射频加热增强热启动子诱导下的自杀基因治疗乳腺癌：面向介入分子影像引导下的基因治疗

发布时间：2018-08-03 08:26

【摘要】：目的：通过研究射频加热技术增强热启动子诱导下的单纯疱疹病毒胸苷激酶／更昔洛韦(herpes simplex virus thymidine kinase/Ganciclovir, HSV-TK/GCV)基因治疗系统对多柔比星耐药性乳腺癌细胞的疗效,为创建介入分子影像引导下的射频加热联合基因治疗新方法奠定基础。方法：体外实验：腔室内四孔板接种人多柔比星耐药性乳腺癌MCF7/A细胞,通过慢病毒将热启动子引导下的HSV-TK基因导入MCF7/A细胞。利用荧光镜检及qPCR技术对基因表达进行检测。将腔室内四孔板置于37-C水浴中,射频加热后加更昔洛韦(10ug/ml)处理细胞。通过检测细胞增殖和凋亡来评估热启动子引导下的基因治疗联合射频加热对耐药性乳腺癌细胞的杀伤效果。体内实验：建立裸鼠乳腺癌模型,并将荷瘤裸鼠分成4组：包括对照组(Control组)；射频加热组(RFH组)；基因治疗组(Phsp-TK组)；及射频加热结合基因治疗组(Phsp-TK+RFH组),通过MRI对肿瘤大小变化进行检测。最后通过病理学技术对照证实MRI的表现。结果：荧光镜检及qPCR结果证实通过慢病毒导入的热启动子诱导的TK基因在射频加热条件下在乳腺癌细胞中有大量表达。PHSp-TK+RFH组贴壁细胞数明显减少,死亡细胞明显增多,与Phsp-TK组及RFH组相比差异显著(p0.01)。流式检测进一步证实Phsp-TK+RFH组肿瘤细胞的凋亡明显增加(p0.05)。在体内实验中,裸鼠荷瘤模型的Phsp-TK+RFH治疗组相比于RFH组及Phsp-TK单处理组其肿瘤体积在处理后有显著缩小。结论：本研究证实通过热启动子诱导下HSV-TK基因在多柔比星耐药性乳腺癌细胞上的有效导入,联合射频加热增强了该基因疗法的疗效,这为创建介入分子影像引导下的耐药性肿瘤局部基因治疗技术奠定了基础。
[Abstract]:Objective: to investigate the effect of radiofrequency heating on doxorubicin resistant breast cancer cells induced by heat promoter induced by herpes simplex virus thymidine kinase / ganciclovir (herpes simplex virus thymidine kinase / Ganciclovirus (HSV-TK/GCV) gene therapy system. It lays a foundation for the establishment of a new method of radiofrequency heating combined with gene therapy under the guidance of interventional molecular imaging. Methods: in vitro experiments: human doxorubicin resistant breast cancer MCF7/A cells were inoculated with four hole plates in the cavity. The HSV-TK gene guided by heat promoter was introduced into MCF7/A cells by lentivirus. Fluorescence microscopy and qPCR technique were used to detect gene expression. The cells were treated with ganciclovir (10ug/ml) after radiofrequency heating. Cell proliferation and apoptosis were detected to evaluate the killing effect of heat promoter guided gene therapy combined with radiofrequency heating on drug-resistant breast cancer cells. The nude mice were divided into four groups: control group (Control group), radiofrequency heating group (RFH group), gene therapy group (Phsp-TK group), and tumor bearing nude mice were divided into 4 groups: control group (Control group), radiofrequency heating group (RFH group), gene therapy group (Phsp-TK group). Radiofrequency heating combined with gene therapy group (Phsp-TK RFH group) was used to detect tumor size by MRI. Finally, MRI findings were confirmed by pathological technique. Results: the results of fluorescence microscopy and qPCR showed that the TK gene induced by the heat promoter introduced by lentivirus in breast cancer cells was significantly reduced in the condition of radiofrequency heating. The number of adherent cells in the group of PHSp-TK RFH was significantly decreased, and the number of dead cells was significantly increased. Compared with Phsp-TK group and RFH group, the difference was significant (p 0.01). Flow cytometry further confirmed that apoptosis of tumor cells in Phsp-TK RFH group was significantly increased (p0.05). In vivo, the tumor volume of Phsp-TK RFH group was significantly smaller than that of RFH group and Phsp-TK single treatment group. Conclusion: this study confirmed that HSV-TK gene was effectively introduced into doxorubicin resistant breast cancer cells induced by heat promoter and combined with radiofrequency heating to enhance the efficacy of this gene therapy. This lays a foundation for the establishment of local gene therapy techniques for drug-resistant tumors under the guidance of interventional molecular imaging.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R450;R737.9

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