摘要
目的 将编码蜂毒素的基因用于肝癌治疗,为蜂毒素的临床应用提供实验依据。 方法 采用细菌内同源重组法构建携蜂毒素基因及甲胎蛋白(AFP)启动子的重组腺病毒,X-gal染色检测其转染效率,MTT法测定其对肝癌细胞的抑制作用,并观察其体外转染对肝癌细胞致瘤能力的影响及肿瘤内注射对荷肝癌裸鼠的抑瘤效果。 结果 蜂毒素基因mRNA在细胞中得到了转录;当感染复数(MOI)为10时,重组腺病毒在体外对BEL-7402人肝癌细胞的转染效率达到100％,体内转染亦有较高的效率。Ad-rAFP-Mel和Ad-rAFP对BEL7402细胞的抑制率分别为66.2％±2.7％和2.9％±2.3％(t=30.83,P<0.01)。Ad-CMV-Mel对BEL7402、SMMC7721及L02细胞的抑制率分别为58.9％±9.6％、65.9％±3.8％和31.7％±1.2％,而Ad-rAFP-Mel对BEL7402、SMMC7721及L02细胞的抑制率分别为66.2％±2.7％、16.1％±6.6％和7.5％±3.3％,与Ad-CMV-Mel组比较t=1.27、P=0.27,t=11.31、P<0.01和t=12.12、P<0.01。重组腺病毒体外转染后,肝癌细胞致瘤能力减弱,肿瘤内注射,裸鼠皮下移植瘤消退。 结论 蜂毒素基因重组腺病毒在体内外对肝癌均有特异性抑制作用,证明动物毒素基因作为抗肿瘤目的基因具有可行性。
Objective To find a new method of treating hepatocellular carcinoma with melittin by way of using the melittin gene. Methods The recombinant adenoviruses carrying the melittin gene and a -fetoprotein (AFP) promoter (Ad-rAFP-Mel) were constructed through a bacterial homologous recombinant system. The efficiency of the adenovirus mediated gene transfer and the inhibition effect of Ad-rAFP-Mel on the proliferation of hepatocarcinoma cells were determined by X-gal staining and MTT assay respectively. The tumorigenicity of hepatocarcinoma cells transfected by Ad-rAFP-Mel and the antitumor effect of Ad-rAFP-Mel on the transplanted tumors in nude mice were detected in vivo. Results The mRNA of the melittin gene was transcripted in HepG2 hepatocellular carcinoma cells transducted by Ad-rAFP-Mel. The efficiency of adenovirus mediated gene transfered to BEL-7402 hepatocarcinoma cells was 100% when the multiplicities of infection (MOI) of Ad-rAFP-Mel was 10 in vitro and was high in vivo as well. The inhibitive rates of Ad-rAFP-Mel and Ad-rAFP for BEL7402 cells were 66.2%±2.7% and 2.9%±2.3% (t = 30.83, P < 0.01) by MTT assay. The inhibitive rates of Ad-CMV-Mel for BEL7402, SMMC7721 and L02 cells were 58.9%±9.6%, 65.9%±3.8%, 31.7%±1.2%, respectively, and those of the Ad-rAFP-Mel were 6.2%±2.7%, 16.1±6.6%, 7.5% ±3.3%, respectively (t = 1.27, P = 0.27; t = 11.31, P < 0.01 and t = 12.12, P < 0.01 vs Ad-CMV-Mel group in same cells). The tumorigenicity rates of hepatocarcinoma cells transfected by Ad-rAFP-Mel were decreased. A significant antine-oplastic effect was detectd on transplanted tumor in nude mice by intratumoral injection of Ad-rAFP-Mel. Conclusion Ad-rAFP-Mel can inhibit specifically the proliferation of AFP-producing human hepatocarcinoma cells in vitro and in vivo. It suggests that animal toxin gene can be used as an interesting antitumor gene.
出处
《中华肝脏病杂志》
CAS
CSCD
2004年第12期741-744,共4页
Chinese Journal of Hepatology
基金
上海市卫生局"百人计划"资助项目(97BR044)国家中医药管理局科学技术研究专项基金(02-03JQ08)
