【摘要】：目的：通過(guò)研究射頻加熱技術(shù)增強(qiáng)熱啟動(dòng)子誘導(dǎo)下的單純皰疹病毒胸苷激酶／更昔洛韋(herpes simplex virus thymidine kinase/Ganciclovir, HSV-TK/GCV)基因治療系統(tǒng)對(duì)多柔比星耐藥性乳腺癌細(xì)胞的療效,為創(chuàng)建介入分子影像引導(dǎo)下的射頻加熱聯(lián)合基因治療新方法奠定基礎(chǔ)。方法：體外實(shí)驗(yàn)：腔室內(nèi)四孔板接種人多柔比星耐藥性乳腺癌MCF7/A細(xì)胞,通過(guò)慢病毒將熱啟動(dòng)子引導(dǎo)下的HSV-TK基因?qū)隡CF7/A細(xì)胞。利用熒光鏡檢及qPCR技術(shù)對(duì)基因表達(dá)進(jìn)行檢測(cè)。將腔室內(nèi)四孔板置于37-C水浴中,射頻加熱后加更昔洛韋(10ug/ml)處理細(xì)胞。通過(guò)檢測(cè)細(xì)胞增殖和凋亡來(lái)評(píng)估熱啟動(dòng)子引導(dǎo)下的基因治療聯(lián)合射頻加熱對(duì)耐藥性乳腺癌細(xì)胞的殺傷效果。體內(nèi)實(shí)驗(yàn)：建立裸鼠乳腺癌模型,并將荷瘤裸鼠分成4組：包括對(duì)照組(Control組)；射頻加熱組(RFH組)；基因治療組(Phsp-TK組)；及射頻加熱結(jié)合基因治療組(Phsp-TK+RFH組),通過(guò)MRI對(duì)腫瘤大小變化進(jìn)行檢測(cè)。最后通過(guò)病理學(xué)技術(shù)對(duì)照證實(shí)MRI的表現(xiàn)。結(jié)果：熒光鏡檢及qPCR結(jié)果證實(shí)通過(guò)慢病毒導(dǎo)入的熱啟動(dòng)子誘導(dǎo)的TK基因在射頻加熱條件下在乳腺癌細(xì)胞中有大量表達(dá)。PHSp-TK+RFH組貼壁細(xì)胞數(shù)明顯減少,死亡細(xì)胞明顯增多,與Phsp-TK組及RFH組相比差異顯著(p0.01)。流式檢測(cè)進(jìn)一步證實(shí)Phsp-TK+RFH組腫瘤細(xì)胞的凋亡明顯增加(p0.05)。在體內(nèi)實(shí)驗(yàn)中,裸鼠荷瘤模型的Phsp-TK+RFH治療組相比于RFH組及Phsp-TK單處理組其腫瘤體積在處理后有顯著縮小。結(jié)論：本研究證實(shí)通過(guò)熱啟動(dòng)子誘導(dǎo)下HSV-TK基因在多柔比星耐藥性乳腺癌細(xì)胞上的有效導(dǎo)入,聯(lián)合射頻加熱增強(qiáng)了該基因療法的療效,這為創(chuàng)建介入分子影像引導(dǎo)下的耐藥性腫瘤局部基因治療技術(shù)奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R450;R737.9

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