【作者】 刁建东；

【导师】 毕黎琦；

【作者基本信息】 吉林大学 ， 内科学， 2009， 博士

【摘要】 乳腺癌是女性最常见的恶性肿瘤之一,在一些城市已是妇女恶性肿瘤发病的首位,成为妇女健康的最大威胁。虽然阿霉素是治疗乳腺癌最有效的化疗药物之一,但大部分晚期乳腺癌患者经过联合化疗后复发率仍很高。因此,寻找解决乳腺癌耐药的新治疗方法至关重要。基因治疗是近年来肿瘤防治的重要研究领域,但其缺乏肿瘤靶向性是限制其疗效的重要原因。利用组织特异性的启动子来介导目的基因,从而限制目的基因只在靶细胞中表达,是肿瘤靶向性基因治疗的一种新的途径,有望解决基因治疗缺乏靶向性的问题。本研究目的是通过构建肿瘤特异性启动子hTERT调控的TRAIL真核表达质粒,探讨其对乳腺癌细胞特异性诱导凋亡的作用。我们构建了hTERT启动子调控的TRAIL真核表达质粒(phTERT-TRAIL-EGFP),用脂质体法把构建的phTERT-TRAIL-EGFP质粒转染乳腺癌细胞MDA-MB-435、MDA-MB-231和人胚肺成纤维细胞WI38,用MTT法检测细胞增殖变化及FCM、TUNEL染色法检测细胞凋亡,并用RT-PCR检测凋亡相关基因(cyclin E、CDK2、p27)mRNA表达变化,Western blot检测凋亡相关蛋白(TRAIL、caspase-3、NF-κB)表达变化。结果显示,重组质粒phTERT-TRAIL-EGFP和pCMV-TRAIL-EGFP抑制了乳腺癌细胞的增殖,诱导了乳腺癌细胞的凋亡,hTERT启动子与强效CMV启动子相比较对TRAIL有相同的表达调控作用,其在诱导乳腺癌细胞凋亡作用上稍优于CMV启动子。凋亡执行因子caspase3的表达上调,而与细胞存活相关的蛋白NF-κB表达则下调;调控细胞周期的cyclin E和CDK2基因表达下调,细胞周期依赖性蛋白激酶抑制因子p27基因表达明显增高。说明重组质粒在hTERT启动子和CMV启动子调控下,表达翻译了TRAIL蛋白,TRAIL通过上调p27的表达而抑制CDK2/CyclinE激酶活性,抑制了乳腺癌细胞的增殖。TRAIL诱导的信号转导系统通过形成死亡信号诱导复合物激活了caspase介导的细胞凋亡途径,抑制了细胞生存通路,最终引起了乳腺癌细胞的凋亡。pCMV-TRAIL-EGFP对人胚肺成纤维细胞的增殖有抑制作用,并能诱导其凋亡,而phTERT-TRAIL-EGFP对人胚肺成纤维细胞的增殖和凋亡没有影响,说明其具有肿瘤靶向性。本研究的创新之处在于,成功构建了hTERT启动子调控的TRAIL真核表达质粒(phTERT-TRAIL-EGFP)和转录活性强的CMV启动子调控的TRAIL真核表达质粒(pCMV-TRAIL-EGFP),并在国内首次研究了其对于乳腺癌细胞MDA-MB-435、MDA-MB-231的诱导凋亡作用,证实了hTERT启动子具有与强效CMV启动子同样的转录活性,能特异性诱导乳腺癌细胞凋亡,而对人胚肺成纤维细胞的增殖和凋亡没有影响。并研究了重组质粒抑制乳腺癌细胞增殖、诱导其凋亡的可能机制,国内外未见相关报道。本研究为乳腺癌的特异性基因治疗提供了新的思路。更多还原

【Abstract】 Breast cancer is the most common malignancy and the second cause of cancer-related deaths among women.Although doxorubicin is one of the most effective cytotoxic agents for breast cancer, most patients with advanced-stage breast cancer will recur after doxorubicin-containing chemotherapy. Therefore, it is very important to identify new strategies to overcome the intrinsic and acquired resistance to chemotherapy. The gene treatment was the recent years tumor preventing and controlling important research area, but it lacks the tumor target tropism is limits its curative effect the important reason. Starts using the tumor specificity solves this problem hopefully.The human telomerase reverse transcriptase (hTERT) promoter, whose gene is active in more than 85% of human cancers but quiescent in most somatic cells, could be used to target proapoptotic genes to cancers.The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) may induce many kinds of tumor cell to apoptosis, but has not affected to the normal cell.In this study, we have created two plasmid s that express the EGFP/TRAIL fusion gene driven by the hTERT promoter regulatory components (phTERT-TRAIL-EGFP and pCMV-TRAIL-EGFP). The plasmids pCMV- EGFP- TRAIL and phTRET-EGFP-TRAIL were transfected into the breast cancer cells MDA-MB-435, MDA-MB-231and human embryonic lung fibroblast WI38 by Lipofectamine 2000. The proliferation,apoptosis of the transfected breast cell were measured by MTT and flow cytometry. The expression of cyclin E, CDK2 and p27 were determined by RT-PCR. The expression of TRAIL ,caspase-3 and NF-κB were determined by Western blot. To investigate the effect of hTERT and CMV promoter which regulated trumor targeting TRAIL on the breast cancer line MDA-MB-435 and MDA-MB-231, but not in normal human cells.Cell viability was determined by an MTT assay: The result showed that cell-killing effects of phTERT-TRAIL-EGFP and pCMV-TRAIL-EGFP were dramatically better than those of pEGFP-C1 and PBS in MDA-MB-435 and MDA-MB-231 cells. The suppression rates of MDA-MB-231 were 36.0% and 38.2%. The suppression rates of MDA-MB-435 were 31.2% and 33.8%. The results indicate the hTERT promoter may display strongly with the CMV promoter similar expression regulation function. The cell-killing effects of pCMV-TRAIL-EGFP was dramatically better than those of pEGFP-C1 and PBS in WI38 cells. The suppression rates of WI38 cells was 36.0%. The cell-killing effects of phTERT-TRAIL was lower than pEGFP-C1 and PBS in WI38 cells. The phTERT-TRAIL-EGFP displayed cancer-specific cytotoxicity.The flow cytometry assay and TUNEL assay: To determine whether phTERT-TRAIL-EGFP and pCMV-TRAIL-EGFP caused apoptosis induction, we quantified the apoptosis in our tested cancer cells after treatment by FCM. The result showed that treatment with phTERT-TRAIL-EGFP and pCMV-TRAIL-EGFP dramatically increased the percentage of apoptotic cells in MDA-MB-435 and MDA-MB-231 cells.In the MDA-MB-231 cells,the apoptotic rate of pCMV-TRAIL was 12.03%,that of phTERT-TRAIL was 13.32%. In the MDA-MB-435 cells,the apoptotic rate of pCMV-TRAIL was 6.32%,that of phTERT-TRAIL was 10.25%.The cells treated with PBS or pEGFP-C1 had only background levels of apoptosis (0.21–1.61%).In WI38 cells, the apoptotic rate of pCMV-TRAIL was 10.46%, the apoptotic rate of phTERT-TRAIL was 4.02%, the apoptotic rates of PBS and pEGFP-C1 was 0.05% and 2.89%. TRAIL-induced apoptosis in tumor cells and the hTERT promoter could drive specific gene expression in tumor cells,but not in normal cells.To explore the molecular mechanism involved in phTERT-TRAIL mediated sensitizing for apoptosis, we investigated the protein expression of TRAIL,capspase3 and NF-κB by Western blot. In the MDA-MB-231 cells MDA-MB-435 cells,the expression of TRAIL is significantly increased after treated with phTERT- TRAIL-EGFP and pCMV-TRAIL-EGFP, whereas the expression of NF-κB is inhibited. In the WI38 cells,the same results were discovered in pCMV-TRAIL-EGFP.The results indicated TRAIL is through increases caspase3 expression induction MDA-MB-231 cells and MDA-MB-435 cells apoptosis, whereas the activeness of is reduces, suppressed it to prevent the function which TRAIL induced apoptosis.The expression of cyclin E, CDK2 and p27 were determined by RT-PCR.The results show that the mRNA expression of cyclin E and CDK2 is remarkably decreased in MDA-MB-231 and MDA-MB-435 which were transfected with pCMV-TRAIL and phTERT-TRAIL, nevertheless the expression of p27 was increased. This results proved recombinant plasmid pCMV-TRAIL and phTERT-TRAIL could increase the expression of p27 kinase activity and inhibition CDK2/CyclinE, thereby inhibiting the proliferation of breast cancer cells.In conclusion, hTERT promoter-driven tumor-specific expression of TRAIL decreased the cellular viability and induced the apoptosis of breast cancer cells MDA-MB-231 and MDA-MB-435, but not normal cells.The hTERT promoter could be demonstrated with the strong CMV promoter to express the same regulation to breast cancer cells. After the recombinant plasmid transfected breast cancer cells,with the hTERT promoter and CMV promoter regulating, the expression of the TRAIL protein is increasd, the expression of p27 kinase activity is upregulating and CDK2/CyclinE is inhibited. Eventually the proliferation of breast cancer cells is inhibited. TRAIL-induced signal transduction system through the activation of caspase, inhibited the cell survival pathway, and ultimately led to apoptosis in breast cancer cells. Together our results demonstrate that hTERT-regulated TRAIL expression is a promising strategy in gene therapy for treatment of breast cancer.更多还原