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目的:观察偶联去唾液酸糖蛋白(AF)的人端粒酶的逆转录酶启动子(hTERTp)驱动的自杀基因(tk/cd)协同表达对肝癌细胞株HepG2的靶向杀伤作用。方法:细胞培养,PCR方法构建AF-pGL3-hTERTp-cd与AF-pGL3-hTFERTp-tk质粒,通过瞬时转染方法联合转染肝癌细胞HepG2和正常肝细胞L-02,MTT法观察药物浓度对肝癌细胞存活率的影响,流式技术观察其对肝癌细胞生长和凋亡的影响。结果:MTT法显示GCV、5-FC对自杀基因协同转染的HepG2细胞有较强的细胞毒作用,联合作用可降低药物浓度。流式结果表明在hTERTTp驱动下自杀基因协同作用于肝癌细胞后细胞总的凋亡率为87%,高于单—AF-pGL3-hTERTp-cd组(77%),以及单一AF-pGL3-hTERTp-tk组(70%),对正常肝细胞L-02的生长影响较小。结论:偶联有AF的hTERTp驱动的自杀基因tk/cd协同表达可增强肝癌细胞的自杀效果,并促使其凋亡的作用。
OBJECTIVE: To observe the targeted killing effect of tk / cd co-expression by human telomerase reverse transcriptase (hTERTp) -dependent suicide gene (HepG2) coupled with asialoglycoprotein (AF) on hepatocellular carcinoma cell line HepG2. Methods: The plasmids of AF-pGL3-hTERTp-cd and AF-pGL3-hTFERTp-tk were constructed by cell culture and transfected into HepG2 cells and normal liver cells by transient transfection method. MTT assay was used to observe the drug concentration Hepatic cancer cell survival rate of flow cytometry to observe the impact of hepatocellular carcinoma cell growth and apoptosis. Results: MTT assay showed that GCV and 5-FC had strong cytotoxic effect on HepG2 cells transfected with suicide gene. The combination of GCV and 5-FC reduced the drug concentration. The results of flow cytometry showed that the apoptosis rate of hepatoma cells was 87%, which was higher than that of the single-AF-pGL3-hTERTp-cd group (77%) and single AF-pGL3-hTERTp -tk group (70%) had little effect on the growth of normal liver cells L-02. CONCLUSION: The hTERTp-driven suicide gene tk / cd co-expression coupled with AF can enhance the effect of hepatocarcinoma cell suicide and promote its apoptosis.