hTERT启动子调控的TRAIL基因对肝癌细胞作用的实验研究
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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。
Study on effects of hTERT promoter regulated tumor targeting TRAIL on hepatocarcinoma cells
Primary 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 plasmid
1.1 Acquisition of hTERT promoter and TRAIL
The 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 plasmid
The 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 detection
Human 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 cells
The 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 cells
4.1 Effects of recombinant plasmids on protein expression in SMMC7721 and HL7702 cells
ELISA 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 cells
Real-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.
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。
Study on effects of hTERT promoter regulated tumor targeting TRAIL on hepatocarcinoma cells
Primary 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 plasmid
1.1 Acquisition of hTERT promoter and TRAIL
The 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 plasmid
The 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 detection
Human 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 cells
The 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 cells
4.1 Effects of recombinant plasmids on protein expression in SMMC7721 and HL7702 cells
ELISA 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 cells
Real-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.
引文
[1] http://www.e79.com.cn/xwzx/zlhy/gjhy/20090212/6961.htrnl.
[2] Barnes MN, Deshane JS, Rosenfeld M, et al. Gene therapy and ovarian cancer: a review [J].Obstet Cynecol, 1997, 89(1): 145-155.
[3] Anderson WF, Blaese RM, Culver K. The ADA human gene therapy clinical protocol: pointsto consider response with clinical protocol [J]. Hum Gene Ther, 1990, l(3):331-362.
[4] Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, et al. Gene therapy of human severecombined immunodeficiency (SCID)-Xl disease [J]. Science, 2000, 288(5466):669-672.
[5] http://www.wiley.co.uk/genmed/clinical.
[6] Vattemi E, Claudio PP. Tumor suppressor genes as cancer therapeutics [J]. Drug News Perspect, 2007, 20(8):511-520.
[7] Rudin CM, Marshall JL, Huang CH, et al. Delivery of a liposomal c-raf-1 antisense oligonucleotide by weekly bolus dosing in patients with advanced solid tumors: a phase I study [J]. Clm Cancer Res, 2004, 10(21):7244-7251.
[8] Moreira J N, Santos A, Simoes S. Bcl-2-targeted antisense therapy (Oblimersen sodium): towards clinical reality [J]. Rev Recent Clin Trials, 2006, l(3):217-235.
[9] Isayeva T, Kumar S, Ponnazhagan S. Anti-angiogenic gene therapy for cancer (review) [J]. Int J Oncol, 2004, 25(2):335-343.
[10] Sawicki JA, Anderson DG, Langer R. Nanoparticle delivery of suicide DNA for epithelial ovarian cancer therapy [J]. Adv Exp MedBiol, 2008, 622:209-219.
[11] Loisel-Meyer S, Foley R, Medin JA. Immuno-gene therapy approaches for cancer: from in vitro studies to clinical trials [J]. Front Biosci, 2008, 13:3202-3214.
[12] Wirth T, Kuhnel F, Kubicka S. Telomerase-dependent gene therapy [J]. Curr Mol Med, 2005, 5(2): 243-251.
[13] Labialle S, Dayan G, Michaud M, et al. Gene therapy of the typical multidrug resistance phenotype of cancers: a new hope [J]. Semin Oncol, 2005, 32(6):583-590.
[14]张夏华，吴广通，李蓉.基因治疗现状与前景[J].药学实践杂志, 2009, 27(1):4-10.
[15] Pan D. In Situ ( In Vivo) Gene Transfer intoMurine BoneMarrow Stem Cells [J]. MethodsM0IB10I, 2009, 506:159-169.
[16] Li Z, Dullmann J, Schiedlmeier B, et al. Murine leukemia induced by retroviral gene marking [J]. Science, 2002, 296(5567):497-499.
[17] Marshall E. Gene therapy a suspect in leukemia-like disease [J]. Science, 2002, 298(5591): 34-35.
[18]陶铭.基因治疗及其研究[J].生物学通报, 2008, 43(4):16-18.
[19] Weber W, Fussenegger M. Novel gene switches [J]. Handb Exp Pharmacol, 2007,(178):73-105.
[20] Nemunaitis JM, Nemunaitis J. Potential of Advexin: a p53 gene-replacement therapy inLi-Fraumeni syndrome. Future Oncol, 2008, 4(6):759-768.
[21] Huang CL, Yokomise H, Miyatake A. Clinical significance of the p53 pathway andassociated gene therapy in non-small cell lung cancers. Future Oncol, 2007, 3(1):83-93.
[22]穆红,王玉亮,刘蓉.野生型p53基因在人肝癌细胞的表达及诱导其凋亡[J].细胞与分子免疫学杂志, 2006, 22(6):758-759.
[23] Wang XW, Yuan JH, Zhang RG, et al. Antihepatoma effect of alphafetop rotein antisensephosphorothioate oligodeoxyribonuc leotides in vitro and in mice [J]. World J Gastroenterol,2001, 7(3):345-351.
[24] Kim D, Rossi J. RNAi mechanisms and applications. Biotechniques, 2008, 44(5):613-616.
[25] Pushparaj PN, Aarthi JJ, Mamkandan J, et al. siRNA, miRNA, and shRNA: in vivo applications [J]. J Dent Res, 2008, 87(ll):992-1003.
[26]李华,王欣璐,杨扬,等. RNAi靶向人端粒酶逆转录酶对人肝癌细胞的生长抑制作用[J].南方医科大学学报, 2008, 28(8):1323-132 .
[27] Qiao J, Doubrovin M, Sauter BV, et al. Tumor-specific transcriptional targeting of suicidegene therapy [J]. Gene Ther, 2002, 9(3): 168-175.
[28]薛刚,程莹,曹永宽,等. IFNγ基因修饰的树突状细胞在肝癌免疫治疗中的应用[J].世界华人消化杂志, 2008, 16(25):2820-2825.
[29] Douglas JT. Cancer gene therapy [J]. Technol Cancer Res TREAT, 2003, 2(l):51-64.
[30] Xiao H, Wu Z, Shen H, et al. In vivo reversal of P-glycoprotein-mediated multidrugresistance by efficient delivery of stealth RNAi [J]. Basic Clin Pharmacol Toxicol, 2008,103(4):342-348.
[31] Wiley SR , Schooley K, Smolak PJ, et al . Identification and characterization of a newmember of TNF family that induce apoptosis [J] . Immunity, 1995, 3(6):673-682.
[32] Pitti RM, Marsters SA, Ruppert S, et al. Induction of apoptosis by Apo2 ligand, a newmember of the tumor necrosis factor cytokine family [J]. J Biol Chem, 1996,271(22):12687-126901.
[33] Walczak H, Miller RE, Ariail K, et al. Tumoricidal activity of tumor necrosis factor relatedapoptosis inducing ligand in vivo [J]. NatMed, 1999, 5(2):157-163.
[34] Gores GJ, Kaufmann SH. Is TRAIL hepatotoxic? [J]. Hepatology, 2001, 34(l):3-6.
[35] Wang P, Song JH, Song DK, et al. Role of death receptor and mitochondrial pathways inconventional chemotherapy drug induction of apoptosis [J]. Cell Signal, 2006,18(9):1528-1535.
[36] Song JH, Wang CX, Song DK, et al. Interferon gamma induces neurite outgrowth byup-regulation of p35 neuron-specific cyclin-dependent kinase 5 activator via activation ofERK1/2 pathway [J]. J Biol Chem, 2005, 280(13):12896-12901.
[37]沈滟,赵大治. TRAIL系统与肿瘤细胞的凋亡[J].河南科技大学学报(医学版), 2006, 24(4): 318-320.
[38] Kagawa S, He C, Gu J, et al. Antitumor activity and bystander effects of tumor necrosisfactor-related apotos is-indue ing ligand (TRAIL) gene [J]. Cancer Res, 2001,61(8):3330-3338.
[39] Lu x, Arbiser JL, West J, et al. Tumor necrosis factor-related apoptos is-indue ing ligand caninduce apoptosis in subsets of premaligant cells [J]. Am J Pathol, 2004, 165(5):1613-1620.
[40] Houdy, Yan Z, Han W. Cloning and characterization of TRAIL cDNA [J]. Chin J Biol ChemMol Bid, 2002, 18(1):27-31.
[41] Spierings DC, de veries EG, Vellenga E, et al. Tissue distribution of the death ligand TRAILand its receptors [J]. Histochem, 2004, 52(6):821-831.
[42] Hymowitz, SG, O'Connell MR Ultsch MH, et al. A unique zinc-binding site revealed by ahigh-resolution X-ray structure of homotrimeric Apo2L/TRAIL [J]. Biochemistry, 2000,39(4): 633-640.
[43] Bodmer JL, Meier P, Tschopp J, et al. Cysteine 230 is essential for the structure and activityof the cytotoxic ligand TRAIL [J]. J Biol Chem, 2000, 27(27):20632-20637.
[44] Kim Y, Seol DW. TRAIL, a mighty apoptosis inducer [J]. Molecules and cells, 2003,15:283-293
[45]马远方. TRAIL及其受体的研究进展[J].河南大学学报, 2007, 26(2):1-5.
[46] Truneh A, Sharma S, Silverman C, et al. Temperature sensitive differential affinity of TRAILfor its receptors DR5 is the highest affinity receptor [J]. J Biol Chem, 2000,275(30):23319-23325.
[47] Degli-esposti MA, Smolak PJ, Walczak H, et al. Cloning and characterization of TRAIL-R3,a novel member of the emerging TRAIL receptor family [J]. J Exp Med, 1997,186(7):1165-1170.
[48] Pan H, Rourke K, Chinnaiyan AM, et al. The receptor for the cytotoxic ligand TRAIL [J].Science, 1997, 276(5309): 111-113.
[49] Simonet WS, Lacey DL, Dunstan CR, et al. Osteoprotegerin: a novel secreted proteininvolved in the regulation of bone density [J]. Cell, 1997, 89(2):309-319.
[50] Holen I, Croucher PI, Hamdy FC, et al. Osteoprotegerin (OPG) is a survival factor for humanprostate cancer cells [J]. Cancer Res, 2002, 62(6):1619-1623.
[51] Shipman CM, Croucher PI. Osteoprotegerin is a soluble decoy receptor for tumor necrosisfactor-related apoptos is-indue ing ligand/Apo2 ligand and can function as a paracrine survivalfactor for human myeloma cells [J]. Cancer Res, 2003, 63(5):912-916.
[52] Medema JP, Scaffidi C, Kischkel FC. FLICE is activated by association with the CD95death-inducing signaling complex(DISC) [J]. EMBO J, 1997, 16(10):2794-804.
[53] Muzio M, Stockwell BR, Stennicke HR. An induced proximity model for Caspase-8activation [J]. J Biol Chem, 1998, 273(5):2926-2930.
[54] Sheridan JP, Marsters SA, Piti RM, et al. Control of TRAIL-induced apoptosis by a family ofsignaling and decoy receptors [J]. Science, 1997, 277: 818-825.
[55] Walczak H, Krammer PH. The CD95 (APO-IfFas) and the TRAIL(AP0-2L) apoptosissystems [J]. Exp Cell Res, 2000, 256(l):58-66.
[56] MacFarlane M. TRAIL-mduced signalling and apoptosis [J]. Toxicol Lett. 2003, 139(2-3):89-97.
[57] Green DR, Kroemer G. The pathophysiology of mitochondrial cell death [J]. Science, 2004,305(5684):626-629.
[58] Barczyk K, Kreuter M, Pryjma J, et al. Serum cytochrome c indicates in vivo apoptosis andcan serve as a prognostic marker during cancer therapy [J]. Int J Cancer, 2005,116(2):167-173.
[59] Jiang X, Wang X. Cytochrome c promotes Caspase-9 activation by inducing nucleotidebinding to Apaf-1 [J]. JBiol Chem, 2000, 275(40):31199-311203.
[60] Deveraux QL, Reed JC. IAP family proteins-suppressors of apoptosis [J]. Genes Dev, 1999,13(3): 239-252.
[61] Du C, Fang M, Li Y, et al. Smac, a mitochondrial protein that promotes cytochromec-dependent Caspase activation by eliminating IAP inhibition [J]. Cell, 2000, 102(l):33-42.
[62] Verhagen AM, Ekert PG, Pakusch M. Identification of DIABLO, a mammalian protein thatpromotes apoptosis by binding to and antagonizing IAP proteins [J]. Cell, 2000,102(l):43-53.
[63] Li S, Zhao Y, He X, et al. Relief of extrinsic pathway inhibition by the Bid-dependentmitochondrial release of Smac in Fas-mediated hepatocyte apoptosis [J]. J Biol Chem, 2002,277(30):26912-26920.
[64] Hu WH, Johnson H, Shu HB. Tumor necrosis factor-related apoptosis-indue ing ligandreceptors signal NF-kappaB and JNK activation and apoptosis through distinct pathways [J].JBiol Chem, 1999, 274(43):30603-30610.
[65] Wajant H, Haas E, Schwenzer R, et al. Inhibition of death receptor-mediated gene inductionby a cycloheximide-sensitive factor occurs at the level of or upstream of Fas-associated deathdomain protein (FADD) [J]. J Biol Chem, 2000, 275(32):24357-24366.
[66] MacFarlane M, Cohen GM, Dickens M. JNK(c-Jun N-terminal kinase) and p38 activation inreceptor-mediated and chemically-induced apoptosis of T-cells: differential requirements forcaspase activation [J]. Biochem J, 2000, 348(l):93-101.
[67] He SQ, Chen XP, Zhao YZ, et al. Expression profiles of TRAIL receptors and their clinicalsignificance in human hepatocellular Carcinoma [J]. Chinese-German J Clin Oncol, 2003,2(1): 25-29.
[68] Walczak H, Miller RE, Ariail K, et al. Tumoricidal activity of tumor necrosis factor-relatedapoptosis-indue ing ligand in vivo [J]. Nat Med, 1999, 5(2): 157-163.
[69] Ashkenazi A, Pai RC, Fong S, et al. Safety and antitumor activity of recombinant solubleApo2 ligand [J]. J Clin Invest, 1999, 104(2):155-162.
[70] Wajant H, Pfizenmaier K, Scheurich P. TNF-related apoptosis inducing ligand (TRAIL) andits receptors in tumor surveillance and cancer therapy [J]. Apoptosis, 2002, 7(5):449-459.
[71] Naka T, Sugamura K, Hylander BL, et al. Effects of tumor necrosis factor-relatedapoptosis-indue ing ligand alone and in combination with chemo therapeutic agents onpatients' colon tumors grown in SCID mice [J]. Cancer Res, 2002, 62(20):5800-5806.
[72] Jo M, Kim TH, Seol DW, et al. Apoptosis induced in normal human hepatocytes by tumornecrosis factor-related apoptosis-indue ing ligand [J]. Nat Med, 2000, 6(5):564-567.
[73] Nitsch R, Bechmann 1, Deisz RA, et al. Human brain-cell death induced by turn our-necrosis-factor-related apoptosis-mducmg ligand (TRAIL) [J]. Lancet, 2000, 356(9232):827-828.
[74] Verhagen AM, Ekert PG, Pakusch M. Identification of DIABLO, a mammalian protein thatpromotes apoptosis by binding to and antagonizing IAP proteins [J]. Cell, 2000,102(l):43-53.
[75] Gilley D, Tanaka H, Herbert BS. Telomere dysfunction in aging and cancer [J]. Int J BiochemCellBiol, 2005, 37(5):1000-1013.
[76] Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activitywith immortal cells and cancer [J]. Science, 1994, 266(5193):2011-2015.
[77] Cech TR. Beginning to understand the end of the chromosome [J]. Cell, 2004, 116(2):273-279.
[78] Chen Z, Koeneman KS, Corey DR. Consequences of telomerase inhibition and combinationtreatments for the proliferation of cancer cells [J]. Cancer Res, 2003, 63(18):5917-5925.
[79] Wong JM, Kusdra L, Collins K. Subnuclear shuttling of human telomerase induced bytransformation and DNA damage [J]. Nat Cell Biol, 2002, 4(9):731-736.
[80] Shammas MA, Koley H, Batchu RB, et al. Telomerase inhibition by siRNA causessenescence and apoptosis in Barrett's adenocarcinoma cells: mechanism and therapeuticpotential [J]. Mol Cancer, 2005, 15(4):24-38.
[81] Hahn WC, Stewart SA, Brooks MW, et al. Inhibition of telomerase limits the growth ofhuman cancer cells [J]. Nat Med, 1999, 5(10): 1164-1170.
[82] Fujiwara T, Tanaka N. Telomerase-specific oncolytic virotherapy for human cancer with thehTERT promoter [J]. Uirusu, 2008, 58(1):11-18.
[83] Wang Y, Huang F, Cai H, et al. Potent antitumor effect of TRAIL mediated by a noveladeno-associated viral vector targeting to telomerase activity for human hepatocellularcarcinoma [J]. Gene Med, 2008, 10(5):518-526.
[84] Li HM, Yu YC, Song TB, et al. Expression of TRAIL gene eukaryotic expression vectormodulated by the human telomerase reverse transcriptase gene core promoter in ovariancancer cell line SKOV3 [J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2007, 23(12): 1106-1109.
[85] Su T, Sun HC, Zheng CY, et al. Targeting transgene effect of adTERT-TRAIL on salivaryadenoid cystic carcinoma cell line SACC-83 [J]. Hua Xi Kou Qiang Yi Xue Za Zhi, 2007,25(2): 202-205.
[86] Chen B, He C, Lao WF, et al. Activity of the TNF-related apoptos is-indue ing ligand geneexpressed from the hTERT promoter on colon cancer cell line HT-29 [J]. Zhejiang Da XueXue Bao Yi Xue Ban, 2006, 35(l):45-49.
[87] Bilsland AE, Anderson CJ, Fletcher-Monaghan AJ, et al. Selective ablation of human cancercells by telomerase-specific adenoviral suicide gene therapy vectors expressing bacterialmtroreductase [J]. Oncogene, 2003, 22(3):370-380.
[88] Gu J, Zhang LD, Huang XF, et al. A novel single tetracycline-regulative adenoviral vector fortumor-specific Bax gene expression and cell killing in vitro and in vivo [J]. Oncogene, 2002,21:4757-4764.
[89] Painter RG, Lanson NA, Jin Z, et al. Conditional expression of a suicide gene by the telomerereverse transcriptase promoter for potential post-therapeutic deletion of tumorigenesis [J].Cancer Sci, 2005, 96(9):607-613.
[90] Yang CQ, Deng ZH, Liu Y, el al. Asialofetum-hTERT-TK/GCV targeted gene therapy and itsbystander effect on HepG2 [J]. Zhonghua Gan Zang Bing Za Zhi, 2008, 16(7):509-513.
[91] Doloff JC, Waxman DJ, Jounaidi Y. hTERT-promoter driven oncolytic adenovirus withE1B-19 kDa and E1B-55 kDa gene deletions [J]. Hum Gene Ther, 2008, 19(12): 1383-1400.
[92] Zhao HC, Zhang Q, Yang Y, et al. p53-expressing conditionally replicative adenovirusCNHK500-p53 against hepatocellular carcinoma in vitro [J]. World J Gastroenterol, 2007,13(5): 683-691.
[93] Li GC, Yang JM, Nie MM, et al. Potent antitumoral effects of a novel gene-viral therapeuticsystem CNHK300-mEndostatin in hepatocellular carcinoma [J]. Chin Med J(Engl), 2005,118(3):179-185.
[94]邓文星,张映.实时荧光定量PCR技术综述[J].生物技术通报, 2007, (5): 93-95.
[95] Tominaga H, IshiyamaM, Ohseto F, et al. A water-soluble tetrazolium salt useful forcolorimetric cell viability assay [J]. Anal Commun, 1999, 36(l):47-50.
[96] Cory AH, Owen TC, Barltrop JA, et al. Use of an aqueous soluble tetrazolium/formazanassay for cell growth assays in culture [J]. Cancer Commun, 1991, 3(7):207-212.
[97] Berridge MV, Tan AS, McCoy KD, et al. The biochemical and cellular basis of cellproliferation assays that use terazolium salts [J]. Biochemica, 1996, 4:15-19.
[98] Moamman T. Rapid colorimetric assay for celluar growth and survial:appication toproliferation cytotoxicity assays [J]. J Immunol Methods, 1983, 65(l-2):55-63.
[99] Denizot F, Lang R. Rapid colorimetric assay for cell growth and surrvial: Modifications tothe tetrazolium dye procedure giving improved sensitivity and reliability [J]. J ImmunolMethods, 1986,89(2):271-277.
[100] Twentyman PR, Luscombe M. A study of some variables in a tetrazolium dye (MTT) bassedassay for cell growth and chemosensitxity [J]. BR J Cancer, 1987, 56(3):279-25.
[101] Meier P, Finch A, Evan G Apoptosis in development. Nature, 2000, 407:796-801
[102]刘建越,邓欣,康林,等. SYBR-Green实时荧光PCR检测转基因番木瓜[J].湖南农业大学学报(自然科学版), 2006, 32(4):371-374.
[103] Morrison TB, Weis JJ, Wittwer CT. Quantification of low-copy transcripts by continuousSYBR-Green monitoring during amplification [J]. Biotechniques, 1998, 24(6):954-962.
[104] Maes L, Van Neste L, Van Damme K, et al. Relation between telomerase activity, hTERTand telomere length for intracranial tumours [J]. Oncol Rep, 2007, 18(6): 1571-1576.
[105] Nishio Y, Nakanishi K, Ozeki Y, et al. Telomere length, telomerase activity, and expressionsof human telomerase mRNA component(hTERC)and human telomerase reversetranscriptase (hTERT) mRNA in pulmonary neuro endocrine tumors [J]. Jpn J Clin Oncol,2007, 37(l):16-22.
[106] Murofushi Y, Nagano S, Kamizono J, et al. Cell cycle-specific changes in hTERT promoteractivity in normal and cancerous cells in adenoviral gene therapy: a promising implicationof telomerase-dependent targeted cancer gene therapy [J]. Int J Oncol, 2006, 29(3):681-688.
[107]宋悦,孔北华,马道新,等.宫颈癌及子宫内膜癌细胞系中启动子活性的研究[J].肿瘤防治杂志, 2003, 10(3):442-443.
[108] Niidome T, Huang L. Gene therapy progress and prospects: nonviral vectors [J]. Gene Ther, 2002, 9(24): 1647-1652.
[109] Ganten TM, Koschny R, Sykora J, et al. Preclinical differentiation between apparently safeand potentially hepatotoxic applications of TRAIL either alone or incombination withchemotherapeutic drugs [J]. Clin Cancer Res, 2006, 12(8):2640-2646.
[110]刘永靖,陈飞虎,于奇,等.腺病毒载体携带人TRAIL基因治疗H446小细胞肺癌的体外实验研究[J].中国药理学通报, 2007, 23(6):755-759.
[111] Kagawa S, He C, Gu J, et al. Antitumor activity and bystander effects of the tumor necrosisfactor-related apoptosis-indue ing ligand (TRAIL) gene [ J]. Cancer Res, 2001,61:3330-3338.
[112] Alao Y, Yamada H, Nakagawa Y. Arsenic-induced apoptosis in malignant cells in vitro.Leuk Lymphoma, 2000, 37:53-63.
[113] Anuradha CD, Kanno S, Hirano S. Oxidative damage of mitochondria is a preliminary stepto caspase3 activation in fluoride-induced apoptosis in HL260 cells. Free Radic Biol Med,2001 ,31:367-373.
[114]李宁丽,路丽明,沈佰华,等.重组人可溶性分子诱导细胞凋亡机理探讨[J].上海第二医科大学学报, 2003, 23(1):10-14.
[115]尹伶,濮德敏,韦立蓓,等. Caspase3在NS-398诱导宫颈癌Hela细胞凋亡中的作用[J].中国肿瘤临床, 2007, 34(12):711-713.
[116] Yan-Guo Ren, Klaus W, Wagner, et al. Differential regulation of the TRAIL death receptors DR4 and DR5 by the signal recognition particle [J]. Mol Biol Cell, 2004, 15(11):5064-5074.
[117] Peter H, Dietmar P, Alexandra K, et al. Perturbation of the tumor necrosis factor-related apoptos is-indue ing ligand cascade in ovarian cancer: over expression of FLIPL and deregulation of the functional receptors DR4 and DR5. Clin Cancer Res, 2005, 11(24 Pt 1): 8585-8591.
[2] Barnes MN, Deshane JS, Rosenfeld M, et al. Gene therapy and ovarian cancer: a review [J].Obstet Cynecol, 1997, 89(1): 145-155.
[3] Anderson WF, Blaese RM, Culver K. The ADA human gene therapy clinical protocol: pointsto consider response with clinical protocol [J]. Hum Gene Ther, 1990, l(3):331-362.
[4] Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, et al. Gene therapy of human severecombined immunodeficiency (SCID)-Xl disease [J]. Science, 2000, 288(5466):669-672.
[5] http://www.wiley.co.uk/genmed/clinical.
[6] Vattemi E, Claudio PP. Tumor suppressor genes as cancer therapeutics [J]. Drug News Perspect, 2007, 20(8):511-520.
[7] Rudin CM, Marshall JL, Huang CH, et al. Delivery of a liposomal c-raf-1 antisense oligonucleotide by weekly bolus dosing in patients with advanced solid tumors: a phase I study [J]. Clm Cancer Res, 2004, 10(21):7244-7251.
[8] Moreira J N, Santos A, Simoes S. Bcl-2-targeted antisense therapy (Oblimersen sodium): towards clinical reality [J]. Rev Recent Clin Trials, 2006, l(3):217-235.
[9] Isayeva T, Kumar S, Ponnazhagan S. Anti-angiogenic gene therapy for cancer (review) [J]. Int J Oncol, 2004, 25(2):335-343.
[10] Sawicki JA, Anderson DG, Langer R. Nanoparticle delivery of suicide DNA for epithelial ovarian cancer therapy [J]. Adv Exp MedBiol, 2008, 622:209-219.
[11] Loisel-Meyer S, Foley R, Medin JA. Immuno-gene therapy approaches for cancer: from in vitro studies to clinical trials [J]. Front Biosci, 2008, 13:3202-3214.
[12] Wirth T, Kuhnel F, Kubicka S. Telomerase-dependent gene therapy [J]. Curr Mol Med, 2005, 5(2): 243-251.
[13] Labialle S, Dayan G, Michaud M, et al. Gene therapy of the typical multidrug resistance phenotype of cancers: a new hope [J]. Semin Oncol, 2005, 32(6):583-590.
[14]张夏华，吴广通，李蓉.基因治疗现状与前景[J].药学实践杂志, 2009, 27(1):4-10.
[15] Pan D. In Situ ( In Vivo) Gene Transfer intoMurine BoneMarrow Stem Cells [J]. MethodsM0IB10I, 2009, 506:159-169.
[16] Li Z, Dullmann J, Schiedlmeier B, et al. Murine leukemia induced by retroviral gene marking [J]. Science, 2002, 296(5567):497-499.
[17] Marshall E. Gene therapy a suspect in leukemia-like disease [J]. Science, 2002, 298(5591): 34-35.
[18]陶铭.基因治疗及其研究[J].生物学通报, 2008, 43(4):16-18.
[19] Weber W, Fussenegger M. Novel gene switches [J]. Handb Exp Pharmacol, 2007,(178):73-105.
[20] Nemunaitis JM, Nemunaitis J. Potential of Advexin: a p53 gene-replacement therapy inLi-Fraumeni syndrome. Future Oncol, 2008, 4(6):759-768.
[21] Huang CL, Yokomise H, Miyatake A. Clinical significance of the p53 pathway andassociated gene therapy in non-small cell lung cancers. Future Oncol, 2007, 3(1):83-93.
[22]穆红,王玉亮,刘蓉.野生型p53基因在人肝癌细胞的表达及诱导其凋亡[J].细胞与分子免疫学杂志, 2006, 22(6):758-759.
[23] Wang XW, Yuan JH, Zhang RG, et al. Antihepatoma effect of alphafetop rotein antisensephosphorothioate oligodeoxyribonuc leotides in vitro and in mice [J]. World J Gastroenterol,2001, 7(3):345-351.
[24] Kim D, Rossi J. RNAi mechanisms and applications. Biotechniques, 2008, 44(5):613-616.
[25] Pushparaj PN, Aarthi JJ, Mamkandan J, et al. siRNA, miRNA, and shRNA: in vivo applications [J]. J Dent Res, 2008, 87(ll):992-1003.
[26]李华,王欣璐,杨扬,等. RNAi靶向人端粒酶逆转录酶对人肝癌细胞的生长抑制作用[J].南方医科大学学报, 2008, 28(8):1323-132 .
[27] Qiao J, Doubrovin M, Sauter BV, et al. Tumor-specific transcriptional targeting of suicidegene therapy [J]. Gene Ther, 2002, 9(3): 168-175.
[28]薛刚,程莹,曹永宽,等. IFNγ基因修饰的树突状细胞在肝癌免疫治疗中的应用[J].世界华人消化杂志, 2008, 16(25):2820-2825.
[29] Douglas JT. Cancer gene therapy [J]. Technol Cancer Res TREAT, 2003, 2(l):51-64.
[30] Xiao H, Wu Z, Shen H, et al. In vivo reversal of P-glycoprotein-mediated multidrugresistance by efficient delivery of stealth RNAi [J]. Basic Clin Pharmacol Toxicol, 2008,103(4):342-348.
[31] Wiley SR , Schooley K, Smolak PJ, et al . Identification and characterization of a newmember of TNF family that induce apoptosis [J] . Immunity, 1995, 3(6):673-682.
[32] Pitti RM, Marsters SA, Ruppert S, et al. Induction of apoptosis by Apo2 ligand, a newmember of the tumor necrosis factor cytokine family [J]. J Biol Chem, 1996,271(22):12687-126901.
[33] Walczak H, Miller RE, Ariail K, et al. Tumoricidal activity of tumor necrosis factor relatedapoptosis inducing ligand in vivo [J]. NatMed, 1999, 5(2):157-163.
[34] Gores GJ, Kaufmann SH. Is TRAIL hepatotoxic? [J]. Hepatology, 2001, 34(l):3-6.
[35] Wang P, Song JH, Song DK, et al. Role of death receptor and mitochondrial pathways inconventional chemotherapy drug induction of apoptosis [J]. Cell Signal, 2006,18(9):1528-1535.
[36] Song JH, Wang CX, Song DK, et al. Interferon gamma induces neurite outgrowth byup-regulation of p35 neuron-specific cyclin-dependent kinase 5 activator via activation ofERK1/2 pathway [J]. J Biol Chem, 2005, 280(13):12896-12901.
[37]沈滟,赵大治. TRAIL系统与肿瘤细胞的凋亡[J].河南科技大学学报(医学版), 2006, 24(4): 318-320.
[38] Kagawa S, He C, Gu J, et al. Antitumor activity and bystander effects of tumor necrosisfactor-related apotos is-indue ing ligand (TRAIL) gene [J]. Cancer Res, 2001,61(8):3330-3338.
[39] Lu x, Arbiser JL, West J, et al. Tumor necrosis factor-related apoptos is-indue ing ligand caninduce apoptosis in subsets of premaligant cells [J]. Am J Pathol, 2004, 165(5):1613-1620.
[40] Houdy, Yan Z, Han W. Cloning and characterization of TRAIL cDNA [J]. Chin J Biol ChemMol Bid, 2002, 18(1):27-31.
[41] Spierings DC, de veries EG, Vellenga E, et al. Tissue distribution of the death ligand TRAILand its receptors [J]. Histochem, 2004, 52(6):821-831.
[42] Hymowitz, SG, O'Connell MR Ultsch MH, et al. A unique zinc-binding site revealed by ahigh-resolution X-ray structure of homotrimeric Apo2L/TRAIL [J]. Biochemistry, 2000,39(4): 633-640.
[43] Bodmer JL, Meier P, Tschopp J, et al. Cysteine 230 is essential for the structure and activityof the cytotoxic ligand TRAIL [J]. J Biol Chem, 2000, 27(27):20632-20637.
[44] Kim Y, Seol DW. TRAIL, a mighty apoptosis inducer [J]. Molecules and cells, 2003,15:283-293
[45]马远方. TRAIL及其受体的研究进展[J].河南大学学报, 2007, 26(2):1-5.
[46] Truneh A, Sharma S, Silverman C, et al. Temperature sensitive differential affinity of TRAILfor its receptors DR5 is the highest affinity receptor [J]. J Biol Chem, 2000,275(30):23319-23325.
[47] Degli-esposti MA, Smolak PJ, Walczak H, et al. Cloning and characterization of TRAIL-R3,a novel member of the emerging TRAIL receptor family [J]. J Exp Med, 1997,186(7):1165-1170.
[48] Pan H, Rourke K, Chinnaiyan AM, et al. The receptor for the cytotoxic ligand TRAIL [J].Science, 1997, 276(5309): 111-113.
[49] Simonet WS, Lacey DL, Dunstan CR, et al. Osteoprotegerin: a novel secreted proteininvolved in the regulation of bone density [J]. Cell, 1997, 89(2):309-319.
[50] Holen I, Croucher PI, Hamdy FC, et al. Osteoprotegerin (OPG) is a survival factor for humanprostate cancer cells [J]. Cancer Res, 2002, 62(6):1619-1623.
[51] Shipman CM, Croucher PI. Osteoprotegerin is a soluble decoy receptor for tumor necrosisfactor-related apoptos is-indue ing ligand/Apo2 ligand and can function as a paracrine survivalfactor for human myeloma cells [J]. Cancer Res, 2003, 63(5):912-916.
[52] Medema JP, Scaffidi C, Kischkel FC. FLICE is activated by association with the CD95death-inducing signaling complex(DISC) [J]. EMBO J, 1997, 16(10):2794-804.
[53] Muzio M, Stockwell BR, Stennicke HR. An induced proximity model for Caspase-8activation [J]. J Biol Chem, 1998, 273(5):2926-2930.
[54] Sheridan JP, Marsters SA, Piti RM, et al. Control of TRAIL-induced apoptosis by a family ofsignaling and decoy receptors [J]. Science, 1997, 277: 818-825.
[55] Walczak H, Krammer PH. The CD95 (APO-IfFas) and the TRAIL(AP0-2L) apoptosissystems [J]. Exp Cell Res, 2000, 256(l):58-66.
[56] MacFarlane M. TRAIL-mduced signalling and apoptosis [J]. Toxicol Lett. 2003, 139(2-3):89-97.
[57] Green DR, Kroemer G. The pathophysiology of mitochondrial cell death [J]. Science, 2004,305(5684):626-629.
[58] Barczyk K, Kreuter M, Pryjma J, et al. Serum cytochrome c indicates in vivo apoptosis andcan serve as a prognostic marker during cancer therapy [J]. Int J Cancer, 2005,116(2):167-173.
[59] Jiang X, Wang X. Cytochrome c promotes Caspase-9 activation by inducing nucleotidebinding to Apaf-1 [J]. JBiol Chem, 2000, 275(40):31199-311203.
[60] Deveraux QL, Reed JC. IAP family proteins-suppressors of apoptosis [J]. Genes Dev, 1999,13(3): 239-252.
[61] Du C, Fang M, Li Y, et al. Smac, a mitochondrial protein that promotes cytochromec-dependent Caspase activation by eliminating IAP inhibition [J]. Cell, 2000, 102(l):33-42.
[62] Verhagen AM, Ekert PG, Pakusch M. Identification of DIABLO, a mammalian protein thatpromotes apoptosis by binding to and antagonizing IAP proteins [J]. Cell, 2000,102(l):43-53.
[63] Li S, Zhao Y, He X, et al. Relief of extrinsic pathway inhibition by the Bid-dependentmitochondrial release of Smac in Fas-mediated hepatocyte apoptosis [J]. J Biol Chem, 2002,277(30):26912-26920.
[64] Hu WH, Johnson H, Shu HB. Tumor necrosis factor-related apoptosis-indue ing ligandreceptors signal NF-kappaB and JNK activation and apoptosis through distinct pathways [J].JBiol Chem, 1999, 274(43):30603-30610.
[65] Wajant H, Haas E, Schwenzer R, et al. Inhibition of death receptor-mediated gene inductionby a cycloheximide-sensitive factor occurs at the level of or upstream of Fas-associated deathdomain protein (FADD) [J]. J Biol Chem, 2000, 275(32):24357-24366.
[66] MacFarlane M, Cohen GM, Dickens M. JNK(c-Jun N-terminal kinase) and p38 activation inreceptor-mediated and chemically-induced apoptosis of T-cells: differential requirements forcaspase activation [J]. Biochem J, 2000, 348(l):93-101.
[67] He SQ, Chen XP, Zhao YZ, et al. Expression profiles of TRAIL receptors and their clinicalsignificance in human hepatocellular Carcinoma [J]. Chinese-German J Clin Oncol, 2003,2(1): 25-29.
[68] Walczak H, Miller RE, Ariail K, et al. Tumoricidal activity of tumor necrosis factor-relatedapoptosis-indue ing ligand in vivo [J]. Nat Med, 1999, 5(2): 157-163.
[69] Ashkenazi A, Pai RC, Fong S, et al. Safety and antitumor activity of recombinant solubleApo2 ligand [J]. J Clin Invest, 1999, 104(2):155-162.
[70] Wajant H, Pfizenmaier K, Scheurich P. TNF-related apoptosis inducing ligand (TRAIL) andits receptors in tumor surveillance and cancer therapy [J]. Apoptosis, 2002, 7(5):449-459.
[71] Naka T, Sugamura K, Hylander BL, et al. Effects of tumor necrosis factor-relatedapoptosis-indue ing ligand alone and in combination with chemo therapeutic agents onpatients' colon tumors grown in SCID mice [J]. Cancer Res, 2002, 62(20):5800-5806.
[72] Jo M, Kim TH, Seol DW, et al. Apoptosis induced in normal human hepatocytes by tumornecrosis factor-related apoptosis-indue ing ligand [J]. Nat Med, 2000, 6(5):564-567.
[73] Nitsch R, Bechmann 1, Deisz RA, et al. Human brain-cell death induced by turn our-necrosis-factor-related apoptosis-mducmg ligand (TRAIL) [J]. Lancet, 2000, 356(9232):827-828.
[74] Verhagen AM, Ekert PG, Pakusch M. Identification of DIABLO, a mammalian protein thatpromotes apoptosis by binding to and antagonizing IAP proteins [J]. Cell, 2000,102(l):43-53.
[75] Gilley D, Tanaka H, Herbert BS. Telomere dysfunction in aging and cancer [J]. Int J BiochemCellBiol, 2005, 37(5):1000-1013.
[76] Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activitywith immortal cells and cancer [J]. Science, 1994, 266(5193):2011-2015.
[77] Cech TR. Beginning to understand the end of the chromosome [J]. Cell, 2004, 116(2):273-279.
[78] Chen Z, Koeneman KS, Corey DR. Consequences of telomerase inhibition and combinationtreatments for the proliferation of cancer cells [J]. Cancer Res, 2003, 63(18):5917-5925.
[79] Wong JM, Kusdra L, Collins K. Subnuclear shuttling of human telomerase induced bytransformation and DNA damage [J]. Nat Cell Biol, 2002, 4(9):731-736.
[80] Shammas MA, Koley H, Batchu RB, et al. Telomerase inhibition by siRNA causessenescence and apoptosis in Barrett's adenocarcinoma cells: mechanism and therapeuticpotential [J]. Mol Cancer, 2005, 15(4):24-38.
[81] Hahn WC, Stewart SA, Brooks MW, et al. Inhibition of telomerase limits the growth ofhuman cancer cells [J]. Nat Med, 1999, 5(10): 1164-1170.
[82] Fujiwara T, Tanaka N. Telomerase-specific oncolytic virotherapy for human cancer with thehTERT promoter [J]. Uirusu, 2008, 58(1):11-18.
[83] Wang Y, Huang F, Cai H, et al. Potent antitumor effect of TRAIL mediated by a noveladeno-associated viral vector targeting to telomerase activity for human hepatocellularcarcinoma [J]. Gene Med, 2008, 10(5):518-526.
[84] Li HM, Yu YC, Song TB, et al. Expression of TRAIL gene eukaryotic expression vectormodulated by the human telomerase reverse transcriptase gene core promoter in ovariancancer cell line SKOV3 [J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2007, 23(12): 1106-1109.
[85] Su T, Sun HC, Zheng CY, et al. Targeting transgene effect of adTERT-TRAIL on salivaryadenoid cystic carcinoma cell line SACC-83 [J]. Hua Xi Kou Qiang Yi Xue Za Zhi, 2007,25(2): 202-205.
[86] Chen B, He C, Lao WF, et al. Activity of the TNF-related apoptos is-indue ing ligand geneexpressed from the hTERT promoter on colon cancer cell line HT-29 [J]. Zhejiang Da XueXue Bao Yi Xue Ban, 2006, 35(l):45-49.
[87] Bilsland AE, Anderson CJ, Fletcher-Monaghan AJ, et al. Selective ablation of human cancercells by telomerase-specific adenoviral suicide gene therapy vectors expressing bacterialmtroreductase [J]. Oncogene, 2003, 22(3):370-380.
[88] Gu J, Zhang LD, Huang XF, et al. A novel single tetracycline-regulative adenoviral vector fortumor-specific Bax gene expression and cell killing in vitro and in vivo [J]. Oncogene, 2002,21:4757-4764.
[89] Painter RG, Lanson NA, Jin Z, et al. Conditional expression of a suicide gene by the telomerereverse transcriptase promoter for potential post-therapeutic deletion of tumorigenesis [J].Cancer Sci, 2005, 96(9):607-613.
[90] Yang CQ, Deng ZH, Liu Y, el al. Asialofetum-hTERT-TK/GCV targeted gene therapy and itsbystander effect on HepG2 [J]. Zhonghua Gan Zang Bing Za Zhi, 2008, 16(7):509-513.
[91] Doloff JC, Waxman DJ, Jounaidi Y. hTERT-promoter driven oncolytic adenovirus withE1B-19 kDa and E1B-55 kDa gene deletions [J]. Hum Gene Ther, 2008, 19(12): 1383-1400.
[92] Zhao HC, Zhang Q, Yang Y, et al. p53-expressing conditionally replicative adenovirusCNHK500-p53 against hepatocellular carcinoma in vitro [J]. World J Gastroenterol, 2007,13(5): 683-691.
[93] Li GC, Yang JM, Nie MM, et al. Potent antitumoral effects of a novel gene-viral therapeuticsystem CNHK300-mEndostatin in hepatocellular carcinoma [J]. Chin Med J(Engl), 2005,118(3):179-185.
[94]邓文星,张映.实时荧光定量PCR技术综述[J].生物技术通报, 2007, (5): 93-95.
[95] Tominaga H, IshiyamaM, Ohseto F, et al. A water-soluble tetrazolium salt useful forcolorimetric cell viability assay [J]. Anal Commun, 1999, 36(l):47-50.
[96] Cory AH, Owen TC, Barltrop JA, et al. Use of an aqueous soluble tetrazolium/formazanassay for cell growth assays in culture [J]. Cancer Commun, 1991, 3(7):207-212.
[97] Berridge MV, Tan AS, McCoy KD, et al. The biochemical and cellular basis of cellproliferation assays that use terazolium salts [J]. Biochemica, 1996, 4:15-19.
[98] Moamman T. Rapid colorimetric assay for celluar growth and survial:appication toproliferation cytotoxicity assays [J]. J Immunol Methods, 1983, 65(l-2):55-63.
[99] Denizot F, Lang R. Rapid colorimetric assay for cell growth and surrvial: Modifications tothe tetrazolium dye procedure giving improved sensitivity and reliability [J]. J ImmunolMethods, 1986,89(2):271-277.
[100] Twentyman PR, Luscombe M. A study of some variables in a tetrazolium dye (MTT) bassedassay for cell growth and chemosensitxity [J]. BR J Cancer, 1987, 56(3):279-25.
[101] Meier P, Finch A, Evan G Apoptosis in development. Nature, 2000, 407:796-801
[102]刘建越,邓欣,康林,等. SYBR-Green实时荧光PCR检测转基因番木瓜[J].湖南农业大学学报(自然科学版), 2006, 32(4):371-374.
[103] Morrison TB, Weis JJ, Wittwer CT. Quantification of low-copy transcripts by continuousSYBR-Green monitoring during amplification [J]. Biotechniques, 1998, 24(6):954-962.
[104] Maes L, Van Neste L, Van Damme K, et al. Relation between telomerase activity, hTERTand telomere length for intracranial tumours [J]. Oncol Rep, 2007, 18(6): 1571-1576.
[105] Nishio Y, Nakanishi K, Ozeki Y, et al. Telomere length, telomerase activity, and expressionsof human telomerase mRNA component(hTERC)and human telomerase reversetranscriptase (hTERT) mRNA in pulmonary neuro endocrine tumors [J]. Jpn J Clin Oncol,2007, 37(l):16-22.
[106] Murofushi Y, Nagano S, Kamizono J, et al. Cell cycle-specific changes in hTERT promoteractivity in normal and cancerous cells in adenoviral gene therapy: a promising implicationof telomerase-dependent targeted cancer gene therapy [J]. Int J Oncol, 2006, 29(3):681-688.
[107]宋悦,孔北华,马道新,等.宫颈癌及子宫内膜癌细胞系中启动子活性的研究[J].肿瘤防治杂志, 2003, 10(3):442-443.
[108] Niidome T, Huang L. Gene therapy progress and prospects: nonviral vectors [J]. Gene Ther, 2002, 9(24): 1647-1652.
[109] Ganten TM, Koschny R, Sykora J, et al. Preclinical differentiation between apparently safeand potentially hepatotoxic applications of TRAIL either alone or incombination withchemotherapeutic drugs [J]. Clin Cancer Res, 2006, 12(8):2640-2646.
[110]刘永靖,陈飞虎,于奇,等.腺病毒载体携带人TRAIL基因治疗H446小细胞肺癌的体外实验研究[J].中国药理学通报, 2007, 23(6):755-759.
[111] Kagawa S, He C, Gu J, et al. Antitumor activity and bystander effects of the tumor necrosisfactor-related apoptosis-indue ing ligand (TRAIL) gene [ J]. Cancer Res, 2001,61:3330-3338.
[112] Alao Y, Yamada H, Nakagawa Y. Arsenic-induced apoptosis in malignant cells in vitro.Leuk Lymphoma, 2000, 37:53-63.
[113] Anuradha CD, Kanno S, Hirano S. Oxidative damage of mitochondria is a preliminary stepto caspase3 activation in fluoride-induced apoptosis in HL260 cells. Free Radic Biol Med,2001 ,31:367-373.
[114]李宁丽,路丽明,沈佰华,等.重组人可溶性分子诱导细胞凋亡机理探讨[J].上海第二医科大学学报, 2003, 23(1):10-14.
[115]尹伶,濮德敏,韦立蓓,等. Caspase3在NS-398诱导宫颈癌Hela细胞凋亡中的作用[J].中国肿瘤临床, 2007, 34(12):711-713.
[116] Yan-Guo Ren, Klaus W, Wagner, et al. Differential regulation of the TRAIL death receptors DR4 and DR5 by the signal recognition particle [J]. Mol Biol Cell, 2004, 15(11):5064-5074.
[117] Peter H, Dietmar P, Alexandra K, et al. Perturbation of the tumor necrosis factor-related apoptos is-indue ing ligand cascade in ovarian cancer: over expression of FLIPL and deregulation of the functional receptors DR4 and DR5. Clin Cancer Res, 2005, 11(24 Pt 1): 8585-8591.