论文标题:自杀基因、血卟啉联合治疗肺癌的实验研究
Empirical Study on Lung Cancer Associate Suicide Gene and Hematoporphyrin
论文作者
论文导师 马忠森,论文学位 博士,论文专业 内科学
论文单位 吉林大学,点击次数 129,论文页数 115页File Size2759K
2007-04-01论文网 http://www.lw23.com/lunwen_744802/
suicide gene; hematoporphyrin; lung cancer; retroviral vector
本研究是将逆转录病毒介导的HSV-TK自杀基因系统与声动力学疗法联合进行了鼠Lewis肺癌细胞、人小细胞肺癌NCI-446细胞及C57BL/6小鼠Lewis肺癌模型的治疗。用MTT法检测细胞存活率,流式细胞仪检测细胞周期及凋亡率,结果表明HSV-TK/GCV自杀基因系统与声动力疗法对鼠Lewis肺癌细胞及人小细胞肺癌细胞NCI-446均有明显杀伤作用,联合治疗对两种肿瘤细胞的杀伤作用明显增强,具有协同作用;两种细胞发生G1期阻滞现象及细胞凋亡率增加,两种疗法分别对荷瘤小鼠有明显的抑制肿瘤生长及延长生存期的作用,二者联合能使肿瘤明显缩小,甚至消失,更加延长荷瘤小鼠的生存期。 本研究将自杀基因与声动力疗法联合治疗肺癌是一全新的想法与设计,两种疗法联合治疗肺癌完全可能提高肺癌的治疗作用及5年生存率,为肺癌的临床治疗提供理论依据。
Objective: Gene therapy in cancer treatment is a hot topic in modern medicine, while suicide gene therapy system is even more popular as it has more advantages. Suicide gene therapy has been studied and reported more in Brain Glioblastoma, Hepatoma, and Colon cancer as well as other gastrointestinal cancers, etc. There’ve been few reported studies in lung cancers. Suicide gene therapy is more advantageous than other gene therapies; however, it is still limited in clinical cancer treatment due to its low transfection rate to cancer cells and weak inducible immune responses to cancer cells. Therefore, combined suicide gene therapy and other cancer treatments are the way to go in future. Hematoporphyrin (HP), a substance isolated from blood, is light sensitive. HP can be easily phagocytized by macrophages in tissue as it has very high affinity to surface protein of cancer cells. It is likely HP prone to accumulation in extrocellular space due to high capillary permeability; poor lymphatic circulation and low pH in extracellular space in cancer tissue. The accumulated HP can be activated by ultrasound and subsequently induced series oxidation reactions, which can damage and kill the cancer cell to help in cancer treatment. The above process has been called Sonodynamic therapy or SDT. A special attention has been paid to SDT as its high tissue selectivity and specificity as well as fewer side effects. My study focused on combination of suicide gene therapy and Sonodynamic therapy to look for effective treatment for lung cancer and to provide theoretical and clinical evidences in lung cancer treatment. Methods: 1.in this study, a plasmid, PLEGFP-N1, carries green florescence protein (GFP) reporter gene from retrovirus was used as vector to construct a new plasmid called plEGFP-N1-TK. Thereafter, plEGFP-N1-TK was transfected into PA317 cell line and selected with G418 and green florescence protein. Viral titer with PA317/TK positive cell lines was measured. 2.Collect supernatant from above cell line with highest viral titer and transfect lung carcinoma cell line from Lewis rat and human small cell lung carcinoma cell line-NCI-446. Select HSV-TK positive Lewis/TK and NCI-446/TK cell lines with G418 and green florescence protein and observe the killing and standby effects from GCV by using MTT method. 3.Mix Lewis, NCI-446 cells with HP in different concentrations and process the mixtures with ultrasound. Observe the killing effect in these cells with MTT method. 4 . Treated these two lung cancer cell lines with combination of HSV-TK/GCV suicide gene system and sonodynamic therapy and observed cell morphological changes under microscope after treatment with different methods; also MTT method was used to detect viability and flow cytometry to evaluate cell cycle and apoptosis. 5.Established mouse animal model for Lewis lung carcinoma and randomly distribute the tumor-bearing mice into different groups. We observed tumor size closely. Killed one group mice after 14 days of treatment and surgically remove the tumors. Weigh the tumor from each animal and make description of morphology of each tumor. Eventually, all the tumor samples were processed for pathology slides and HE stain. One group of mice continued to be observed for life span. Result: 1.Establishment of plEGFP-N1-TK recombinant vector 2.Establishment of cell line PA317/TK: Transfected plEGFP-N1-TK into PA317 cell line and screened the positive clone with G418 and florescence protein. 3.Collected supernatant from PA 317/TK cell culture and infected NIH3T3 cell line with the supernatant. The viral titer was 4×104 cfu/ml. 4.After treatment of both Lewis/TK and NCI-446/TK cell lines with either HSV-TK/GCV suicide gene system or ultrasound, we found that both HSV-TK/GCV suicide gene system and sonodynamic therapy were effective in cell killing in both Lewis lung carcinoma and human small cell lung carcinoma. And the combination of these two therapies had synchronizing effect. The combination of sonodynamic therapy and suicide gene system most likely causes the blocking of G1 phase and induce apoptosis of tumor cells. 5.In vivo experience, We observesd that HSV-TK/GCV suicide gene system or sonodynamic therapy could inhibit the proliferation of Lewis cell in human small cell lung carcinoma respectively,but the tumors were not significant smaller than control group by using neither of two therapies.However, the tumor have been significiant atrophied or disappeared and the tumor-bearing mice could survive for more days with combination of these two therapies. Therefore, HSV-TK/GCV suicide gene system had a synergetic anti-tumor effect with sonodynamic therapy in vivo. Conclusion: 1.Establishment the therapeutic alliance method of HSV-TK/GCV suicide gene system and sonodynamic therapy on mouse Lewis lung carcinoma cell line、the small cell lung carcinoma cell of human NCI-446 and the C57BL/6 mouse animal model with Lewis lung carcinoma, there are not reported that the therapeutic alliance of the HSV-TK suicide gene and the sonodynamic therapy on lung cancer in domestic and foreign. 2.The HSV-TK/GCV suicide gene system has killing effect on both mouse Lewis lung cancer cells line and human small lung carcinoma cells line . The bystander effect could be observated in HSV-TK/GCV system. 3.SDT has the very good killing effect on both the mouse Lewis lung cancer cells line and the human small lung cancer cells line. 4. the therapeutic alliance of HSV-TK/GCV suicide gene system and sonodynamic therapy on Lewis and NCI-446 is synergetic. 5. The SDT and the suicide gene treatment all display the proportion increase of the G0-G1 phase cell to the cell cycle and the G1 phase stoppage obviously , the apoptosis rate are increased obviously . The proportion increase of the G0-G1 phase cell to the cell cycle and the apoptosis rate are more increased obviously after the therapeutic alliance of HSV-TK/GCV suicide gene system and sonodynamic therapy. G1 phase stoppage and apoptosis of tumor cells are main effect of combination of sonodynamic therapy and suicide gene system. 6. Both HSV-TK/GCV suicide gene system and sonodynamic therapy could inhibit the proliferation of Lewis cell in mice and prolong the life span of the tumor-bearing mice. Furthermore, these two therapies have synergetic effect for that the tumor have been significiant atrophied or disappeared and the tumor-bearing mice could survive for more days with combination of these two therapies.
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