hTERT非端粒酶依赖途径调控喉鳞癌细胞凋亡的研究

发布时间：2018-04-28 20:51

本文选题：人端粒酶逆转录酶 + 前列腺凋亡反应因子　；参考：《武汉大学》2012年博士论文

【摘要】：目的：应用RNA干扰技术沉默人端粒酶逆转录酶(hTERT)前后,检测喉癌细胞及移植瘤组织中hTERT表达含量与端粒酶活性及前列腺凋亡反应因子(Par-4)表达相关性,探讨人端粒酶逆转录酶不依赖其催化活性的抗凋亡作用及其机制,为喉癌的基因治疗提供新的思路。方法：构建靶向人端粒酶逆转录酶mRNA的质粒p-EGFP-shTERT,将质粒转染体外培养的喉癌Hep-2细胞,应用TRAP-PCR法检测检测转染后0h、12h、24h、48h四个时间点喉癌Hep-2细胞端粒酶活性,流式细胞术检测转染后四个时间点细胞凋亡程度,Western-blot检测hTERT蛋白及Par-4蛋白表达；建立喉癌移植瘤裸鼠动物模型,采用瘤体内多点注射方式将质粒p-EGFP-shTERT导入移植瘤内,采用量子点超敏荧光免疫技术检测质粒转染前以及转染后6d、10d、14d移植瘤组织内hTERT和Par-4两种蛋白表达情况,方差分析、卡方检验及Spearman相关性检验分析实验结果。结果：质粒成功转染Hep-2细胞24h后端粒酶活性开始下降,48h后端粒酶活性被抑制；质粒转染12h后Hep-2细胞即有显著凋亡,随时间延长,凋亡率逐渐增高；hTERT蛋白表达随转染时间延长逐渐降低,Par-4蛋白表达逐渐增加；体内实验中发现,质粒转染前,hTERT蛋白在喉癌Hep-2细胞裸鼠移植瘤中呈高表达,而且胞核、胞浆内均有表达,但Par-4蛋白仅在胞浆表达,质粒转染14天后,hTERT表达降低,胞核内表达下调明显,而Par-4在胞核表达增多,Speraman等级相关分析发现,移植瘤中Par-4和hTERT在质粒p-EGFP-shTERT转染前后表达呈负相关(p0.05,r=-0.908)。结论：应用RNA干扰抑制hTERT表达过程中,我们发现质粒转染24h后,端粒酶活性仅有轻度降低,48小时后端粒酶活性才被抑制,然而质粒转染Hep-2细胞12h后,就有凋亡反应出现,24h后凋亡显著,因此推测,RNA干扰抑制hTERT诱导的凋亡反应并不依赖端粒酶活性的抑制。同样,hTERT介导的肿瘤细胞抗凋亡作用除端粒酶激活途径外,还存在其它非端粒酶途径；抑制hTERT蛋白表达的同时,我们还发现,前列腺凋亡反应因子(Par-4)表达上调,且质粒转染前后两种蛋白表达部位发生改变,提示Par-4可能参与hTERT不依赖其催化活性的抗凋亡途径,hTERT可能在胞浆中与Par-4相互作用,抑制了Par-4转入胞核,从而抑制了细胞凋亡。
[Abstract]:Objective: to detect the correlation between the expression of hTERT and the expression of telomerase activity and prostate apoptosis-response factor Par-4 in laryngeal carcinoma cells and transplanted tumor tissues before and after the silencing of human telomerase reverse transcriptase (hTERT) by RNA interference technique. To explore the antiapoptotic effect of human telomerase reverse transcriptase independent of its catalytic activity and its mechanism, and to provide a new idea for gene therapy of laryngeal carcinoma. Methods: the plasmid p-EGFP-shTERTtargeting human telomerase reverse transcriptase (mRNA) was constructed and transfected into Hep-2 cells of laryngeal carcinoma in vitro. The telomerase activity of Hep-2 cells was detected by TRAP-PCR assay at four time points (0 h, 12 h, 24 h and 48 h) after transfection. The expression of hTERT protein and Par-4 protein was detected by Western-blot at four time points after transfection, and the mouse model of laryngeal carcinoma transplantation tumor was established. The plasmid p-EGFP-shTERT was introduced into the transplanted tumor by multi-point injection in vivo. The expression of hTERT and Par-4 in tumor tissues before and 6 days after transfection were detected by quantum dot hypersensitive fluorescence immunoassay. The results of variance analysis, chi-square test and Spearman correlation test were analyzed. Results: the telomerase activity of Hep-2 cells was decreased after 24 hours of plasmid transfection, and the telomerase activity was inhibited after 12 hours of plasmid transfection, and the apoptosis of Hep-2 cells was observed after 12 hours of plasmid transfection, and the telomerase activity was prolonged with time. The expression of hTERT protein decreased gradually with the extension of transfection time, the expression of hTERT protein increased gradually in vivo, and the expression of hTERT protein was found to be highly expressed in the transplanted tumor of Hep-2 cell line of laryngeal carcinoma, and its nucleus was also found in vivo. However, the expression of Par-4 protein was only expressed in the cytoplasm. After 14 days of transfection, the expression of hTERT was decreased and the expression of hTERT was down-regulated in the nucleus. The expression of Par-4 in the nucleus was increased by Speraman rank correlation analysis. There was a negative correlation between the expression of Par-4 and hTERT in the transplanted tumor before and after transfection of plasmid p-EGFP-shTERT. Conclusion: during the inhibition of hTERT expression by RNA interference, we found that telomerase activity was inhibited only after 24 hours of transfection, but only after a slight decrease of 48 hours. However, 12 hours after transfection of the plasmid into Hep-2 cells, telomerase activity was inhibited. After 24 hours of apoptosis, we speculate that the inhibition of hTERT induced apoptosis by hTERT does not depend on the inhibition of telomerase activity. In addition to telomerase activation pathway, there are other non-telomerase pathways in tumor cells mediated by hTERT, while inhibiting the expression of hTERT protein, we also found that the expression of prostatic apoptosis-responsive factor Par-4) was up-regulated. The changes of two protein expression sites before and after plasmid transfection suggest that Par-4 may participate in the antiapoptotic pathway of hTERT independent of its catalytic activity. HTERT may interact with Par-4 in the cytoplasm and inhibit the transfer of Par-4 into the nucleus, thus inhibiting the apoptosis of the cells.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.65

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