本文選題：P_(HSP)-TK基因　切入點(diǎn)：胰腺癌　出處：《浙江大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究目的:通過對(duì)射頻加熱(RFH)增強(qiáng)熱休克蛋白啟動(dòng)子(PHSP)引導(dǎo)下的單純皰疹病毒胸苷激酶基因(PHSP-TK)治療胰腺癌療效的研究,及大鼠骨髓間充質(zhì)干細(xì)胞(rBM-MSC)介導(dǎo)的PHSP-TK基因治療胰腺癌的研究,為進(jìn)一步探索分子影像引導(dǎo)下的射頻加熱結(jié)合干細(xì)胞介導(dǎo)的新型腫瘤治療方法奠定基礎(chǔ)。研究方法:利用攜有PHSP-TK基因的慢病毒轉(zhuǎn)染人胰腺癌細(xì)胞株(CFPAC-1、BXPC-3)及rBM-MSC細(xì)胞,得到CFPAC-1-TK、BXPC-3-TK及rBM-MSC-TK細(xì)胞,利用倒置熒光顯微鏡及流式細(xì)胞術(shù)對(duì)轉(zhuǎn)染效率進(jìn)行檢測(cè)。射頻加熱增強(qiáng)PHSP-TK基因治療胰腺癌療效實(shí)驗(yàn),實(shí)驗(yàn)分組與處理:①胰腺癌對(duì)照組(Control組)、②胰腺癌射頻加熱組(RFH組)、③25%胰腺癌細(xì)胞轉(zhuǎn)染PHSP-TK基因組(25%PHSP-TK組)、④25%胰腺癌細(xì)胞轉(zhuǎn)染PHSP-TK基因聯(lián)合射頻加熱組(25%PHSP-TK+RFH組)、⑤50%胰腺癌細(xì)胞轉(zhuǎn)染PHSP-TK基因組(50%PHSP-TK組)、⑥50%胰腺癌細(xì)胞轉(zhuǎn)染PHSP-TK基因聯(lián)合射頻加熱組(50%PHSP-TK+RFH組)、⑦75%胰腺癌細(xì)胞轉(zhuǎn)染PHSP-TK基因組(75%PHSP-TK組)、⑧75%胰腺癌細(xì)胞轉(zhuǎn)染PHSP-TK基因聯(lián)合射頻加熱組(75%PHSP-TK+RFH組)。以rBM-MSC攜PHSP-TK基因治療胰腺癌實(shí)驗(yàn),實(shí)驗(yàn)分組與處理:①胰腺癌對(duì)照組(Control組)、②胰腺癌射頻加熱組(RFH組)、③胰腺癌與rBM-MSC1比1混合培養(yǎng)組(胰腺癌+rBM-MSC組)、④胰腺癌與rBM-MSC-TK 1比1混合培養(yǎng)組(胰腺癌+rBM-MSC-TK組)、⑤胰腺癌與rBM-MSC-TK 1比1混合培養(yǎng)聯(lián)合射頻加熱組(胰腺癌+rBM-MSC-TK+RFH組)。通過檢測(cè)細(xì)胞增殖及凋亡來評(píng)估PHSP-TK基因聯(lián)合射頻加熱治療對(duì)胰腺癌的療效,同時(shí)評(píng)估射頻加熱結(jié)合基于rBM-MSC運(yùn)載PHSP-TK基因治療胰腺癌的療效。結(jié)果:倒置熒光顯微鏡鏡檢及流式細(xì)胞術(shù)結(jié)果證實(shí)利用慢病毒導(dǎo)入的PHSP-TK基因在人胰腺癌細(xì)胞株CFPAC-1、BXPC-3及rBM-MSC細(xì)胞中高表達(dá),在射頻加熱條件下PHSP-TK基因表達(dá)水平顯著提高(P0.001)。胰腺癌細(xì)胞增殖及凋亡實(shí)驗(yàn)結(jié)果:CCK8結(jié)果顯示PHSP-TK+RFH組較PHSP-TK組、RFH組細(xì)胞存活率低(P0.05)。進(jìn)一步凋亡檢測(cè)結(jié)果證實(shí):PHSP-TK+RFH組較PHSP-TK組、RFH組細(xì)胞死亡率高(P0.05)。rBM-MSC實(shí)驗(yàn)結(jié)果:①rBM-MSC實(shí)現(xiàn)了原代分離,分離獲得的rBM-MSC細(xì)胞呈梭形,表面抗原鑒定CD29、CD54、CD90和CD45陽性率分別為96.59%、94.91%、99.43%和3.21%,可以向成骨及成脂方向分化。②CCK8結(jié)果顯示:胰腺癌+rBM-MSC-TK+RFH組較Control組、胰腺癌+rBM-MSC-TK組、RFH組細(xì)胞存活率低(P0.05);胰腺癌+rBM-MSC-TK組較Control組、RFH組細(xì)胞存活率低(P0.05)。③細(xì)胞凋亡實(shí)驗(yàn)結(jié)果證實(shí):胰腺癌+rBM-MSC-TK+RFH組較胰腺癌+rBM-MSC-TK組細(xì)胞死亡率高(P0.05)。結(jié)論:實(shí)驗(yàn)證實(shí)外源PHSP-TK基因可以在人胰腺癌細(xì)胞及rBM-MSC上的有效導(dǎo)入、高豐度表達(dá),射頻加熱增強(qiáng)了間充質(zhì)干細(xì)胞介導(dǎo)的PHSP-TK基因治療胰腺癌的殺傷效果,這為進(jìn)一步建立一種分子影像引導(dǎo)下的干細(xì)胞介導(dǎo)的新型腫瘤治療方法奠定了基礎(chǔ)。
[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.
【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】：2017
【分類號(hào)】：R735.9

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