靶向性反向半胱氨酸天冬氨酸蛋白酶3重组腺病毒诱导肝癌细胞凋亡的实验研究

Targeted adenovirus containing r-caspase-3 gene induces apoptosis of hepatocellular carcinoma cells in vitro

目的观察靶向性反向半胱氨酸天冬氨酸蛋白酶3（r—caspase-3）重组腺病毒体外诱导肝癌细胞凋亡的效果和特异性。方法构建甲胎蛋白（AFP）增强子和白蛋白（ALB）启动子腺病毒载体（pAdTrack．EATP．PALB），亚克隆r—caspase-3至载体pAdTrack—EAFP-PALB,PmeI线性化pAdTrack—EAFP．PALB／r—easpase．3，电转化至BJ／AdEasy菌，获得重组腺病毒骨架pAdeasy—EnrP-PALB／r．caspase一3。PacI酶切后脂质体转染AD293细胞进行包装、扩增、获得病毒。绿色荧光蛋白（GFP）监测病毒滴度和感染效率；RT-PCR和Western印迹法检测r-caspase．3在HepG2细胞中的表达；流式细胞术检测其特异性诱导肝癌细胞凋亡的作用。结果穿梭载体pAdTrack．EAFP-PALB／r—caspase-3酶切、测序正确。穿梭载体、pAdeasy-1载体同源重组后PCR及PacI酶切鉴定结果表明pAdeasy．E＆FP-PALB／r—caspase-3重组成功。转染AD293细胞后可观察到GFP的表达。病毒感染HepG2细胞总DNA经RT—PCR和Western印迹均可检测到目的基因的表达，证实Ad-E—FP-PAL／dr—caspase-3病毒颗粒包装成功；靶向性r—easpase-3重组腺病毒感染各组细胞24h后，各组凋亡指数分别为：HepG细胞48．2％、7721细胞17．7％、L-02细胞7．3％、MDA—MB-231细胞0％。结论成功构建了靶向性Ad．EAFP-PALB／r—caspase-3重组腺病毒，体外实验表明其具有凋亡诱导靶向性，为进一步研究靶向性r—caspase-3基因治疗肝细胞肝癌及其生物学功能提供了依据。

Objective To investigate the effect and specificity of adenovirus containing r-caspase-3 gene on apoptosis of hepatocellular carcinoma cells. Methods The vector α-fetoprotein enhancer-albumin promotor （pAdTraek-EArv-PALB） was constructed and the r-caspase-3 gene was subcloned into the vector. The linearized shuttle plasmid was homogenously recombined with AdEasy-l in BJ5183 cells. The candidate clone was analyzed by restriction endonuclease digestion and sequencing, and then pAdeasy-EAFP-PALS/r- caspase-3 vector was digested with Pac I and transfected into AD293 cells for packaging and amplifying. Infection titer and rate of recombinant virus was monitored by green fluorescent protein （GFP） expression. The expression of r-caspase-3 was detected by RT-PCR and Western-blot. The apoptosis of HepG2, 7721, L- 02 and MDA- MB- 231 cells was detected by FCM method, Results The sequence of shuttle vector pAdTrack-EArp-PALB/r-caspase-3 was correct after digestion by restriction endonuclease. Vector pAdeasy-EAvv- PALB/r-caspase- 3 was identificated by Pac I restriction digestion and PCR. The expression of GFP was observed in the transfected AD293 cells. The expression of r-caspase-3 gene was detected in the infected HepG2 cells by RT-PCR and Western-blot. 3＇he cells were infected with recombinant r-caspase-3 after 24 hours, and their apoptotic index were as follows： HepG ceils, 48.2%; 7721 cells, 17.7%; L-02 ceils, 7.3%; MDA- MB- 231 cells, 0%. Conclusion The recombinant of hepatocellular carcinoma- targeting adenovirus containing r- caspase- 3 gene is constructed successfully and can induce the targeted apoptosis of hepatocellular carcinoma cells, which provides the evidences for future research in hepatocellular carcinoma.