应用PCR法从木薯的DNA中扩增出lis基因,将其克隆至pcDNA3.1(+)质粒中,构建重组质粒pcDNA3.1/lis。通过脂质体法将其转染人肝癌细胞系HepG2,进行G418筛选,获得稳定转染的肝癌细胞系HepG2/lis,通过免疫荧光染色、RT-PCR和Western blot法鉴定lis在细胞中的表达;采用Lambert法对细胞内lis酶的活性进行分析;采用MTT法观察稳定转染细胞系生长曲线,流式细胞仪分析细胞周期,裸鼠成瘤实验分析其体内成瘤特性。结果 lis基因能稳定整合于真核细胞中,并包装出具备β-葡萄糖苷酶活性的蛋白。lis在细胞中的稳定表达对细胞形态、生长特性、细胞周期、体内成瘤性等生物学特征并无明显影响。结论成功构建含lis基因的稳定转染肝癌细胞系,为将lis自杀基因系统应用于肝癌的治疗提供了实验基础。
Objective To construct a stable transfection hepatoma cell line containing the cassava linamarase (lis) gene and study its biological characteristics. Methods Lis gene was amplified from cassava DNA by PCR and cloned into pcDNA3.1 (+) plasmid to construct recombinant plasmid pcDNA3.1 / lis. The recombinant plasmids were transfected into HepG2 cells by lipofectamine. The cells were screened by G418 to obtain HepG2 / lis hepatoma cells stably transfected. The expression of lis was identified by immunofluorescent staining, RT-PCR and Western blot ; Lambert method was used to analyze the activity of intracellular lis enzyme; the growth curve of stable transfected cell line was observed by MTT method; the cell cycle was analyzed by flow cytometry; the tumorigenicity in vivo was analyzed by nude mice. Results The lis gene was stably integrated into eukaryotic cells and packaged with proteins that had beta-glucosidase activity. Stable expression of lis in cells had no significant effect on biological characteristics such as cell morphology, growth characteristics, cell cycle and in vivo tumorigenicity. Conclusion The stable transfection of hepatoma cell line with lis gene was successfully constructed and provided an experimental basis for the application of lis suicide gene system in the treatment of liver cancer.