本文選題：二聚化 + 自殺基因　； 參考：《新鄉(xiāng)醫(yī)學(xué)院》2015年碩士論文

【摘要】：背景：基因修飾T細(xì)胞免疫治療越來(lái)越顯示出其優(yōu)越性,在癌癥患者的治療上起到了積極的作用。然而,這種“超自然”T淋巴細(xì)胞的調(diào)控是臨床基因和細(xì)胞治療需要解決的關(guān)鍵問(wèn)題,利用自殺基因系統(tǒng)調(diào)控基因修飾的細(xì)胞是基因治療的安全措施之一。研究顯示,iCasp9自殺基因系統(tǒng)能夠高效快速的誘導(dǎo)細(xì)胞凋亡,并且iCasp9幾乎全部是人源性的,所以轉(zhuǎn)導(dǎo)細(xì)胞引起免疫反應(yīng)的風(fēng)險(xiǎn)大大降低,因此,該系統(tǒng)受到了人們的廣泛關(guān)注。目的：建立可誘導(dǎo)的Caspase9自殺基因系統(tǒng)誘導(dǎo)Jurkat T細(xì)胞,利用陽(yáng)性化合物誘導(dǎo)細(xì)胞凋亡,獲得T細(xì)胞條件消融的相關(guān)數(shù)據(jù),為基因修飾T細(xì)胞的免疫治療提供安全保障。方法：1、利用分子克隆技術(shù)將目的基因(FKBP12/Caspase9)克隆入慢病毒載體pCDH-CMV-MCS-EFl-puro中,構(gòu)建piCasp9表達(dá)質(zhì)粒,三質(zhì)粒(piCasp9、pVSVG、 pDe18.9)磷酸鈣法共轉(zhuǎn)染293T細(xì)胞制備慢病毒(LV-KLC9),并進(jìn)行滴度測(cè)定。2、用病毒液感染Jurkat T細(xì)胞,經(jīng)puromycin篩選擴(kuò)增。Western blot檢測(cè)目的基因在細(xì)胞中的表達(dá)；利用Annexin V染色法對(duì)AP20187誘導(dǎo)二聚體介導(dǎo)的細(xì)胞凋亡情況進(jìn)行分析。結(jié)果：1、成功構(gòu)建piCasp9表達(dá)質(zhì)粒,三質(zhì)粒共轉(zhuǎn)染293T細(xì)胞制備的慢病毒LV-KLC9具有高效性,72h后95%的293T細(xì)胞可以觀察到綠色熒光。采用逐孔稀釋滴度測(cè)定法測(cè)定病毒滴度,病毒滴度可達(dá)3.0×107 IU/ml。2、病毒感染Jurkat T細(xì)胞的效率達(dá)65%；Western blot檢測(cè)發(fā)現(xiàn)約在49kD處出現(xiàn)明顯的特異條帶,而對(duì)照組無(wú)此條帶,說(shuō)明慢病毒將融合基因?qū)隞urkat細(xì)胞,并在細(xì)胞中成功表達(dá)。給予10 nM AP20187對(duì)細(xì)胞進(jìn)行凋亡誘導(dǎo),并于不同時(shí)間點(diǎn)檢測(cè)細(xì)胞凋亡,流式細(xì)胞術(shù)結(jié)果顯示：細(xì)胞凋亡率隨著時(shí)間的延長(zhǎng)逐漸增加,表現(xiàn)為時(shí)間依賴(lài)性的細(xì)胞死亡。結(jié)論：成功建立了iCasp9自殺基因系統(tǒng)可誘導(dǎo)細(xì)胞凋亡生物效應(yīng)的細(xì)胞模型,體外給予AP20187后,攜帶目的基因的Jurkat細(xì)胞呈現(xiàn)時(shí)問(wèn)依賴(lài)性的凋亡,這有望為基因修飾T細(xì)胞的免疫治療提供安全保障。
[Abstract]:Background: gene-modified T-cell immunotherapy (GMT) has shown its advantages and played an active role in the treatment of cancer patients. However, the regulation of supernatural T lymphocytes is a key problem in clinical gene and cell therapy. It is one of the safe measures for gene therapy to regulate the genetically modified cells by using suicide gene system. Studies have shown that iCasp9 suicide gene system can induce apoptosis efficiently and rapidly, and iCasp9 is almost all human origin, so the risk of immune response caused by transduction cells is greatly reduced. Therefore, the system has attracted wide attention. Aim: to establish an inducible Caspase9 suicide gene system to induce Jurkat T cells, to induce apoptosis by using positive compounds, and to obtain the relevant data of T cell conditioned ablation, so as to provide a safe guarantee for the immunotherapy of genetically modified T cells. Methods the target gene (FKBP12 / Caspase9) was cloned into lentivirus vector pCDH-CMV-MCS-EFl-puro by molecular cloning technique, and the piCasp9 expression plasmid was constructed. Three plasmids (piCasp9 pVSVG, pDe18.9) calcium phosphate were co-transfected into 293T cells to prepare lentivirus LV-KLC9. The expression of the target gene in the cells was detected by puromycin screening and amplification, and the apoptosis induced by AP20187 was analyzed by Annexin V staining. Results the piCasp9 expression plasmid was constructed successfully. The lentivirus LV-KLC9 prepared by co-transfection of the three plasmids into 293T cells had high efficiency. After 72 hours, 95% of the 293T cells could be observed green fluorescence. The virus titer was determined by one-hole dilution titer assay. The virus titer reached 3.0 脳 10 ~ 7 IU / ml 路2. The efficiency of virus infection in Jurkat T cells was 65%. Western blot detection showed that there were obvious specific bands in 49kD, but there was no specific band in the control group. The results showed that lentivirus could transfer the fusion gene into Jurkat cells and express it successfully. Apoptosis was induced by 10 nm AP20187, and apoptosis was detected at different time points. The results of flow cytometry showed that the rate of apoptosis increased with time, and showed time dependent cell death. Conclusion: the iCasp9 suicide gene system was successfully established to induce the biological effect of apoptosis. After AP20187 was given in vitro, the Jurkat cells carrying the target gene showed time-dependent apoptosis. This is expected to provide a safe guarantee for the immunotherapy of genetically modified T cells.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R730.51

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