【作者】 江建新；

【导师】 秦仁义；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 第一部分蛋白质组学分析重组腺病毒介导hSSTR2基因转染对胰腺癌细胞蛋白表达的影响目的探讨hSSTR2基因转染对胰腺癌细胞Panc-1蛋白表达的影响；寻找新的胰腺癌敏感治疗靶点。方法利用腺病毒载体Ad．CMV．eGFP．hSSTR2．将hSSTR2全长cDMA导入胰腺癌细胞Panc-1，用RT-PCR及Western Blot分别检测hSSTR2在mRNA水平及蛋白水平上的表达。采用荧光差异蛋白组学(2D-DIGE)技术分离并筛选转染hSSTR2的实验组、空载体对照组以及空白组胰腺癌细胞之间差异表达蛋白；用反射式基质辅助激光解吸附电离串联飞行时间质谱(MALD1-TOF/TOF)技术对差异蛋白进行鉴定。采用Western Blot验证波形蛋白、PKM2、SEPT11的差异表达；采用免疫组化检测上述三种蛋白在胰腺癌组织中的表达。结果hSSTR2成功的转染了胰腺癌细胞，建立了hSSTR2阴性和阳性表达胰腺癌细胞Panc-1荧光差异蛋白表达图谱。经DeCyder v6．5软件分机，其得到21个有统计学意义的差异蛋白质点；选择1．3倍以上的差异点18个，经质谱鉴定得到13个蛋白质，在转染hSSTR2的胰腺癌细胞中低表达蛋白为GMP合酶、磷酸化应激诱导蛋白、谷氨酸脱氢酶、Septin-11、波形蛋白、异柠檬酸脱氢酶α亚基、线粒体内膜易位酶；高表达蛋白为真核延长因子(?)α(?)、丙酮酸激酶异构体M2型、烯酰CoA水合酶、转录调节因子1-β、Mitofilin、HSP105。结论实验筛选的差异蛋白功能涉及到糖、脂肪以及核酸代谢，细胞生长调节和细胞凋亡等过程。对这些蛋白质功能的进一步验证，将有助于解析缺失hSSTR2基因表达胰腺癌细胞生长、侵袭转移的分子机制，从而为寻找新的胰腺癌敏感治疗靶点奠定基础。第二部分Lv-VIM-shRNA的构建和鉴定目的构建人VIM基因RNA干扰(RNAi)慢病毒表达载体，并评价其在胰腺癌细胞中的干扰效果。方法利用在线软件设计3条人VIM基因shRNA序列，并选用一条文献报道序列，合成、退火形成双链寡核酸后克隆到pGCL-GFP／U6载体的黏性末端，将连接产物转化到DH5α感受态细胞，经PCR筛选阳性克隆、测序鉴定。实时定量PCR和Western Blot用于在转染了过表达质粒pEGFP-N1-hVIM的293T细胞中筛选有效的pGCL-GFP／U6-shRNA，将其和pHelper1．0、pHelper2．0共转染293T细胞，包装产生慢病毒颗粒并测定病毒滴度。Real-time PCR和Western Blot在胰腺癌细胞Panc-1中验证慢病毒(Lv-VIM-shRNA)干扰Vim mRNA和波形蛋白表达效果。结果成功构建携带VIM基因干扰序列的慢病毒表达载体，病毒滴度为2×10~9TU／ml。在胰腺癌细胞Panc-1中验证其干扰VIM基因效果，Vim mRNA和Vimentin表达明显下调。结论成功构建出针对人VIM基因RNA干扰(RNAi)慢病毒载体，体外感染胰腺癌细胞Panc-1后可有效抑制VIM基因和蛋白的表达。为研究VIM在胰腺癌中涉及肿瘤发生和进展的信号转导机途径以及寻找新的治疗靶点提供一种研究工具。更多还原

【Abstract】 PartⅠEffection of Differential Proteins Expression in PancreaticCancer Cells Transfected with Ad.CMV.eGFP.hSSTR2 by ProteomeAnalysisObjective To study the effection of differential proteins expression in pancreatic cancercells Panc-1 transfered hSSTR2 gene in vitro and search new sensitive therapeutic targetsof pancreatic cancer.Methods The full length hSSTR2 cDNA was introduced into pancreatic cancer cell linePanc-1 by adenovirus mediated transfection and stable expression of RNA and protein ofhSSTR2 were detected by RT-PCR and Westen blot.Comprehensive analyses of proteinswere focused on total protein spots exhibiting statistical alternations among theexperimental group,vector control and mock control by 2D-DIGE analysis.Proteinidentification was done by peptide mass finger printing with matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry(MALDI—TOF/TOF).Western blotwas performed to verify the differential expression of vimentin,PKM2 and SEPT11.Immunohistochemistry was performed to detect the expression of vimentin,PKM2 andSEPT11 in pancreatinc carcer tissues.Results hSSTR2 gene was transfected into Panc-1 pancreatic cancer cells in vitro successfully,and fluorescence difference protein expression patterns were establishedbetween hSSTR2 negative and positive expression of pancreatic cancer cells Panc-1.ByDeCyder v6.5 software analysis,a total of 21 have been statistically significant differencesin protein spots.There had the difference 18 points (greater then 1.3-fold) and these proteinspots was identified by mass spectrometry for 13 proteins.Proteins found to have lowerabundance levels in pancreatic cancer cells Panc-1 with hSSTR2 positive expressioncompared to negative expression included GMP synthase,Stress induced phosphoprotein1,Glutamate dehydrogenase 1,Septin-11,vimentin,Isocitrate dehydrogenase [NAD] subunitalpha,Import inner membrane translocase subunit TIM50.Proteins found to have increasedabundance levels in Panc-1 pancreatic cancer cells with hSSTR2 negative expressioncompared to positive expression included Elongation factor 1-alpha 1,Isoform M2 ofPyruvate kinase isozymes M1/M2,Enoyl-CoA hydratase tripartite motif-containing 28protein,Isoform 1 of Mitochondrial inner membrane protein,Heat shock protein 105 kDa.Conclusion The function of screening difference proteins involved in the process ofmetabolism of sugar,fat and nucleic acid,and the regulation of cell growth.It was beenhelpful for revealing the molecular mechanisms of growth,invasion and metastasis ofpancreatic cancer cell which missed hSSTR2 gene expression to further validate theirfunctions.PartⅡConstruction and Identification of Lv-VIM-shRNAObjective To construct a lentiviral expression vector for RNA interference (RNAi) ofhuman VIM gene;and assess its gene silencing effect in pancreatic cancer cell line Panc-1.Methods Three pairs of human VIM gene short hairpin RNA (shRNA) sequences weredesigned following the procedure of a software available on-line and one pair came fromdocument.After synthesis and annealing,four double-stranded oligonucleotides (dsOligo) were cloned into the pGCL-GFP/U6 plasmid,which were subsequently confirmed by PCRand DNA sequencing analysis.Real-time PCR and Western Blot were used to screen theeffective pGCL-GFP-shRNA plasmid in 293T cells,then the most effective one was packedinto the recombinant lentivirus Lv-VIM-shRNA with lentiviral packing materials pHelper 1.0 and pHelper 2.0 in 293T cells.The titer of lentivirus was determined by hole-by-dilutiontiter assay.The silencing effect of Lv-V1M-shRNA in Panc-1 cells was validated byreal-time PCR and Western Blot.Results An effective Lv-VIM-shRNA was successfully constructed.The titer of lentiviruswas determined on 2×10~9TU/ml.The expression of VIM mRNA and vimentin wasdown-regluated in the Panc-1 cells infected with Lv-VIM-shRNA.Conclusion An effective Lv-VIM-shRNA could inhibit the expression of VIM gene inPanc-1 cells in vitro,which provides a tool for investigating the role of VIM gene in thesignaling pathway involved in tumorigenesis and progression of pancreatic cancer andsearching new therapitic targets.更多还原