藤黄酸对人胃腺癌细胞BGC-823细胞端粒酶逆转录酶和AKt蛋白磷酸化表达的影响  

Effect of gambogic acid on AKt and human telomerase reverse transcriptase(hTERT) in human gastric carcinoma BGC-823 cells

作　　者：许毅[1] 黄新恩[2] 陈森清[2] 马国建[2] 张晓梅[2] 朱明[2] 俞军[2] 

机构地区：[1]江苏省中医院药学部,江苏省南京市210029 [2]江苏省肿瘤医院科研科,江苏省南京市210009

出　　处：《实用老年医学》2017年第2期118-122,共5页Practical Geriatrics

基　　金：江苏省科技厅资助项目(BK20161598);南京市科技局资助项目(201605006);江苏省肿瘤医院重点课题(ZK201603)

摘　　要：目的探讨藤黄酸(GA)对人胃癌细胞BGC-823端粒酶活性和端粒酶逆转录酶(hTERT)mRNA、AKt蛋白磷酸化表达的影响。方法将处于对数生长期的BGC-823细胞制成细胞悬液,加入96孔酶标板内,加入不同浓度的GA孵育24、48、72 h后,收集细胞,采用MTT比色法测定半抑制浓度(IC50)值;分别采用聚合酶链式反应(PCR)、酶联免疫反应(ELISA)法检测端粒酶的活性和逆转录PCR(RT-PCR)法检测hTERT mRNA的表达;通过免疫共沉淀(IP)法获得hTERT与磷酸化AKt(p-AKt)蛋白的结合体,然后用Western blot法检测p-AKt蛋白表达的变化。结果 GA可以明显抑制胃癌细胞BGC-823的端粒酶活性和hTERT mRNA的表达,呈一定的时效、量效关系;进一步研究发现,GA能够抑制hTERT和p-AKt蛋白的结合,且作用呈时间依赖性,进而影响p-AKt参与的hTERT的活化,从而完成对hTERT转录后的调控,抑制端粒酶的活性。结论 GA可明显抑制胃癌细胞BGC-823端粒酶活性和hTERT的表达,GA对BGC-823细胞生长抑制的作用机制可能与GA影响p-AKt参与的hTERT的活化,从而完成对hTERT转录后的调控有关。Objective To explore the effect of Gambogic acid（ GA） on p-AKt and human telomerase reverse transcriptace（ hTERT） in human gambogic acidstric carcinoma cells. Methods To investigate the inhibitiny effects of GA on human grastric carcinoma cells BGC-823,we observed the cells treated with GA by invert-microscope. MTT assay was applied to study the growth inhibition of human grastric carcinoma cells BGC-823 in vitro. The activity of telomerase and the expression of hTERT were detected by PCR-ELISA and RT-PCR. Furthermore,the immunoprecipitation assay was used to detect the affinity of hTERT and p-AKt in BGC-823 induced by GA treatment. And then protein level of p-AKt was determined by western blot analysis. Results GA decreased the expression of telomerase activity and hTERT at time-dependent and concentration-dependent manner. Then,further investigation showed that the affinity of hTERT and p-AKt was inhibited by GA treatment via the down-regulation of p-AKt expression in significant time-and concentration-dependent manner. Then the activity of telomerase was inhibited. Conclusions These results show that GA can inhibit the proliferation of human grastric carcinona cells BGC-823. The mechanism may be that GA inhibits the posttranscriptional activation of hTERT in an p-AKt dependent manner in BGC-823 cells and finally,and affect the activity of telomerase.

关 键 词：人胃癌细胞BGC-823 藤黄酸 端粒酶 端粒酶逆转录酶 磷酸化AKT 

分 类 号：R735.2[医药卫生—肿瘤]