【作者】 师义；

【导师】 王昆华；

【作者基本信息】 昆明医科大学 ， 外科学， 2012， 硕士

【摘要】 目的为了寻找胰腺癌基因治疗新方法,利用基因克隆、慢病毒载体构建、细胞转染等方法使人CDld基因稳定转染胰腺癌细胞株PANC-1,为后期以CDld-NKT为桥梁,研究细胞免疫原性变化、免疫上下游通路奠定基础。方法利用基因克隆技术克隆出人CDld基因,连接在pEASY-Tl simple cloning vector上,构建成pEASY-Tl simple-CDld质粒,再进行序列测定；利用基因重组技术将人CDld基因连接慢病毒载体PCDH-CMV-MCS-EFl-copGFP,进行病毒包装、滴度测定后浓缩纯化；通过慢病毒转染预实验确定MOI,进行人CDld重组慢病毒载体转染胰腺癌PANC-1细胞株。结果克隆出人CD1d基因,成功构建pEASY-Tl simple-CDld质粒,测序结果显示与人类基因库中CD1d基因序列一致；成功构建慢病毒载体质粒PCDH-CMV-MCS-EFl-copGFP-CDld,经病毒包装后得到携带CD1d的重组慢病毒颗粒Lenti-hCDld,浓缩后病毒滴度达到4.4×108TU/ml；重组慢病毒颗粒Lenti-hCDld成功转染胰腺癌细胞株PANC-1。结论可以通过基因克隆、慢病毒载体构建、细胞转染等方法稳定转染CD1d基因至胰腺癌细胞株PANC-1,这为后期以CDld-NKT为桥梁,进行细胞免疫原性变化、免疫上下游通路的研究和胰腺癌的基因治疗奠定了基础。更多还原

【Abstract】 Objective Use of gene clone, lentiviral vector construction, cell transfection methods to make people CDld gene stability transfection pancreatic cancer cell lines PANC-1, provide a new perspective to explore the pancreatic cancer gene therapy, lay the foundation of studying the cells immunogenicity change, upstream and downstream immune pathways using the CDld-NKT as the bridge.Methods Human CDld gene was cloned by cloning technology,which connected to the pEASY-Tl simple cloning vector,and then sequenced;By recombinant technology.human CDld gene was enclosed to lentiviral vector PCDH-CMV-MCS-EFl-copGFP,which was concentrated and purificated after determining the concentration;MOI was determined by pre-test of lentiviral transfection.PANC-1cell line was transfected by lentivirus-mediated hCDld.Results Human CDld gene was successfully cloned and constructed into pEASY-Tl-simple-CDld plasmid.It was showed that the cloned hCDld gene is identical with the human genebank by sequencry;Lentivirus vector(PCDH-CMV-MCS-EFl-copGFP-CDld)was successfullyconstructed,and the mature Lenti-hCDld was packaged,and the virus titer of which concentrated to4.4xlO8TU/ml;Lenti-hCDld stably transfected pancreatic cancer cell line PANC-1.Conclusion Human CDld gene can be transfected by gene clone, lentiviral vector construction, cell transfection methods to pancreatic cancer cell lines PANC-1, lay the foundation of studying the cells immunogenicity change, upstream and downstream immune pathways using the CDld-NKT as the bridge, provide a new perspective to explore the pancreatic cancer gene therapy.更多还原