超声微泡造影剂增强TRAIL基因转染肝癌细胞的实验研究

Ultrasound microbubble contrast agent enhances TRAIL gene transfection into hepatocellular carcinoma cells

目的 探讨采用超声辐照联合微泡造影剂增强重组表达质粒pIRES—EGFP—sTRAIL转染肝癌细胞的有效性。方法 构建携带肿瘤坏死因子相关细胞凋亡诱导配体（TRAIL）基因及绿色荧光蛋白（GFP）的真核表达质粒pIRES—EGFP—sTRAIL，转染肝癌细胞HepG2，分为超声辐照联合微泡造影剂转染组、脂质体转染组、单纯微泡造影剂转染组及对照组。荧光显微镜检测GFP以评价转染效率，四甲基偶氮唑盐微量酶反应比色法（MTT）检测细胞生长抑制率，Hoechst33258荧光染色观察细胞形态变化，流式细胞术检测细胞凋亡率，Westernblot检测TRAIL凋亡途径中关键凋亡分子caspase-8和caspase-3的蛋白变化。结果 超声辐照联合微泡造影剂可增强pIRES—EGFP—sTRAIL有效转染HepG2细胞，其转染效率与脂质体转染组、单纯微泡造影剂组及对照组比较差异均有统计学意义（P〈0．05）；携带sTRAIL基因的表达质粒能够激活caspase通路，促进肝癌细胞凋亡，有效抑制肝癌细胞生长。结论 构建的真核表达载体pIRES-EGFP—sTRAIL在肝癌细胞中能有效表达，联合超声微泡造影剂及低强度超声，可显著增强sTRAIL对肝癌细胞的增殖抑制及诱导凋亡作用。

Objective To evaluate the impact of the recombined plasmid vector with enhanced green fluorescent protein（EGFP） encoding soluble tumor necrosis factor related apoptosis inducing ligand （pIRES- EGFP-sTRAIL） on proliferation and apoptosis of human hepatocellular carcinoma cell line HepG2, and investigate the feasibility and efficiency of the transfection of pIRES-EGFP-sTRAIL into HepG2 by ultrasound microbubble contrast agent. Methods pIRES-EGFP-sTRAIL was constructed and transfected into HepG2 cells by using different types of mediated methods ： microbubble echocontrast agent combining appropriate dose of ultrasound irradiation, liposome method, microbubble echocontrast agent only or blank medium treatment. Transfection efficiency was evaluated by EGFP-expressed cell count; proliferation-inhibiting rate and the apoptosis rate of HepG2 cells were determined by MTT method and flow cytometry analysis ; changes of cell morphology were examined by microscopy with Hoechst33258 dyeing; expression of caspase-8 and caspase-3 was detected by Western blot. Results Ultrasound microbubble enhanced pIRES-EGFP-sTRAIL uptake by HepG2 cells, and the transfection efficiency was significantly higher in ultrasound microbubble group than that in other groups（ P 〈 0.05 ） ; pIRES-EGFP-sTRAIL effectively inhibited HepG2 cell proliferation and induced cell apoptosis by triggering caspase cascade. Both the inhibiting rate and apoptosis rate were significantly higher in ultrasound microbubble group than those in other groups（ P 〈 0.05）. Conclusion pIRES-EGFP-sTRAIL expresses effectively in HepG2 cells, sTRAIL has a potential role on the inhibiting proliferation and inducing apoptosis of HepG2 cells by triggering caspase cascade, and this role can be enhanced by the administration of low- intensity ultrasound and microbubble echocontrast agent.