【作者】 刘文松；

【导师】 秦仁义；

【作者基本信息】 华中科技大学 ， 普通外科， 2009， 博士

【摘要】 胰腺癌是预后极差的人体恶性肿瘤之一。胰腺癌患者早期症状不明显，确诊时多数已属晚期，往往已失去手术时机，五年生存率不到5％。包括化疗在内的综合治疗是提高胰腺癌患者生存率的关键，但现有的化疗药物大多对胰腺癌不敏感。吉西他滨是一种新型的嘧啶类抗代谢药物，是目前治疗胰腺癌的主要药物之一，它能缓解临床症状，然而并不能明显延长患者生存时间，化疗耐药是导致治疗失败的主要原因。因此，解决胰腺癌细胞的化疗耐药已经成为临床上迫切需要解决的实际问题。近年来随着分子生物学研究的深入发展，以凋亡抑制蛋白为靶点的基因治疗备受关注，目前的实验研究已经取得了初步的成效和进展，可能为有效提高胰腺癌治疗效果提供了一个新的方向。研究表明，Survivin作为一种新发现的凋亡抑制蛋白(inhibitor of apoptosisproteins,IAPs)，具有抗凋亡和调节细胞周期的双重功能，在人类众多恶性肿瘤中广泛表达，而在正常组织中不表达或低表达。Survivin是至今克隆出的最小IAP，定位于染色体17q25，其基因全长14．7kb，在G2/M期特异性表达，编码16．5kb的蛋白。Survivin的表达与胰腺癌患者的预后、复发以及化疗耐药密切相关。Survivin主要通过其羧基端α-螺旋结构与有丝分裂的纺锤体微管结合，再通过BIR结构与caspase3和caspase7特异性地结合在一起，直接抑制凋亡终末效应器caspase3和caspase7的活性，阻断各种刺激诱导的细胞凋亡过程。Survivin亦可抑制细胞色素C从线粒体释放，阻断caspase激活，从而在上游阻断凋亡信号的传导抑制细胞凋亡。近年来研究发现阻断Survivin表达可抑制多种肿瘤细胞的生长。亦有学者实验证实，靶向抑制Survivin表达可增强胰腺癌细胞的放疗敏感性。由此我们结合Survivin的特性考虑，对Survivin基因进行阻断有可能会成为增强胰腺癌对吉西他滨化疗敏感性的一种新方法，而目前国内外文献尚未见相关报道。在本研究中，我们首先探讨Survivin在胰腺癌组织中的表达及与Bcl-2相关性研究，然后检测化疗诱导胰腺癌细胞凋亡Survivin表达的变化。构建靶向Survivin基因的siRNA真核表达载体，将构建的siRNA质粒通过脂质体导入到人胰腺癌Panc-1和BxPC3细胞中并筛选稳定转染的细胞系，观察其对survivin的mRNA和蛋白表达抑制作用，以及对胰腺癌细胞增殖、细胞周期、细胞侵袭力和对化疗药物吉西他滨的敏感性的影响。实验结果为今后利用靶向Survivin基因的siRNA增强胰腺癌细胞的化疗敏感性提供强有力的证据。本实验共分为以下四部分。第一部分Survivin在胰腺癌组织中的表达及与Bcl-2相关性研究目的本研究旨在检测人胰腺癌组织中Survivin的表达，分析其与临床病理特征的关系及与Bcl-2表达的相关性，探讨其在胰腺癌发生发展中的作用。材料和方法应用免疫组织化学SP法检测50例胰腺癌标本和14例正常胰腺组织中Survivin的表达，分析其与临床病理特征的关系，探讨Survivin与Bcl-2表达的相关性。结果1．50例胰腺癌标本中有31例出现Survivin阳性表达，阳性率为62％，而14例正常胰腺组织中均未检测到Survivin表达(P＜0．05)。2．Survivin蛋白的表达与肿瘤分化程度、临床分期有关(P＜0．05)，与患者性别、肿瘤部位及有无淋巴结转移无显著相关性(P＞0．05)。3．Bcl-2表达阳性、阴性组中Survivin的阳性表达率分别为77．8％和43．5％，两组比较差异有统计学意义(P＜0．05)。结论1．Survivin在胰腺癌组织中的高表达提示其对胰腺肿瘤的发生、发展可能起重要作用，Survivin与Bcl-2的表达呈正相关，二者可能发挥协同作用。2．Survivin的高表达可作为胰腺癌预后不良的生物学指标。第二部分化疗诱导胰腺癌细胞凋亡Survivin表达的变化及意义目的研究紫杉体体外化疗对胰腺癌细胞凋亡抑制蛋白survivin基因mRNA和蛋白表达的影响。材料和方法1．培养人胰腺癌细胞株SW1990，加入低浓度的紫杉醇(PA)，用流式细胞仪分别检测对照组和加药后24h、48h的细胞凋亡率。2．PT-PCR和免疫印迹技术(western-blot)检测survivin基因mRNA和蛋白的表达结果1．给予SW1990胰腺癌细胞低浓度的PA诱导后，对照组和加药后24h和48h胰腺癌细胞凋亡率(％)分别是2．59±0．35，14．75±1．29，22．65±2．80(P＜0．05)。2．加药后24h和48h胰腺癌细胞survivin mRNA分别比对照组提高了1．1倍和2．9倍(P＜0．05)；蛋白分别提高了1．3倍和3．6倍(P＜0．05)。结论紫杉醇化疗可促使胰腺癌细胞内survivin基因mRNA和蛋白的表达增加，这可能是胰腺癌细胞对化疗药物产生耐药性的因素之一。第三部分靶向Survivin基因的siRNA真核表达载体的构建及鉴定目的第一部分和第二部分显示，胰腺癌高表达Survivin蛋白，而且化疗可促使胰腺癌细胞内Survivin基因mRNA和蛋白的表达增加，本部分拟构建并鉴定针对survivin基因的siRNA真核表达载体，为下一步胰腺癌基因治疗奠定基础。材料和方法1．根据Survivin基因cDNA序列，在线设计2个针对目的基因的siRNA靶序列。2．定向克隆至真核表达载体psiRNA-hHlneo上。3．依次用酶切法和测序法对重组体进行鉴定。结果1．酶切鉴定，凝胶电泳分析，重组载体酶切后呈线性，电泳呈2700bp一条带，与预期结果符合。2．测序结果表明，目的序列插入正确。结论本研究成功构建了针对survivin的siRNA真核表达载体，可能为胰腺癌基因治疗提供一种新的治疗载体。第四部分靶向Survivin的siRNA对胰腺癌细胞增殖及对吉西他滨化疗敏感性影响目的本研究拟利用构建的靶向survivin基因的siRNA真核表达载体，稳定转染胰腺癌细胞株，探讨其对胰腺癌细胞增殖和对吉西他滨化疗敏感性的影响。材料和方法1．以psiRNA-survivin转染胰腺癌细胞Panc-1和BxPC3后，经G418筛选，建立稳定转染的细胞系。2．采用RT-PCR、Western Blot检测稳定转染后细胞survivin的mRNA和蛋白表达变化。3．体外培养各组胰腺癌细胞，绘制细胞生长曲线。4．流式细胞仪检测各组胰腺癌细胞的细胞周期。5．Transwell侵袭室方法检测各组胰腺癌细胞体外侵袭力。6．吉西他滨作用48h后，MTT检测各组细胞的增殖活性。7．吉西他滨作用48h后，流式细胞仪检测各组细胞凋亡指数。结果1．成功建立稳定转染psiRNA-survivin的Panc-1和BxPC3细胞系。2．重组质粒稳定转染胰腺癌细胞系后，Panc-1和BxPC3细胞survivin的mRNA分别下调了68．52％和64．32％(P＜0．05)，survivin的蛋白表达分别下调了76．68％和74．38％(P＜0．05)。3．与对照组相比，转染psiRNA-survivin组胰腺癌细胞增殖缓慢，生长曲线十分平缓，差异具有统计学意义(P＜0．05)。4．与对照组相比，转染psiRNA-survivin组胰腺癌细胞发生G0/G1期阻滞，G2/M期细胞减少，差异具有统计学意义(P＜0．05)。5．实验组胰腺癌细胞侵袭数目比对照组明显减少(P＜0．05)。6．psiRNA-survivin能显著增强吉西他滨对胰腺癌细胞的增殖抑制作用(P＜0．05)。7．psiRNA-survivin能显著增强吉西他滨对胰腺癌细胞的凋亡诱导作用(P＜0．05)。结论稳定转染靶向Survivin的siRNA后，胰腺癌细胞survivin的mRNA和蛋白表达量的明显下调。转染psiRNA-survivin组胰腺癌细胞增殖缓慢，生长曲线十分平缓，细胞侵袭力明显下降，并可明显增强吉西他宾对胰腺癌细胞的增殖抑制和凋亡诱导作用，这表明siRNA可能通过阻断survivin的表达而阻断其抗凋亡作用，抑制胰腺癌细胞增殖，并增强胰腺癌对吉西他滨的化疗敏感性。本次实验结果提示靶向survivin的siRNA可以作为克服肿瘤化疗耐药的一种有效方法，其与吉西他滨联合应用可能成为今后临床治疗胰腺癌值得探索的新途径。更多还原

【Abstract】 Pancreatic cancer is among the most. human malignant tumor for poor prognosis.Unfortunately the early symptom of pancreatic cancer is not clear, because of advancedstage when final diagnosis was taken, the 5-year survival rate remains less than 5%. Thecombined therapy including chemotherapy is critical to increase the survival rate ofpancreatic cancer patient, but this tumor is insensitive to most of the chemotherapeutics.Gemcitabine is a new-type miazines antimetabolite, and it has become the standardfirst-line chemotherapeutic agent for advanced and metastatic pancreatic cancer, withsignificant clinical benefit, but still has marginal survival advantage. Chemoresistance is themain reason for the failure of treatment. Thus, the development of an effective treatment tosolve the chemoresistance of pancreatic cancer remains an urgent task. With thedevelopment of molecular bioresearch, gene therapy targeting inhibitor of apoptosis proteingot much attention, and it possibly provide a new trend to effectively treat pancreaticcancer.Previous studies have shown that survivin is the newfound member of the inhibitors ofapoptosis protein (inhibitor of apoptosis protein, IAP) family, has dual function in theregulation of cell cycle and the inhibition of apoptosis. To date, the overexpression ofsurvivin has been reported in various human malignancies, but not or to be low in mostnormal adult tissues. Survivin is the smallest IAP which has been cloned hithreto, and islocated on chromosome 17q25. Its gene total length is 14.7kb, and is expressed specificlyin G2/M phase coding a 16.5kb protein. The expression of survivin has close correlation tothe prognosis, recurrence and chemoresistance of pancreatic cancer. Survivin primarylybind to mitotic spindle microtubule byαhelical structure in C-terminus, then its BIRstructure bind specificly to caspase-3 and caspase-7, and directly inhibit terminal effectorcaspase-3 and caspase-7 activity, so it can block various kinds of stimulus inducingapoptosis procedure. Survivin also can inhibit the cytochrome C release from mitochondria, breakdown the caspase activation, and so it can block the conduction of apoptosis signaland inhibit cell apoptosis at upstream. In the recent years, some reports showed thatblockdown of survivin can inhibit the growth of various tumor cells. Some researcher foundthat inhibition of survivin can enhance the radiosensitivity of pancreatic cancer cells.Therefore concerning the character of survivin, we think that the regulation of survivinexpression could be a possible new treatment for the chemosensitization of humanpancreatic cancer to Gemcitabine, yet such investigation has not been performed until nowin the world.In the present study, we firstly detect the expression of survivin and bcl-2 in pancreaticcarcinoma and analyze their significance, and then study the alteration of survivin geneexpression in pancreatic cancer cell after chemotherapy in virto. A short interfering RNA(siRNA) plasmid expression vector against survivin was constructed and transfected stablyinto Panc-1 and BxPC3 cells. The changes of survivin expression and cell cycle distributionfollowing RNAi and the role of siRNA in inducing tumor cell apoptosis and enhancing itschemosensitivity to gemcitabine were investigated. Together, these data provide strongevidence for the potential use of survivin-targeted RNAi as a novel way to chemosensitizehuman pancreatic cancer cells. Our study was divided into four parts below. Part One The expression of survivin and its relationship withbcl-2 in pancreatic carcinomaObjectiveTo investigate the expression and associativity of survivin and bcl-2 in tissues of humanpancreatic carcinoma, and study their role in the development of pancreatic cancer.MethodsThe expression of survivin and bcl-2 was detected by SP immunohistochemistry inpancreatic tissues from 50 patients with pancreatic carcinoma and that from 14 patientswith normal pancreas, and analyze their relation with clinicopathologic feature.ResultsSurvivin was expressed in 31 of 50 pancreatic carcinoma(62%). In contract, expressionof survivin in nomal pancreatic tissues was not detectable(p＜0.05). The survivin expressionwas correlated with histological grades and clinical stages(p＜0.05), and was not significantlycorrelated with sex, position of tumor or lymph node matastasis status(p＞0.05). Theexpression rate of survivin was 77.8% and 43.5% in positive and negative group of bcl-2expression respectively, and there was significant difference between two groups(p＜0.05).ConclusionA high expression of survivin in pancreatic carcinoma suggests that survivin may playan important role in the development of unfavourable prognosis. The expression of survivinhas positive correlation with bcl-2, and it may be a biological parameter to unfavourableprognosis. Part Two The alteration and significance of survivin expression inpancreatic cancer cell apoptosis induced by chemotherapyObjectiveTo study the alteration of survivin gene expression in pancreatic cancer cell afterchemotherapy(PA) in virto.MethodsCultivate the pancreatic cancer cell SW 1990 in virto, and give low concentration of PAto these cells, then alteration of survivin mRNA and protein expression was evaluated byPT-PCR and western-blot in pancreatic cancer cells which had been cultured with PA for24h、48h and control group respectively. Meanwhile, SW1990 cell apoptosis rate wasdetected by flow cytometry.ResultsThe apoptosis rate(%) of SW1990 cell of control group and in low concentration PAfor 24h、48h were 2.59±0.35,14.75±1.29,22.65±2.80( p＜0.05); Survivin mRNA levelwas increased 1.1 and 2.9 multiple(p＜0.05), Survivin protein level was increased 1.3 and3.6 multiple at 24h and 48h respectively(p＜0.05).ConclusionChemotherapy of PA can increase the expression of survivin in pancreatic cancer cell,which suggest that survivin may correlate with the chemoresistence of pancreaticcarcinoma cell. Part Three The construction and identification of siRNA eukaryoticexpression vector targeting survivin seneObjectiveTo construct and identify the siRNA eukaryotic expression vector targeting survivin gene,which could be applied to explore further gene therapy to pancreatic carcinoma.MethodsAccording to the cDNA sequence of survivin gene, two siRNA target sequence weredesigned on intemet, and then psiRNA1 and psiRNA2 were constructed respectively byeukaryotic expression vector psiRNA-hH1 neo. The constructed recombinant was identified byendonuclease digestion and DNA sequencing.ResultsThe results of endonuclease digestion and DNA sequencing suggested, the sequence ofinserted fragment was correct.ConclusionEukaryotic expression vector of siRNA targeting survivin was successfully constructedin this study, and should be a new effective vector for gene therapy of pancreaticcarcinoma. Part Four Effects of survivin siRNA on proliferation of humanpancreatic cancer cells and chemosensitivity to gemcitabineObjectiveTo construct the siRNA eukaryotic expression vector targeting survivin gene, andinvestigate the effects of survivin siRNA on proliferation of human pancreatic cancer cellsand chemosensitivity to gemcitabine.MethodsThe siRNA eukaryotic expression vector targeting survivin gene was constructed.Panc-1 and BxPC3 cells were transfected with constructed siRNA vector and then selectedby G418, and we got the stable transfected cells. The expression of survivin mRNA andprotein among the stable transfected cells and the untransfected cells was detected bysemiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Westernblot, respectively. The cell growth curve was drawn, and the cell cycle distribution wasmeasured by flow cytometry. The invasiveness of pancreatic cancer cells in vitro wasassayed using transwell cell culture chambers. Panc-1 and BxPC3 cells or transfected cellswere treated by Gemcitabine for 48h, and then the growth inhibition rates was measured byMTT assay and cell apoptosis rate was detected by flow cytometry.ResultsAfter the recombinant plasmid psiRNA-survivin transfected stably to pancreatic cancerline cells, survivin mRNA level were reduced by 68.52% and 64.32% respectively in stablytransfected Panc-1 and BxPC3 cells comparing with control group(P＜0.05), and survivinprotein level were reduced by 76. 68% and 74. 38% (P＜0.05) respectively, and the cellgrowth curve became much slower, many cells were blocked in the G0/G1 phase 68.72±3.21% (P＜0.05). The number of invasived cells in the experimental group was far less thanin control(P＜0.05). Further more, the growth inhibition rates and apoptosis of these stablytransfected cells were significantly increased after treatment by gemcitabine(P＜0.05).Conclusion The constructed siRNA eukaryotic expression vector targeting survivin could decreasesurvivin expression, inhibit the growth and cell invasiveness of pancreatic cancer cellssignificantly, and enhance the chemosensitivity to gemcitabine. Therefore, the inhibition ofsurvivin expression could be a possible new treatment for the chemosensitization of humanpancreatic cancer.更多还原