本文選題：人端粒酶逆轉(zhuǎn)錄酶 + 前列腺凋亡反應(yīng)因子��； 參考：《武漢大學(xué)》2012年博士論文

【摘要】：目的：應(yīng)用RNA干擾技術(shù)沉默人端粒酶逆轉(zhuǎn)錄酶(hTERT)前后,檢測(cè)喉癌細(xì)胞及移植瘤組織中hTERT表達(dá)含量與端粒酶活性及前列腺凋亡反應(yīng)因子(Par-4)表達(dá)相關(guān)性,探討人端粒酶逆轉(zhuǎn)錄酶不依賴其催化活性的抗凋亡作用及其機(jī)制,為喉癌的基因治療提供新的思路。 方法：構(gòu)建靶向人端粒酶逆轉(zhuǎn)錄酶mRNA的質(zhì)粒p-EGFP-shTERT,將質(zhì)粒轉(zhuǎn)染體外培養(yǎng)的喉癌Hep-2細(xì)胞,應(yīng)用TRAP-PCR法檢測(cè)檢測(cè)轉(zhuǎn)染后0h、12h、24h、48h四個(gè)時(shí)間點(diǎn)喉癌Hep-2細(xì)胞端粒酶活性,流式細(xì)胞術(shù)檢測(cè)轉(zhuǎn)染后四個(gè)時(shí)間點(diǎn)細(xì)胞凋亡程度,Western-blot檢測(cè)hTERT蛋白及Par-4蛋白表達(dá)；建立喉癌移植瘤裸鼠動(dòng)物模型,采用瘤體內(nèi)多點(diǎn)注射方式將質(zhì)粒p-EGFP-shTERT導(dǎo)入移植瘤內(nèi),采用量子點(diǎn)超敏熒光免疫技術(shù)檢測(cè)質(zhì)粒轉(zhuǎn)染前以及轉(zhuǎn)染后6d、10d、14d移植瘤組織內(nèi)hTERT和Par-4兩種蛋白表達(dá)情況,方差分析、卡方檢驗(yàn)及Spearman相關(guān)性檢驗(yàn)分析實(shí)驗(yàn)結(jié)果。 結(jié)果：質(zhì)粒成功轉(zhuǎn)染Hep-2細(xì)胞24h后端粒酶活性開始下降,48h后端粒酶活性被抑制；質(zhì)粒轉(zhuǎn)染12h后Hep-2細(xì)胞即有顯著凋亡,隨時(shí)間延長(zhǎng),凋亡率逐漸增高；hTERT蛋白表達(dá)隨轉(zhuǎn)染時(shí)間延長(zhǎng)逐漸降低,Par-4蛋白表達(dá)逐漸增加；體內(nèi)實(shí)驗(yàn)中發(fā)現(xiàn),質(zhì)粒轉(zhuǎn)染前,hTERT蛋白在喉癌Hep-2細(xì)胞裸鼠移植瘤中呈高表達(dá),而且胞核、胞漿內(nèi)均有表達(dá),但Par-4蛋白僅在胞漿表達(dá),質(zhì)粒轉(zhuǎn)染14天后,hTERT表達(dá)降低,胞核內(nèi)表達(dá)下調(diào)明顯,而Par-4在胞核表達(dá)增多,Speraman等級(jí)相關(guān)分析發(fā)現(xiàn),移植瘤中Par-4和hTERT在質(zhì)粒p-EGFP-shTERT轉(zhuǎn)染前后表達(dá)呈負(fù)相關(guān)(p0.05,r=-0.908)。 結(jié)論：應(yīng)用RNA干擾抑制hTERT表達(dá)過(guò)程中,我們發(fā)現(xiàn)質(zhì)粒轉(zhuǎn)染24h后,端粒酶活性僅有輕度降低,48小時(shí)后端粒酶活性才被抑制,然而質(zhì)粒轉(zhuǎn)染Hep-2細(xì)胞12h后,就有凋亡反應(yīng)出現(xiàn),24h后凋亡顯著,因此推測(cè),RNA干擾抑制hTERT誘導(dǎo)的凋亡反應(yīng)并不依賴端粒酶活性的抑制。同樣,hTERT介導(dǎo)的腫瘤細(xì)胞抗凋亡作用除端粒酶激活途徑外,還存在其它非端粒酶途徑；抑制hTERT蛋白表達(dá)的同時(shí),我們還發(fā)現(xiàn),前列腺凋亡反應(yīng)因子(Par-4)表達(dá)上調(diào),且質(zhì)粒轉(zhuǎn)染前后兩種蛋白表達(dá)部位發(fā)生改變,提示Par-4可能參與hTERT不依賴其催化活性的抗凋亡途徑,hTERT可能在胞漿中與Par-4相互作用,抑制了Par-4轉(zhuǎn)入胞核,從而抑制了細(xì)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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.65

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本文編號(hào)：1816822