【作者】 宋禾；

【导师】 董明；

【作者基本信息】 中国医科大学 ， 外科学， 2016， 博士

【摘要】 目的胰腺癌生物学行为恶劣,90%的患者在初诊一年之内死亡,预后极差,5年生存率不足5%,中位生存时间不足半年。30年来病人预后没有得到明显改善,研究新型靶点治疗尤为关键。TRPV6是一种高选择钙离子通道蛋白。在众多肿瘤组织中,起到参与肿瘤发生发展的作用。TRPV6 m RNA和蛋白的表达是在前列腺癌组织中强烈表达,在结肠癌,乳腺癌、甲状腺癌、甲状旁腺癌和卵巢癌上亦高有表达,而在食管癌、肾癌及非小细胞肺癌中低表达。现在尚不能完全明确TRPV6起到癌基因或抑癌基因的作用。本研究探讨TRPV6钙离子通道的表达对胰腺癌患者预后的影响及干扰该基因是否抑制体外胰腺癌细胞增殖,促进凋亡为治疗胰腺癌提供新的治疗方向。方法收集2006年1月至2014年1月中国医科大学附属第一医院具有完整临床病理学资料的胰腺导管腺癌手术切除标本76对,均有癌旁组织作为对照。另有6对胰腺癌及配对的癌旁组织新鲜标本。(1)免疫组织化学(IHC)、免疫印迹(WB)检测76例配对的胰腺癌和癌旁组织中TRPV6表达水平,探讨其表达的临床病理学意义及与患者预后的关系。(2)细胞培养:AsPC-1、BxPC-3、Capan-2、SW1990、PANC-1细胞按常规培养。(3)Western blot检测TRPV6蛋白在胰腺癌细胞和组织中表达水平。(4)CCK8法检测沉默TRPV6对胰腺癌细胞(Capan-2、SW1990)的生长抑制作用。(5)流式细胞仪检测沉默TRPV6对胰腺癌细胞(Capan-2、SW1990)凋亡及细胞周期的影响。(6)Transwell实验检测胰腺癌细胞(Capan-2、SW1990)侵袭和迁移能力(7)Western blot检测沉默TRPV6对细胞增殖、凋亡等信号通路相关靶蛋白表达的影响。(8)沉默TRPV6联合三种化疗药物做细胞毒性检测。结果1、免疫组化结果:TRPV6在癌组织中阳性表达率为57.9%(44/76),在76例配对胰腺癌组织表达高于癌旁组织(t=3.039;P=0.003)。TRPV6主要呈胞浆表达。在Western blot发现TRPV6的蛋白在癌中表达比癌旁组织要高。2、TRPV6蛋白表达与临床病理学参数关系:TRPV6表达与肿瘤大小(χ~2=5.076,P=0.024)及TNM分期(χ~2=4.791,P=0.029)呈正相关,但是与年龄、性别、分化程度、淋巴结转移、神经浸润、血管侵袭、肿瘤标记物、肝转移无明显相关性。单因素分析发现,TRPV6阳性表达的胰腺癌患者预后更差(P=0.003)。此外,TNM分期(P=0.016)及血管侵袭(P=0.012)同样是影响胰腺癌的预后因素。多因素分析发现:TRPV6阳性表达(P=0.029)和血管侵袭(P=0.033)是影响胰腺癌患者预后的独立危险因素。3、检测TRPV6在各株胰腺癌细胞株中(AsPC-1、BxPC-3、Capan-2、SW1990、PANC-1)的表达情况,其中Capan-2、SW1990较高表达,因此,选择Capan-2、SW1990作为后续TRPV6干扰实验的胰腺癌细胞系。同时设计三条干扰序列,采用Western blot方法检测其干扰效率,结果发现TRPV6干扰后,TRPV6-siRNA1,TRPV6-siRNA2,TRPV6-siRNA3三组的表达水平分别为对照组的42.1%±3.4(p<0.001),63.5%±2.8,84.3%±2.8(p<0.05)。其中干扰序列1的效果最佳,用于后续试验。在CCK8实验中,我们选择Capan-2、SW1990二组细胞株。下调TRPV6对Capan-2、SW1990细胞的生长抑制作用明确,下调TRPV6对Capan-2在24小时、48小时、72小时的细胞增殖抑制率分别为34.6±2.3%、34.4±2.8%、37.9±1.9%。对SW1990在24小时、48小时、72小时的细胞增殖抑制率分别为52.1±3.2%、36.4±2.9%、44.2±2.1%。我们采用流式细胞仪对Capan-2、SW1990细胞的凋亡进行检测分析。在下调TRPV6后,Capan-2凋亡的细胞比率24.12±2.03%较对照组5.88±0.78%有显著增多(p(27)0.05);SW1990凋亡的细胞比率20.89±2.56%较对照组6.22±0.54%有显著增多(p(27)0.05)。在Capan-2和SW1990细胞中,转染TRPV6-si RNA1与转染NC相比可增加G0/G1期细胞,减少S期细胞比例,(p(27)0.05)。结合增殖实验结果,说明下调TRPV6可促进胰腺癌癌细胞停滞于G1期。迁移和侵袭实验发现:在Capan-2细胞中,转染TRPV6-Si的细胞与阴性对照组(Neg.Cont)相比,体外迁移和侵袭能力均有不同程度的下降,差异具有统计学意义(迁移,115±6 VS.48±4,p<0.05;侵袭,92±5VS.43±3,p<0.01);在SW1990细胞中,转染TRPV6-Si抑制其表达后细胞迁移侵袭能力明显强于其阴性对照组,差异具有统计学意义(迁移,152±5 VS.72±3,p<0.001;侵袭,140±6VS.80±4,p<0.01)。进一步深入研究发现:在下调TRPV6后,PCNA,Bcl-2,MMP9,Numb蛋白表达明显下调,而BAX,E-cadherin的表达则明显上调,β-catenin无明显变化。在下调TRPV6后,分别联合吉西他滨、奥沙利铂和顺铂对胰腺癌Capan-2进行细胞毒性检测。发现只有与奥沙利铂联合作用时,可显著增强奥沙利铂化疗药物的敏感性。结论1、TRPV6在胰腺癌组织表达高于癌旁组织。TRPV6表达高表达与肿瘤大小、TNM分期呈正相关。TRPV6表达、TNM分期、血管侵袭是影响胰腺癌的预后因素。TRPV6表达和血管侵袭是影响胰腺癌患者预后的独立危险因素。2、沉默TRPV6可以抑制胰腺癌细胞的增殖,促进凋亡,使细胞在G1期阻滞,抑制胰腺癌细胞的侵袭和转移。提高奥沙利铂的药物敏感性。3、TRPV6通过调控PCNA,Bcl-2,BAX,E-cadherin,MMP9影响胰腺癌细胞的生物学行为。更多还原

【Abstract】 ObjectivePancreatic cancer is the highly frequently occuring malignant disease with the poor prognosis,and more than 90% patients will come to death in 1 year and 5-year survival rate is less than 5%,and prognosis cound not be elvated in the past 30 years.TRPV6 is the highly selected calcium channel protein,which could participate in the development of various tumors.The aim of study is to elucidate the influence of TRPV6 on the pancreatic cancer,and role and the related mechanism of TRPV6 in the proliferation and apoptosis of pancreatic cancerMethodsFrom Jan.2006 to Jan.2014,there were 76 pairs of sample of pancreatic ductal adenocarcinoma tissue and adjacent noncancerous tissue plus 6 pairs of pancreatic cancer samples and its counterpart adjacent noncancerous tissue to detect.(1)IHC and WB were used to detect the expression of TRPV6 to investigate the raltionship between cliniopathological parameter and prognosis.(2)Cell culture,AsPC-1,Bx PC-3,Capan-2,sw1990,PANC-1 were cultured.(3)WB was used to detect the protein expression of TRPV6 in the various pancreatic cancer cells.(4)Cell Counting Kit-8(CCK8)was used to detect the proliferative inhibitory effect of TRPV6-Si in pancreatic cancer cells(Capan-2,sw1990).(5)Flow cytometry(FCM)was to detect the pancreatic cancer cell cycle and the apoptosis after use of TRPV6-SiRNA.(6)Transwell was to detect the migration and invasion of pancreatic cancer cell(7)Western blot was to detect the target protein expression of its related proliferative and apoptotic signaling pathway.(8)Combination of TRPV6-SiRNA and 3 differentchemical drug to detect the sensitivity of drug.Results(1)IHC: TRPV6 was highly detected in malignant tissues.Positive expression of TRPV6 was related to the tumor size and TNM stage.(2)Kaplan-Meier analysis showed that a possitive expression of TRPV6 was associated with the poor prognosis.Multivariate analysis showed vessel invasion and TRPV6 were the independent risk factors correlated with the prognosis.WB: the protein expression of the pancreatic cancer is significantly higher than the counterpart normal tissue in 6 cases.(3)CCK8: the growth inhibitory effect is definite by TRPV6-SiRNA(4)FCM: the rate of apoptosis effect is promoted by TRPV6-SiRNA;G1 was arrested in the cell cycle.(5)TRPV6-SiRNA inhibited the migration and invasion of pancreatic cancer cell(6)TRPV6-SiRNA decreased the level of Numb、the antiapoptotic protein bcl-2、MMP9,and increased the level of the proapoptotic protein Bax、E-cadherin(7)Si-TRPV6 could increase the sensitity of oxalipalitin.ConclusionThe tumor size and TNM stage were associated with the expression of the TRPV6 in pancreatic cancer,the patients could get the better prognosis with lower TRPV6 expression.The TRPV6-SiRNA may inhibit the proliferation of the pancreatic cancer cell,induce apoptosis,arrest G1 and inhibit the invasion and migration.TRPV6-SiRNA could increase the sensitity of oxalipalitin.TRPV6 can regulate the biological behavior of pancreatic cancer cell via PCNA,Bcl-2,BAX,E-cadherin,MMP9.更多还原