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

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-EAFP-PALB),亚克隆r-caspase-3至载体pAdTrack-EAFP-PALP,Pme Ⅰ线性化pAdTrack-EAFP-PALB/r-caspase-3,电转化至BJ/AdEasy菌,获得重组腺病毒骨架pAdeasy-EAFP-PALB/r-caspase-3.Pac Ⅰ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Pac Ⅰ酶切鉴定结果表明pAdeasy-EAFP-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-EAFP-PALB/r-caspase-3病毒颗粒包装成功;靶向性r-caspase-3重组腺病毒感染各组细胞24 h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.

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

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 (pAdTrack-EAFP-PALB) was constructed and the r-caspase-3 gene was subcloned into the vector. The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183 cells. The candidate clone was analyzed by restriction endonuclease digestion and sequencing, and then pAdeasy-EAFP-PALB/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-EAFP-PALB/r-caspase-3 was correct after digestion by restriction endonuclease. Vector pAdeasy-EAFP-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. The cells were infected with recombinant r-caspase-3 after 24 hours, and their apoptotic index were as follows: HepG cells, 48.2%; 7721 cells, 17.7%; L-02 cells, 7.3%;M DA- 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.