CAJ | 학술논문

人核糖体DNA打靶载体pHr是一种由中南大学医学遗传学国家重点实验室开发构建的针对人类基因组的同源重组质粒载体.利用pHr构建了一种mda-7/GFP融合基因的人源基因表达载体pHr-CMG,并研究了其在肝癌细胞系Bel-7402中的作用.利用荧光显微镜、RT-PCR和Western blotting检测了mda-7/GFP融合基因的表达;利用细胞周期分析、MTT和Hoechst33258染色研究了其在细胞中的作用.结果显示,pHr-CMG载体能在Bel-7402细胞中有效表达MDA-7/GFP融合蛋白,进而抑制细胞增殖和诱导细胞凋亡,推测其可能是由载体表达了mda-7基因引起细胞在G2/M期累积所导致的.同时,实验结果证实了人核糖体DNA打靶载体系统以及pHr-CMG表达载体的有效性,为其在进一步基因治疗研究中的应用提供了理论和实验基础.
인핵당체DNA타파재체pHr시일충유중남대학의학유전학국가중점실험실개발구건적침대인류기인조적동원중조질립재체.이용pHr구건료일충mda-7/GFP융합기인적인원기인표체재체pHr-CMG,병연구료기재간암세포계Bel-7402중적작용.이용형광현미경、RT-PCR화Western blotting검측료mda-7/GFP융합기인적표체;이용세포주기분석、MTT화Hoechst33258염색연구료기재세포중적작용.결과현시,pHr-CMG재체능재Bel-7402세포중유효표체MDA-7/GFP융합단백,진이억제세포증식화유도세포조망,추측기가능시유재체표체료mda-7기인인기세포재G2/M기루적소도치적.동시,실험결과증실료인핵당체DNA타파재체계통이급pHr-CMG표체재체적유효성,위기재진일보기인치료연구중적응용제공료이론화실험기출.
Human ribosomal DNA (hrDNA) targeting vector pHr is a homologous recombinant plasmid for human genome which developed in the State Key Laboratory of Medical Genetics. pHr was used to construct a recombinant plasmid pHr-CMG expressing mda-7/GFP fusion gene and its efficacy in the hepatocellular carcinoma cell line Bel-7402 was investigated. The expression of mda-7/GFP fusion gene was detected by fluorescent microscope, RT-PCR and Western blotting, and its function was detected by cell-cycle analyses, MTT assay and Hoechst33258 staining. The results demonstrated that pHr-CMG vector could express MDA-7/GFP fusion protein effectually and the mda-7 gene could induce cell apoptosis and proliferation suppression in Bel-7402 cell line, which might be caused by the G2/M cell cycle arrest. These results also suggested that human ribosomal DNA targeting vector system and the pHr-CMG vector may be applied in further gene therapy researches for hepatocellular carcinoma.