【作者】 朱东明；

【导师】 李德春；

【作者基本信息】 苏州大学 ， 普通外科学， 2008， 博士

【摘要】 第一部分EPAS1、VEGF在胰腺癌中表达的意义及相关性目的研究胰腺癌组织中缺氧诱导因子2(EPAS1/HIF-2α)、血管内皮生长因子(VEGF)和微血管密度(MVD)的表达及与临床病理特征之间的关系。方法应用逆转录聚合酶链反应(RT-PCR)、免疫印迹(western blotting)、免疫组化(IHC)S-P法检测60例胰腺癌及相应正常胰腺组织中EPAS1、VEGF mRNA和蛋白的表达和分布,同时检测MVD作为判断血管生成的指标,分析其间的相关性以及与肿瘤临床病理特征之间的关系。结果EPAS1、VEGF和MVD在胰腺癌组织中的表达明显高于正常胰腺组织, VEGF在mRNA和蛋白水平均增高(t=6.10,P=0.0003;t=98.41,P=0.0001),但EPAS1只在蛋白水平增高(t=22.51,P=0.0001)。此外,三者之间的表达具有显著相关性(EPAS1和VEGF之间,r=0.73583,P=0.0041;VEGF和MVD之间,r=0.85783, P= 0.0001;EPAS1和MVD间,r=0.64062,P=0.0003), EPAS1和VEGF的阳性表达与胰腺癌的TNM分期、肿瘤大小有关。结论胰腺癌组织中EPAS1和VEGF呈过量表达,且EPAS1的表达与VEGF及MVD呈显著正相关。EPAS1可通过上调VEGF表达来促进胰腺癌血管生成从而在胰腺癌的发生发展中起重要作用。第二部分靶向EPAS1的小干扰RNA的设计与功能筛选目的构建编码EPAS1 mRNA的shRNA质粒表达载体,并筛选出基因沉默效果最明显的shRNA质粒表达载体。方法根据EPAS1的基因序列设计三条shRNA作为RNA干扰靶点,分别构建3个shRNA质粒表达载体并通过PCR和测序进行鉴定。经鉴定正确后分别稳定转染PANC-1细胞,实时荧光定量PCR(RQ-PCR)和western blotting分别从mRNA和蛋白质水平检测抑制效果。结果构建的质粒表达载体经PCR鉴定均可扩增出预期条带,测序证明质粒构建成功。靶向EPAS1基因的shRNA对稳定转染的PANC-1细胞中EPAS1 mRNA和蛋白质表达均有抑制作用:在常氧状态下,EPAS1-shRNA-1、EPAS1-shRNA-2和EPAS1-shRNA-3对EPAS1 mRNA表达的抑制率分别为67.5%、46.8%、47.9%(P<0.05),在缺氧状态下的抑制率分别为86.6%、55.1%、56.4%(P<0.05);在常氧状态下,EPAS1-shRNA-1、EPAS1-shRNA-2、EPAS1-shRNA-3对EPAS1蛋白表达的抑制率分别为48.8%、18.9%、28.9%(P<0.05),在缺氧状态下的抑制率分别为73.6%、40.0%、37.2%(P<0.05)。其中以EPAS1-shRNA-1的抑制作用最明显。结论成功构建了靶向EPAS1基因的shRNA质粒表达载体.其中抑制效果最明显的shRNA质粒表达载体为pGCsi-U6/Neo/GFP/EPAS1-shRNA-1质粒。第三部分rAAV2-EGFP-U6-EPAS1-siRNA的构建与鉴定目的构建并制备携带靶向EPAS1基因的shRNA的2型重组腺相关病毒载体。方法将PCR法扩增所得EGFP-U6-EPAS1-siRNA片段插入载体质粒pSNAV2.0- lacz-α的EcoR I和Sal I酶切位点,构建重组质粒pSNAV2.0-EGFP-U6-EPAS1- siRNA。以HSV1-rc/ΔUL2为辅毒,包装rAAV2-EGFP-U6-EPAS1-siRNA病毒。对所获病毒载体进行SDS-PAGE、PCR鉴定分析和滴度测定。结果SDS-PAGE、PCR鉴定分析结果表明靶向EPAS1基因的shRNA片段成功包装入rAAV2载体,病毒纯度在95%以上。点杂交法测定rAAV2基因组滴度约为1×1012v.g. /L。结论成功制备了rAAV2-EGFP-U6-EPAS1-siRNA病毒载体。第四部分EPAS1-siRNA对人胰腺癌细胞生物学行为影响的体外研究目的观察由rAAV2携带的EPAS1-siRNA对体外培养的人胰腺癌细胞的抑制作用。方法将rAAV2-EGFP-U6-EPAS1-siRNA转染入人胰腺癌细胞株PANC-1中,并测定转染效率。分别在常氧和缺氧状态下进行细胞培养。RT-PCR、IHC S-P法、western blotting检测EPAS1和VEGF mRNA和蛋白表达,MTT法检测PANC-1增殖能力,Giemsa染色、Hoechst 33342和PI双染、Annexin V-FITC/PI双染和DNA Ladder检测细胞凋亡,ABC-ELISA检测分泌性VEGF水平, CAM试验检测血管生成,CAM成瘤试验检测肿瘤生长能力。结果rAAV2-EGFP-U6-EPAS1-siRNA被成功地转染入PANC-1细胞中,FCM测定转染效率在40%左右。与阴性对照组和空白对照组比较,实验组PANC-1细胞的EPAS1和VEGF mRNA的表达明显受到抑制(EPAS1 mRNA:常氧状态下,F=44.64,P=0.0002;缺氧状态下,F=53.33,P=0.0012。VEGF mRNA:常氧状态下,F=25.36,P=0.0012;缺氧状态下,F=56.62,P=0.0001)。同样的结果见于蛋白表达的检测中(EPAS1蛋白:常氧状态下,F=46.12,P=0.0002;缺氧状态下,F=602.11,P=0.0001。VEGF蛋白:常氧状态下,F=46.69,P=0.0002;缺氧状态下,F=64.56,P=0.0001)。同时,实验组细胞增殖能力下降(F=83.85,P=0.0001),凋亡率增高(在缺氧状态下,F=124.98, P=0.0001),分泌性VEGF水平下降(在缺氧状态下,F=9.83,P=0.0128)。实验组CAM的血管计数明显少于对照组(常氧状态下F=29.04,P=0.0008,缺氧状态下,F=88.01,P=0.0001),CAM移植瘤生长受抑制,肿瘤体积小于对照组(F=64.04, P=0.0001)。结论在体外,rAAV2-EGFP-U6-EPAS1-siRNA能抑制人胰腺癌细胞PANC-1的EPAS1和VEGF基因表达,并能抑制细胞增殖,促进凋亡,抑制CAM血管形成与移植瘤生长。第五部分EPAS1-siRNA对人胰腺癌抑制作用的体内研究目的在整体水平研究rAAV2-EGFP-U6-EPAS1-siRNA对人胰腺癌生长、转移和浸润行为的抑制作用。方法建立人胰腺癌裸鼠皮下移植瘤模型、腹腔转移模型和原位移植模型。在皮下模型中,按1×1012v.g./kg标准在瘤内分别注射rAAV2-EGFP-U6-EPAS1-siRNA、rAAV2-EGFP和NS,观察抑瘤效果,IHC S-P法检测EPAS1、VEGF蛋白表达及MVD值,TUNEL和透射电镜检测凋亡。在腹腔转移模型中,按1×1012v.g./kg标准分别腹腔注射rAAV2 -EGFP-U6-EPAS1-siRNA、rAAV2-EGFP和NS,计数种植瘤数目。在原位模型中,按1×1012v.g./kg标准分别腹腔注射rAAV2-EGFP-U6-EPAS1-siRNA、rAAV2-EGFP和NS,观察抑瘤效果及转移情况。结果实验组皮下移植瘤生长明显受抑制,体积和重量明显小于阴性对照组和空白对照组(体积,F=171.09,P=0.0001;重量,F=199.50,P=0.0001),EPAS1、VEGF蛋白表达弱于对照组(EPAS1,H=3.18,P=0.2043;VEGF,H=6.16,P =0.046), MVD值明显小于对照组(F=55.42,P=0.0001),凋亡指数明显小于对照组(F= 286.57,P=0.0001)。实验组裸鼠腹腔种植瘤数目明显少于对照组(F=52.23,P=0.0001)。实验组裸鼠原位移植瘤的体积和重量明显小于对照组(体积,F=27.52,P=0.0001;重量, F=102.41, P=0.0001),远处转移率是16.7%,而对照组裸鼠远处转移率是50%。结论在体内实验中,rAAV2-EGFP-U6-EPAS1-siRNA能明显抑制人胰腺癌的生长与转移,促进凋亡,减少新生血管形成,显示一定的抑瘤效应。更多还原

【Abstract】 PartⅠThe significance and correlation of EPAS1 and VEGF expression in pancreatic carcinomaObjective To investigate the expressions of Endothelia PAS domain protein1 （EPAS1）,vascular endothelial growth factor（VEGF） and microvessel density（MVD） as well as the relationships among them,and the relationships to the clinicopathologic features of human pancreatic carcinoma.Methods In 60 cases of resected specimens of pancreatic carcinoma and their corresponding normal pancreatic tissues,RT-PCR was used to detect the expression levels of EPAS1 and VEGF mRNAs,western blotting and immunohistochemical SP method were used to examine the expression levels and distributions of EPAS1 and VEGF proteins respectively. MVD regarded as a marker of angiogenesis was explored also.The correlations among them and their relationships to the clinicopathologic characteristics of human pancreatic carcinoma were analyzed.Results Higher expressions of EPAS1,VEGF and MVD were detected in pancreatic carcinoma than in normal pancreatic tissue,for VEGF both at mRNA and protein level（t=6.10,P=0.0003; t=98.41,P=0.0001）, for EPAS1 only at protein level （t= 22.51,P=0.0001）. Furthermore, significant correlations were observed among them （between EPAS1 and VEGF, r=0.73583,P=0.0041; between VEGF and MVD, r= 0.85783, P=0.0001; between EPAS1 and MVD, r=0.64062, P=0.0003）, the positive expressions of EPAS1 and VEGF were closely related with TNM staging and tumor size of pancreatic carcinoma.Conclusions Both EPAS1 and VEGF are overexpressed in pancreatic carcinoma, and the expression of EPAS1 is directly correlated with that of VEGF and the value of MVD significantly. EPAS1 may be involved in the angiogenesis of pancreatic carcinoma by upregulating the expression of VEGF, and play an important role in the carcinogenesis and aggression in pancreatic carcinoma.PartⅡDesign and screening of the small interfering RNA targeting EPAS1 geneObjective To construct a plasmid expression vector coding for the short hairpin RNA （shRNA） targeting EPAS1 mRNA.Methods Three plasmid expression vectors coding for shRNA targeting EPAS1 gene sequence were constructed.The recombinant plasmids were identified by PCR and sequencing, and then transfected stably into PANC-1 cells respectively. The EPAS1 gene silencing effect was detected by quantitative RT-PCR and western blotting.Results The expected bands were amplified from the plasmids coding for shRNA by PCR,then sequencing confirmed that the EPAS1-shRNA plasmids were successfully constructed. Transfection of PANC-1 cells with shRNA plasmids resulted in an inhibition of EPAS1 mRNA and protein expressions respectively.For EPAS1-shRNA-1, EPAS1- shRNA-2 and EPAS1-shRNA-3,the inhibitory rates of EPAS1 mRNA expression were 67.5%, 46.8% and 47.9% in normoxic condition （P<0.05）,as 86.6%, 55.1% and 56.4% in hypoxic condition （P<0.05）;And the inhibitory rates of EPAS1 protein expression were 48.8% , 18.9 % and 28.9% respectively in normoxic condition （P<0.05）,compared with 73.6%, 40.0% and 37.2% respectively in hypoxic condition （P<0.05）.The most potent effect of silencing EPAS1 gene expression was conducted by EPAS1-shRNA-1.Conclusions The plasmid expression vectors coding for shRNA targeting EPAS1 mRNA have been constructed successfully, of which pGCsi-U6/Neo/GFP/EPAS1- shRNA-1 effectively silences EPAS1 gene the most in PANC-1 cells.PartⅢConstruction and identification of rAAV2-EGFP-U6-EPAS1-siRNAObjective To construct and prepare the recombinant adeno-associated virus serotype 2 （rAAV2） vector for expressing short hairpin RNA（shRNA） targeting EPAS1 mRNA. Methods By inserting the sequence that contained EGFP-U6-EPAS1-siRNA into the EcoR I and Sal I site of vector plasmid pSNAV2.0-lacz-α,we constructed the recombinant vector of plasmid pSNAV2.0-EGFP-U6-EPAS1-siRNA.The recombinant vector of plasmid,BHK cell lines and helper virus HSV1-rc/ΔUL2 were used for the package of rAAV2-EGFP-U6-EPAS1-siRNA vector.The viral purity,identity,titer of rAAV2 viral stock were analyzed by the methods of SDS-PAGE,PCR and dot-blot respectively.Results The results of SDS-PAGE and PCR indicated that the shRNA fragment targeting EPAS1 was successfully packaged into rAAV2,and the viral purity of rAAV2 was above 95%.The titer of rAAV2 was approximately 1×1012v.g. /L.Conclusions The viral vector of rAAV2-EGFP-U6-EPAS1-siRNA is successfully constructed and prepared.Part IV Study of the impact of biological behaviors on human pancreatic carcinoma cells by EPAS1-siRNA in vitroObjective To observe the inhibitory effects of EPAS1-siRNA mediated by rAAV2 on human pancreatic carcinoma cells cultured in vitro.Methods rAAV2-EGFP-U6-EPAS1-siRNA was transfected into human pancreatic carcinoma cell line PANC-1, and the efficiency of transfection was detected. The cells were cultured under normoxic and hypoxic conditions respectively.Then RT-PCR, immunohistochemical SP method and western blotting were used to detect EPAS1, VEGF mRNA and protein expression. While MTT assay was used to determine proliferation rate of PANC-1. Detection of apoptosis was manipulated by the means of Giemsa staining, Hoechst 33342/PI double staining, Annexin V-FITC/PI double staining and DNA Ladder.And secretory VEGF level was measured by ABC-ELISA.Furthermore, CAM assay was used to detect angiogenesis in CAM.At last,a transplanted tumor model of pancreatic carcinoma on CAM was established to check the growth ability of tumor.Results rAAV2-EGFP-U6-EPAS1-siRNA was successfully transfected into PANC-1 cells, and the transfection efficiency detected by FCM was about 40%. Expressions of EPAS1 and VEGF mRNA were significantly inhibited in PANC-1 cells of experimental group,compared with in that of negative control group or blank control group （for EPAS1 mRNA: in normoxic condition, F=44.64, P=0.0002; in hypoxic condition, F=53.33, P=0.0012. for VEGF mRNA: in normoxic condition, F=25.36, P=0.0012; in hypoxic condition,F=56.62, P=0.0001）. The same results were found as detecting their protein expressions （for EPAS1 protein: in normoxic state, F=46.12,P=0.0002; in hypoxic state, F=602.11,P=0.0001. for VEGF protein: in normoxic state, F=46.69, P=0.0002; in hypoxic state, F=64.56, P=0.0001）.Meanwhile,in experimental group cells, ability of proliferation was inhibited（F=83.85, P=0.0001）, apoptotic rate increased （in hypoxic state, F=124.98, P=0.0001）, and secretory VEGF level decreased（in hypoxic state,F=9.83, P=0.0128）.The count of newly formed blood vessels of experimental group was significanly less than that of control groups（in normoxic condition, F=29.04, P=0.0008; in hypoxic condition, F=88.01, P=0.0001）. Lastly, growth of transplanted tumor on CAM was depressed, as tumor of experimental group was smaller than that of control groups （F=64.04, P=0.0001）.Conclusions rAAV2-EGFP-U6-EPAS1-siRNA can inhibit expressions of EPAS1 and VEGF gene, cell proliferation, and promote apoptosis in human pancreatic carcinoma cell of PANC-1 in vitro. It can also inhibit angiogenesis and growth of transplanted tumor on CAM.Part V Research for inhibition human pancreatic carcinoma by EPAS1-siRNA in vivoObjective To research inhibition of biological behaviors of human pancreatic carcinoma such as growth,metastasis and invasion by rAAV2-EGFP-U6-EPAS1-siRNA in vivo.Methods Human pancreatic carcinoma transplanted subcutaneously in nude mouse,peritoneal metastasis model of human pancreatic carcinoma and model of surgical orthotopic implantation of human pancreatic carcinoma were established firstly. rAAV2- EGFP-U6-EPAS1-siRNA, rAAV2-EGFP and NS were intratumorally injected with 1×1012 v.g./kg respectively in subcutaneous xenograft model, inhibitory effect of tumor growth was observed , EPAS1 and VEGF protein expressions as well as value of MVD were detected by immunohistochemical SP method, TUNEL staining and transmission electron microscopy were used to determine apoptosis. rAAV2-EGFP-U6-EPAS1-siRNA, rAAV2- EGFP and NS were intraperitoneal injected with 1×10¹² v.g./kg respectively in peritoneal metastasis model of nude mouse, the number of metastases was counted. rAAV2-EGFP- U6-EPAS1-siRNA, rAAV2-EGFP and NS were intraperitoneal injected with 1×10¹²v.g./kg respectively in the model of surgical orthotopic implantation, inhibitory effect of tumor growth and metastasis of tumor were observed.Results Growth rate of subcutaneous xenograft significantly decreased in experimental group , the tumor’s size and weight of experimental group were significantly lesser than those of negative control group and blank control group （for volume, F=171.09, P=0.0001; for weight, F=199.50, P=0.0001）.For experimental group, expressions of EPAS1 and VEGF proteins were weaker than those of control groups （for EPAS1, H=3.18, P=0.2043; for VEGF, H=6.16, P=0.046）, values of MVD and apoptotic index（AI） were significantly lesser than those of control groups （for MVD, F=55.42, P=0.0001; for AI, F=286.57, P=0.0001）. And number of peritoneal seeding was less in experimental group than in control groups（F=52.23, P=0.0001）. The size and weight of orthotopic implantation in experimental group were significantly less than those in control groups （for volume, F=27.52, P=0.0001; for weight, F=102.41, P=0.0001）. While distant metastasis rate was 16.7% in experimental group, compared with 50% in control groups.Conclusions rAAV2-EGFP-U6-EPAS1-siRNA can dramatically inhibit growth and metastasis, promote apoptosis and reduce neovascularization of human pancreatic carcinoma in vivo.更多还原