【作者】 李金华；

【导师】 李娟； 龚守良；

【作者基本信息】 吉林大学 ， 劳动卫生与环境卫生学， 2010， 硕士

【摘要】 原发性肝癌是人类最常见的恶性肿瘤之一,其发生、发展和转移是一个涉及多基因调控的复杂过程。目前,原发性肝癌的主要治疗方法包括手术切除、肝移植术、放射治疗、生物治疗、全身化学疗法、激素治疗和综合治疗等,然而这些方法的治疗效果都不理想。基因治疗作为肿瘤治疗的一种新手段,近年来在肝癌治疗方面取得了很大的进展,有望成为肝癌治疗的新途径,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,探讨其对肝癌细胞特异性诱导凋亡的作用。本研究构建了hTERT启动子调控的TRAIL真核表达质粒(pshuttle-hTERT和pshuttle-hTERT-TRAIL),脂质体转染肝癌细胞(SMMC7721)和人正常肝细胞(HL7702),检测了细胞增殖、细胞凋亡和细胞周期进程的变化,以及凋亡相关基因(TRAIL和DR5)mRNA和蛋白(TRAIL和caspase-3)的表达变化。实验结果显示,重组质粒pshuttle-TRAIL和pshuttle-hTERT-TRAIL对人肝癌细胞SMMC-7721具有增殖抑制作用和凋亡诱导作用,且hTERT启动子调控的TRAIL基因在诱导肝癌细胞凋亡作用上优于TRAIL基因单独作用;重组质粒pshuttle-TRAIL和pshuttle-hTERT-TRAIL不影响人正常肝细胞HL 7702的增殖和凋亡,说明其具有肿瘤靶向性,其研究结果为提高肿瘤基因治疗提供了新的治疗方案,为肝癌的特异性基因治疗提供了理论基础和实验依据。更多还原

【Abstract】 Study on effects of hTERT promoter regulated tumor targeting TRAIL on hepatocarcinoma cellsPrimary hepatic carcinoma (PHC) is one of the most common malignant tumors among human beings, its genesis, development and metastasis is a complicated process involved in multiple genes regulation. The primary ways for PHC therapy include surgical resection, radiotherapy, chemotherapy, and so on. As a new means of oncotherapy, the gene treatment has achieved the advancement in hepatoma at the recent years, but lacking the tumor target tropism is the important reason of limiting its curative effect. Human telmoerase reserse transcriptase (hTERT) promoter belongs to a specific promoter of tumor cells, and it could kill telomerase positive tumor cells selectively and has no effect on telomerase negative normal cells. The tumor necrosis factor-related apopto sis-indue ing ligand (TRAIL) may only induce the apoptosis in many kinds of tumor cells, transformant cells and virus infected cells, while exhibit little or no toxicity in normal cells. These results suggest that TRAIL has very strong potential effect for anticancer therapy. In this study, hTERT promoter regulated tumor targeting TRAIL expression plasmid was constructed, which induces the apoptosis in hepatoma carcinoma cells was explored for developing a new way of cancer therapy.1 Construction and identification of recombinant plasmid1.1 Acquisition of hTERT promoter and TRAILThe specific PCR primers were designed and synthesized according to the sequence of hTERT promoter gene which was cloned and amplified from cDNA of 293T cells as the template. TRAIL gene was cloned and amplified from cDNA of placent as the template, and the specific PCR primers were designed and synthesized according to the sequence of TRAIL gene. The two genes were identified by cleavage of endonucleases and sequencing process. The results of identification confirmed that the sequence of the cloned genes were identical to that published on Genbank.1.2 Construction of recombinant plasmidThe hTERT promoter and TRAIL genes were ligated to pshuttle vector to construct the single-gene recombinant plasmids, pshuttle-TRAIL and pshuttle-hTERT-TRAIL with the technique of genetic engineering. The two recombinant plasmids were identified by cleavage of endonucleases and PCR, and confirmed that the two recombinant plasmids were correct.2 Experimental grouping and index detectionHuman hepatoma carcinoma cell (SMMC7721) and human normal hepatocyte (HL7702) were used. The experiment was divided into four groups which were the control, pshuttle, pshuttle-TRAIL and pshuttle-hTERT-TRAIL groups. In the time-course experiment, the selected time points were 4, 8, 16, 24 and 48 h. Real-time PCR was used to detect the expression of TRAIL and DR5 mRNA, ELISA and spectrophotometry were used to detecte the expressions of TRAIL protein and caspase-3 protein levels respectively. MTT assay was used to detect the cell proliferation, and flow cytometry to cell cycle and apoptosis.3 Effects on biological behaviour of recombinant plasmids in SMMC7721 and HL7702 cellsThe cell proliferation of hepatocarcinoma cells (P < 0.01 ~ P < 0.001) from 16 h after transfection in the recombinant plasmid groups (pshuttle-TRAIL and pshuttle-hTERT-TRAIL) was inhibited significantly, and the growth rates of the SMMC7721 cells transfected with pshuttle-hTERT-TRAIL declined significantly as compared with those in the pshuttle-TRAIL group from 24 h after transfection (P < 0.05). As compared with that in the control group, the SMMC7721 cell apoptotic percentages in the pshuttle-TRAIL and pshuttle-hTERT-TRAIL groups increased significantly, especially that in the pshuttle-hTERT-TRAIL group was significantly higher than that in the pshuttle-TRAIL group (P < 0.05, P < 0.001) 8 and 24 h after transfection. The percentage of each phase cells in cell cycle was detected by FCM. The results showed that as compared with that in the control and pshuttle groups respectively, the percentage of S phase cells increased significantly in pshuttle-TRAIL and pshuttle-hTERT-TRAIL groups (P < 0.05, P < 0.001) 8 h after transfection, and as compared with that in the pshuttle-TRAIL group, the apoptotic percentage in the pshuttle-hTERT-TRAIL group increased significantly (P < 0.01). The percentage of G2/M phase cells increased significantly in the pshuttle-hTERT-TRAIL group 24 h after transfection as compared with that in the control and pshuttle groups respectively (P < 0.01), and as compared with that in the pshuttle-TRAIL group, the apoptotic percentage in the pshuttle-hTERT-TRAIL group increased significantly (P< 0.001).As compared with that in the control group, HL7702 cell proliferation in pshuttle and recombinant plasmids groups did not change significantly, the percentages of apoptotic and each phase cells 8 and 24 h after transfection also did not.Above-mentioned results showed that the hTERT promoter regulated tumor targeting TRAIL expression plasmid could inhibit cell proliferation, promote cell apoptosis, induce S phase delay and the G2 phase arrest in SMMC7721 cells, especially superior to that of pshuttle-TRAIL. Furthermore, it showed no effects on cell proliferation, apoptosis and cell cycle progression in HL7702 cells.4 Mechanism of apoptosis induced by recombinant plasmids in SMMC7721 and HL7702 cells4.1 Effects of recombinant plasmids on protein expression in SMMC7721 and HL7702 cellsELISA was used to detected TRAIL protein expression and spectrophotometry was used to detect caspase-3 protein expression. The results showed that TRAIL protein expression of recombinant palsmids in SMMC7721 cells increased. The TRAIL protein expression in pshuttle-TRAIL reached the peak value 4 h after transfection, while that in pshuttle-hTERT-TRAIL reached the peak value at 8 h. As compared with that in the pshuttle-TRAIL group, the TRAIL protein expression in the pshuttle-hTERT-TRAIL group 8, 16 and 24 h increased significantly (P < 0.01). Caspase-3 protein expression in pshuttle-TRAIL and pshuttle-hTERT-TRAIL groups 8 and 24 h after transfection increased significantly as compared with that of the control group (P < 0.001), especially that in pshuttle-hTERT-TRAIL group was significantly higher than that in pshuttle-TRAIL group (P < 0.01, P < 0.001).The TRAIL protein expression showed significantly no difference in recombinant plasmid groups as compared with the control group at different times after transfection in HL7702 cells. And the caspase-3 protein expression was no difference in recombinant plasmid group as compared with the control group 8 and 24 h after transfection.Above-mentioned results showed that the hTERT promoter regulated tumor targeting TRAIL expression plasmid could increase the TRAIL and caspase-3 protein expressions in SMMC7721 cells, especially superior to that in pshuttle-TRAIL, and did no effects on the TRAIL and caspase-3 protein expressions in HL7702 cells. These results suggest that the apoptosis in hepatoma carcinoma cells induced by hTERT promoter-driven TRAIL is related to the activation of caspase-3 medicated apoptosis pathway.4.2 Effects of recombinant plasmids on gene mRNA expression of in SMMC7721 and HL7702 cellsReal-time PCR was used to detect the TRAIL and DR5 mRNA expressions. The TRAIL mRNA expression increased significantly in pshuttle-TRAIL group 4, 8, 16 and 48 h after transfection in SMMC-7721 cells as compared with that in the control and pshuttle groups (P < 0.01, P < 0.001). The TRAIL mRNA expression in pshuttle-hTERT-TRAIL group reached the peak 8 h after transfection, and showed significant increase at different times after transfection as compared with that in other 3 groups (P < 0.001). The DR5 mRNA expression increased significantly in pshuttle-TRAIL group as compared with that in the control and pshuttle groups 16 h after transfection in SMMC-7721 cells (P < 0.05). And the DR5 mRNA expression increased significantly in pshuttle-hTERT-TRAIL group 16, 24 and 48 h after transfection as compared with that of the other 3 groups (P < 0.01, P < 0.001). The TRAIL and DR5 mRNA expression showed no difference in recombinant plasmid groups as compared with that in the control group at different times after transfection in HL7702 cells.Above-mentioned results showed that the hTERT promoter regulated tumor targeting TRAIL expression plasmid could increase the TRAIL and DR5 mRNA expressions in SMMC7721 cells, especially superior to that of pshuttle-TRAIL, and showed no effects on the TRAIL and DR5 mRNA expressions in HL7702 cells. These results exhibited that under the regulation of hTERT promoter, TRAIL triggers the external pathway of cell apoptosis through the interaction with DR5.In summary, hTERT promoter-driven TRAIL (pshuttle-hTERT-TRAIL) could inhibit cell proliferation and promote cell apoptosis in SMMC7721 cells, especially superior to that in pshuttle-TRAIL. However, it showed no effects on cell proliferation and apoptosis in HL7702 cells. Recombinant plasmid pshuttle-hTERT-TRAIL specifically expressed TRAIL in the tumor cells and led to a cancer specific cytotoxic effect. The cell apoptotic mechanism involved in the process of tumor cell death. Under the regulation of hTERT promoter, TRAIL exerts its function by binding with its receptors-DR5, then expresses and translates TRAIL protein, and consequently inhibits cell proliferation. TRAIL activates signal transduction system, which activates the key downstream effectors of apoptosis such as caspase-3, finally induces apoptosis in hepatoma carcinoma cells. Taken together, this research suggests that hTERT promoter-driven TRAIL could be a new way for hepatoma therapy, and provides original idea and experimental base for targeted gene therapy.更多还原