中国病理生理杂志
中國病理生理雜誌
중국병리생리잡지
CHINESE JOURNAL OF PATHOPHYSIOLOGY
2007年
8期
1488-1494
,共7页
周鸿科%杨冬华%汤绍辉%黄卫
週鴻科%楊鼕華%湯紹輝%黃衛
주홍과%양동화%탕소휘%황위
胰岛素样生长因子Ⅱ%绿色荧光蛋白%癌,肝细胞%基因疗法
胰島素樣生長因子Ⅱ%綠色熒光蛋白%癌,肝細胞%基因療法
이도소양생장인자Ⅱ%록색형광단백%암,간세포%기인요법
Insulin -like growth factor Ⅱ%Green fluorescent protein%Carcinoma,hepatocellular%Gene therapy
目的:构建人胰岛素样生长因子Ⅱ基因(IGF-Ⅱ)P3启动子驱动单纯疱疹病毒胸苷激酶(tk)与增强型绿色荧光蛋白(EGFP)融合基因穿梭质粒,观察其在肝癌细胞系HepG2及宫颈癌细胞系HeLa细胞中的表达及其作用.方法:应用基因重组技术,构建IGF-Ⅱ P3启动子驱动EGFP与tk融合表达穿梭质粒载体,脂质体转染技术将其转入HepG2及HeLa中,48 h后荧光显微镜下观察荧光表达,RT-PCR法检测tk和EGFP mRNA表达,四甲基偶氮唑蓝(MTT)实验检测转染融合蛋白载体后给予不同浓度的GCV对HepG2及HeLa细胞毒作用.结果:酶切鉴定和测序分析证实重组质粒构建成功;转染此穿梭质粒的细胞中仅有HepG2细胞检测到绿色荧光;RT-PCR检测到tk和EGFP的mRNA仅在HepG2细胞中表达;GCV对转染了携此质粒载体的HepG2细胞有选择性细胞毒作用.结论:成功构建IGF-Ⅱ P3启动子驱动携带tk与EGFP融合基因穿梭质粒载体,转染后对人肝癌细胞系HepG2有选择性杀伤作用,为进一步靶向性肝癌腺病毒基因治疗奠定了基础.
目的:構建人胰島素樣生長因子Ⅱ基因(IGF-Ⅱ)P3啟動子驅動單純皰疹病毒胸苷激酶(tk)與增彊型綠色熒光蛋白(EGFP)融閤基因穿梭質粒,觀察其在肝癌細胞繫HepG2及宮頸癌細胞繫HeLa細胞中的錶達及其作用.方法:應用基因重組技術,構建IGF-Ⅱ P3啟動子驅動EGFP與tk融閤錶達穿梭質粒載體,脂質體轉染技術將其轉入HepG2及HeLa中,48 h後熒光顯微鏡下觀察熒光錶達,RT-PCR法檢測tk和EGFP mRNA錶達,四甲基偶氮唑藍(MTT)實驗檢測轉染融閤蛋白載體後給予不同濃度的GCV對HepG2及HeLa細胞毒作用.結果:酶切鑒定和測序分析證實重組質粒構建成功;轉染此穿梭質粒的細胞中僅有HepG2細胞檢測到綠色熒光;RT-PCR檢測到tk和EGFP的mRNA僅在HepG2細胞中錶達;GCV對轉染瞭攜此質粒載體的HepG2細胞有選擇性細胞毒作用.結論:成功構建IGF-Ⅱ P3啟動子驅動攜帶tk與EGFP融閤基因穿梭質粒載體,轉染後對人肝癌細胞繫HepG2有選擇性殺傷作用,為進一步靶嚮性肝癌腺病毒基因治療奠定瞭基礎.
목적:구건인이도소양생장인자Ⅱ기인(IGF-Ⅱ)P3계동자구동단순포진병독흉감격매(tk)여증강형록색형광단백(EGFP)융합기인천사질립,관찰기재간암세포계HepG2급궁경암세포계HeLa세포중적표체급기작용.방법:응용기인중조기술,구건IGF-Ⅱ P3계동자구동EGFP여tk융합표체천사질립재체,지질체전염기술장기전입HepG2급HeLa중,48 h후형광현미경하관찰형광표체,RT-PCR법검측tk화EGFP mRNA표체,사갑기우담서람(MTT)실험검측전염융합단백재체후급여불동농도적GCV대HepG2급HeLa세포독작용.결과:매절감정화측서분석증실중조질립구건성공;전염차천사질립적세포중부유HepG2세포검측도록색형광;RT-PCR검측도tk화EGFP적mRNA부재HepG2세포중표체;GCV대전염료휴차질립재체적HepG2세포유선택성세포독작용.결론:성공구건IGF-Ⅱ P3계동자구동휴대tk여EGFP융합기인천사질립재체,전염후대인간암세포계HepG2유선택성살상작용,위진일보파향성간암선병독기인치료전정료기출.
AIM: To construct the shuttle plasmid vector for thymidine kinase (tk) and EGFP fusion protein gene driven by IGF - Ⅱ P3 promoter, and investigate the specific killing effect of the HSV - tk/GCV system on hepatocellular carcinoma(HCC) cells in vitro. METHODS: Recombinant shuttle plasmid vector was constructed by techniques of genetic recombination and screening, and identified by restriction digestion and sequencing analysis. Then the recombinant shuttle plasmid was transfected into HepG2 and HeLa cells by techniques of lipofectamine transfection and its expression was detected by fluorescence microscope and RT -PCR. Cell killing after ganciclovir(GCV) application was determined by MTT. RESULTS: Identification of pDC316 -tkEGFP- P3 by enzyme digestion and sequencing analysis showed that the length, inserted location and direction of the target genes which were inserted into the recombinant were correct. It was found that enhanced green fluorescence protein could only be seen in HepG2 cells, but not in HeLa cells. The results of RT -PCR showed that only two bands could be seen in the samples of pDC316 -tkEGFP- P3 transfected HepG2 cells. The MTT test showed the selective cytotoxicity of GCV to the transfected HepG2 cells. CONCLUSION: The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF - Ⅱ P3 promoter has been constructed successfully and its specific expression in HepG2 cells provided a sound basis for targeted gene therapy for HCC.
