敲低AKT1基因增强裸鼠胃癌移植瘤对阿霉素的敏感性

Knockdown of AKT1 gene enhances the sensitivity of gastric cancer xenografts of nude mice to doxorubicin

目的研究敲低AKT1基因对裸鼠胃癌移植瘤阿霉素敏感性的影响。方法构建AKT1基因RNA干扰(RNAi)的慢病毒载体p GCSIL-sh AKT1-GFP,转染HEK293T细胞,以p GCSIL-sh CON-GFP为空载体组。获得慢病毒颗粒后,感染胃癌BGC-823细胞,采用Western blot法检测感染后BGC-823细胞中AKT1蛋白水平。皮下注射慢病毒感染的BGC-823细胞构建裸鼠胃癌移植瘤模型,使用阿霉素(DOX)进行处理,观察处理后移植瘤体积大小并绘制肿瘤生长曲线,采用HE染色观察移植瘤组织病变情况,采用组织原位末端转移酶标记技术(TUNEL)检测移植瘤组织中细胞凋亡情况。结果成功构建AKT1基因RNAi的慢病毒载体并成功感染胃癌BGC-823细胞,感染后胃癌BGC-823细胞中AKT1蛋白水平明显降低。敲低AKT1的BGC-823细胞裸鼠移植后,移植瘤生长减慢、移植瘤细胞凋亡增加,并可增强DOX对移植瘤生长的抑制作用。结论敲低AKT1基因可增强BGC-823细胞裸鼠移植瘤对DOX的敏感性。

Objective To investigate the effect of lentivirus-mediated shRNA targeting AKT1 gene on the sensitivity of gastric cancer xenografts to doxorubicin. Methods To establish the lentiviral vector of AKT1 RNA interference（ RNAi）,the vector of p GCSIL-sh AKT1-GFP was infected into HEK293 T cells,and meanwhile,the empty vector p GCSIL-sh CON-GFP was assigned as a blank group,and then the viruses were harvested. BGC-823 gastric cancer cells were infected with the viruses. The protein expression of AKT1 was detected by Western blotting. The gastric cancer xenografts in nude mice were constructed using BGC-823 cells infected with the viruses,followed by the administration of doxorubicin. The size of tumor was evaluated and the growth curve of the tumor was drawn; HE staining was used to observe the pathological conditions of the xenografts; and the apoptosis of xenografts was examined by TUNEL assay. Results The recombinant plasmid of LV-sh AKT1 was successfully constructed,and then transfected into HEK293 T cells to produce high-titer lentivirus with a titer of 5 × 108 TU/m L. The expression of AKT1 protein in gastric cancer BGC-823 cells was significantly down-regulated after successfully infected with the LV-sh AKT1. Nude mice xenografts experiment showed that AKT1 gene silencing inhibited the growth of tumor xenografts,and also enhanced the inhibitory effect of doxorubicin on the growth of tumor xenografts. TUNEL staining showed that AKT1 gene silencing promoted the apoptosis of tumor xenograft cells,and the effect was more obvious when combined with doxorubicin. Conclusion AKT1 gene silencing can enhance the sensitivity of gastric cancer xenografts to doxorubicin through promoting cell apoptosis.