超声微泡造影剂增强重组腺相关病毒rAAV—sTRAIL感染肝癌细胞的实验观察

Adeno-associated virus mediated tumor necrosis factor related apoptosis inducing ligand gene transfected into hepatocellular carcinoma cells by ultrasound microbubble intensifier： an experimental study

目的探讨携带可溶性肿瘤坏死因子相关凋亡诱导配体（sTRAIL）基因的重组腺相关病毒载体rAAV-sTRAIL对肝癌细胞的增殖抑制及诱导凋亡作用，以及采用超声辐照联合微泡造影剂介导rAAV-sTRAIL转染肝癌细胞的有效性。方法单纯重组腺相关病毒rAAV-sTRAIL感染肝癌细胞株HepG2，以及利用超声辐照联合微泡造影剂介导重组腺相关病毒rAAV-sTRAIL感染HepG2细胞，分别以RT-PCR方法和Westen印迹法检测细胞内sTRAIL基因mRNA及蛋白表达量，噻唑蓝法检测细胞的增殖抑制率，流式细胞术检测细胞凋亡率。结果重组腺相关病毒rAAV-sTRAIL可有效转染HepG2细胞，sTRAIL mRNA表达水平在微泡造影剂组高于单纯病毒组，二者差异有统计学意义（P〈0．05）；rAAV-sTRAIL可抑制HepG2细胞增殖，其抑制率在微泡造影剂组高于单纯病毒组，二者差异有统计学意义（P〈0．05）；rAAV-sTRAIL可诱导HepG2细胞凋亡，其凋亡率在微泡造影剂组高于单纯病毒组，二者差异有统计学意义（P〈0．05）。结论构建出的重组腺相关病毒rAAV-sTRAIL在肝癌细胞中能有效表达，sTRAIL基因对肝癌细胞有明显的抑制增殖和促凋亡作用；联合超声微泡造影剂及低强度超声，可有效提高腺相关病毒载体在肝癌细胞中的感染率，从而增强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 adeno-associated virus encoding soluble tumor necrosis factor related apoptosis inducing ligand gene （rAAV-sTRAIL） on proliferation and apoptosis of human hepatocellular carcinoma （HCC） cells, and to investigate the feasibility and efficiency of transfection of rAAV-sTRAIL into human HCC cells by ultrasound microbubble intensifier. Methods Human HCC cells of the line HepG2 were transfected with rAAV-sTRAIL or rAAV-sTRAIL combined with microbubble echocontrast agent and appropriate dose of ultrasound irradiation. RT-PCR and Western blotting were used to detect the mRNA and protein expression of sTRAIL gene. MTT method was used to detect the proliferation inhibition rate, and the apoptosis rate of the HepG2 cells was evaluated by flow cytometry. Results The expression levels of sTRAIL mRNA and protein were higher in the rAAV-TRAIL combined with ultrasound microbubble group than in the rAAV-sTRAIL group （ both P 〈 0.05 ）. The proliferation inhibition rate of the rAAV-TRAIL combined with ultrasound microbubble group was significantly higher than that of the rAAV-sTRAIL group （ P 〈 0. 05 ）. The apoptotic effect of the rAAV-TRAIL combined with ultrasound microbubble group was greater than that of the rAAV-sTRAIL group （ P 〈 0.05 ）. Conclusion TRAIL has a potential role to inhibit e proliferation and induce apoptosis of human hepatocellular carcinoma cells. Ultrasound and microbubble echocontrast agent increase the transfection rate of rAAV vector into HCC cells.