射频加热增强间充质干细胞介导的热休克蛋白启动子引导下胸苷激酶基因治疗胰腺癌疗效的研究

发布时间：2018-03-03 11:34

本文选题：P_(HSP)-TK基因　切入点：胰腺癌　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的:通过对射频加热(RFH)增强热休克蛋白启动子(PHSP)引导下的单纯疱疹病毒胸苷激酶基因(PHSP-TK)治疗胰腺癌疗效的研究,及大鼠骨髓间充质干细胞(rBM-MSC)介导的PHSP-TK基因治疗胰腺癌的研究,为进一步探索分子影像引导下的射频加热结合干细胞介导的新型肿瘤治疗方法奠定基础。研究方法:利用携有PHSP-TK基因的慢病毒转染人胰腺癌细胞株(CFPAC-1、BXPC-3)及rBM-MSC细胞,得到CFPAC-1-TK、BXPC-3-TK及rBM-MSC-TK细胞,利用倒置荧光显微镜及流式细胞术对转染效率进行检测。射频加热增强PHSP-TK基因治疗胰腺癌疗效实验,实验分组与处理:①胰腺癌对照组(Control组)、②胰腺癌射频加热组(RFH组)、③25%胰腺癌细胞转染PHSP-TK基因组(25%PHSP-TK组)、④25%胰腺癌细胞转染PHSP-TK基因联合射频加热组(25%PHSP-TK+RFH组)、⑤50%胰腺癌细胞转染PHSP-TK基因组(50%PHSP-TK组)、⑥50%胰腺癌细胞转染PHSP-TK基因联合射频加热组(50%PHSP-TK+RFH组)、⑦75%胰腺癌细胞转染PHSP-TK基因组(75%PHSP-TK组)、⑧75%胰腺癌细胞转染PHSP-TK基因联合射频加热组(75%PHSP-TK+RFH组)。以rBM-MSC携PHSP-TK基因治疗胰腺癌实验,实验分组与处理:①胰腺癌对照组(Control组)、②胰腺癌射频加热组(RFH组)、③胰腺癌与rBM-MSC1比1混合培养组(胰腺癌+rBM-MSC组)、④胰腺癌与rBM-MSC-TK 1比1混合培养组(胰腺癌+rBM-MSC-TK组)、⑤胰腺癌与rBM-MSC-TK 1比1混合培养联合射频加热组(胰腺癌+rBM-MSC-TK+RFH组)。通过检测细胞增殖及凋亡来评估PHSP-TK基因联合射频加热治疗对胰腺癌的疗效,同时评估射频加热结合基于rBM-MSC运载PHSP-TK基因治疗胰腺癌的疗效。结果:倒置荧光显微镜镜检及流式细胞术结果证实利用慢病毒导入的PHSP-TK基因在人胰腺癌细胞株CFPAC-1、BXPC-3及rBM-MSC细胞中高表达,在射频加热条件下PHSP-TK基因表达水平显著提高(P0.001)。胰腺癌细胞增殖及凋亡实验结果:CCK8结果显示PHSP-TK+RFH组较PHSP-TK组、RFH组细胞存活率低(P0.05)。进一步凋亡检测结果证实:PHSP-TK+RFH组较PHSP-TK组、RFH组细胞死亡率高(P0.05)。rBM-MSC实验结果:①rBM-MSC实现了原代分离,分离获得的rBM-MSC细胞呈梭形,表面抗原鉴定CD29、CD54、CD90和CD45阳性率分别为96.59%、94.91%、99.43%和3.21%,可以向成骨及成脂方向分化。②CCK8结果显示:胰腺癌+rBM-MSC-TK+RFH组较Control组、胰腺癌+rBM-MSC-TK组、RFH组细胞存活率低(P0.05);胰腺癌+rBM-MSC-TK组较Control组、RFH组细胞存活率低(P0.05)。③细胞凋亡实验结果证实:胰腺癌+rBM-MSC-TK+RFH组较胰腺癌+rBM-MSC-TK组细胞死亡率高(P0.05)。结论:实验证实外源PHSP-TK基因可以在人胰腺癌细胞及rBM-MSC上的有效导入、高丰度表达,射频加热增强了间充质干细胞介导的PHSP-TK基因治疗胰腺癌的杀伤效果,这为进一步建立一种分子影像引导下的干细胞介导的新型肿瘤治疗方法奠定了基础。
[Abstract]:Objective: to study the effect of radiofrequency heating (RFH) on the enhancement of herpes simplex virus thymidine kinase gene (PHSP-TK) under the guidance of heat shock protein promoter PHSPin in the treatment of pancreatic cancer. And rat bone marrow mesenchymal stem cells / rBM-MSC-mediated PHSP-TK gene therapy for pancreatic cancer, Methods: lentivirus carrying PHSP-TK gene was used to transfect human pancreatic cancer cell line CFPAC-1 BXPC-3) and rBM-MSC cells. CFPAC-1-TKTK BXPC-3-TK and rBM-MSC-TK cells were obtained. The transfection efficiency was detected by inverted fluorescence microscope and flow cytometry. Radiofrequency heating enhanced the efficacy of PHSP-TK gene in the treatment of pancreatic cancer. The experiment was divided into two groups: control group, control group, control group, control group, control group, control group, control group, radiofrequency radiofrequency (RF) heating group. 32.5% of pancreatic cancer cells were transfected into PHSP-TK genome and 42% of pancreatic cancer cells were transfected into PHSP-TK gene and radiofrequency radiofrequency (RF) heating group. 55% of pancreatic cancer cells were transfected into PHSP-TK RFH group and 55% of Pancreatic carcinoma cells were transfected into PHSP-TK RFH group. PHSP-TK genome 50% pancreatic cancer cells were transfected with PHSP-TK gene in PHSP-TK group combined with radiofrequency radiofrequency heating (RFH) group. 75% of PHSP-TK cells were transfected with PHSP-TK gene and 75% of PHSP-TK cells in radiofrequency radiofrequency (RF) heating group. The effect of rBM-MSC with PHSP-TK gene on pancreatic cancer was studied. The experiment was divided into two groups: control group (control group), radiofrequency radiofrequency heating group (RFH group) and rBM-MSC1 ratio 1 group (rBM-MSC group) and rBM-MSC-TK 1: 1 group (Pancreatic carcinoma rBM-MSC-TK group). Adenocarcinoma and rBM-MSC-TK 1: 1 mixed culture combined with radiofrequency heating (rBM-MSC-TK RFH group) was used to evaluate the effect of PHSP-TK gene combined with radiofrequency heating on pancreatic cancer by detecting cell proliferation and apoptosis. The efficacy of radiofrequency heating combined with rBM-MSC carrying PHSP-TK gene in the treatment of pancreatic cancer was also evaluated. Results: reverse fluorescence microscopy and flow cytometry showed that lentivirus transfected PHSP-TK gene was used in human pancreatic cancer cell line CFPAC-1 and BXPC-3. High expression in rBM-MSC cells, The expression of PHSP-TK gene increased significantly under radiofrequency heating. The results of cell proliferation and apoptosis of pancreatic cancer cell line: CCK8 showed that the survival rate of PHSP-TK RFH group was lower than that of PHSP-TK group. Further apoptosis assay confirmed that the cell survival rate of PHSP-TK RFH group was lower than that of PHSP-TK group. The cell death rate of RFH group was higher than that of P0.05. rBM-MSC. Results: 1 rBM-MSC was isolated in primary culture. The positive rates of CD29, CD54, CD90 and CD45 were 96.599.91% and 3.21%, respectively. The results showed that the rBM-MSC-TK RFH group of pancreatic cancer was more likely to differentiate into osteogenesis and adipogenic direction than that from Control group, and the positive rates of CD29 and CD54 + CD90 and CD45 were 96.59% and 94.91% and 3.21%, respectively. The results showed that the rBM-MSC-TK RFH group was more positive than the Control group. The cell survival rate of pancreatic cancer rBM-MSC-TK group was lower than that of Control group, and the cell survival rate of pancreatic cancer rBM-MSC-TK group was lower than that of Control group. The results showed that the cell death rate of pancreatic carcinoma rBM-MSC-TK RFH group was higher than that of pancreatic carcinoma rBM-MSC-TK group (P 0.05). Conclusion: the results showed that the cell death rate of pancreatic cancer rBM-MSC-TK RFH group was higher than that of pancreatic cancer rBM-MSC-TK group. It is confirmed that exogenous PHSP-TK gene can be effectively introduced into human pancreatic cancer cells and rBM-MSC. High abundance expression and radiofrequency heating enhanced the killing effect of mesenchymal stem cell mediated PHSP-TK gene therapy for pancreatic cancer, which laid a foundation for the further establishment of a new method of cancer therapy mediated by stem cells under the guidance of molecular image.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9

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