【作者】 彭涛；

【导师】 王春友；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 胰腺癌是一种临床表现隐匿、发展迅速、预后很差的消化道恶性肿瘤，其早期诊断困难，治疗方法有限，因而死亡率很高。在西方国家，胰腺癌的死亡率已由第5位升至第4位。在我国，胰腺癌是消化道恶性肿瘤的三大死亡原因之一，85％的患者就诊时已属晚期，手术切除率仅10％～15％。5年生存率仅为1％～5％，目前化疗仍是胰腺癌辅助治疗的重要手段之一。吉西他滨是新一代阿糖胞苷类似物，可以明显改善晚期胰腺癌患者的疾病相关症状和生活质量，于1996年被美国食品与药品监督管理局（FDA）批准取代5-Fu作为抗胰腺癌一线药物，并被视作临床研究的“金标准”。1997年一项关于吉西他滨的Ⅲ期临床试验显示，该药可明显改善患者症状、延长患者中位生存期并使患者获益。但肿瘤的耐药性是影响化疗效果、疾病预后的最大障碍。如何提高吉西他滨介导的胰腺癌化疗敏感性成为当前研究的热点。近年以来，已有多项研究显示PI3K／Akt信号转导网络系统与肿瘤化疗耐药关系密切。多药耐药基因（MDR1）家族是研究最早的公认的肿瘤多药耐药相关基因之一。多药耐药基因的过度表达导致肿瘤细胞抗凋亡能力增强，从而降低化疗药物的细胞毒作用。而这一过程受多种因素影响，其中PI3K／Akt发挥了重要作用。有研究表明PI3K／Akt信号转导网络系统通过上调（multidrug resistance-related protein 1，MRP1）MRP1的表达而导致肿瘤细胞产生耐药，MRP1水平与PI3K／Akt表达在AML细胞中呈正相关性。另有研究利用PI3K抑制剂wortmannin抑制Akt的磷酸化之后，MRP1的水平也随之下降。Abdul-Ghani的研究也表明PI3K／Akt可以上调MRP1的水平，从而使细胞的凋亡减少，而PI3K／Akt的抑制剂LY294002可以使细胞凋亡率大幅度提高。这些结果提示干扰沉默AKT基因表达可能成为临床上增加胰腺癌化疗敏感性的一种方法。按照以上设想并在导师王春友教授的指导和支持下，我在普外实验室开展了RNA干扰AKT2对胰腺癌吉西他滨化疗敏感性的影响及相关机制研究。为此，我们通过（1）RNA干扰胰腺癌细胞AKT2的表达对吉西他滨敏感性的体外实验，（2）RNA干扰AKT2对胰腺癌裸鼠移植瘤的吉西他滨敏感性的体内实验，这两部分来观察RNA干扰AKT2对胰腺癌吉西他滨化疗敏感性的影响。然后通过（3）建立胰腺癌吉西他滨耐药细胞系，（4） RNA干扰AKT2表达逆转胰腺癌细胞耐药的实验研究，这两部分来初步探讨RNA干扰AKT2对改变胰腺癌耐药性的相关机制。通过以上实验为进一步阐明胰腺癌化疗耐药的分子生物学机理，从而在分子水平上寻找其中的关键基因并开发相应靶向治疗，达到提高胰腺癌化疗疗效提供理论基础。第一部分PANC-1细胞中AKT2的表达对吉西他滨敏感性的实验研究目的：探讨胰腺癌细胞Panc-1中AKT2的表达对吉西他滨敏感性的影响。方法：体外将AKT2特异性siRNA表达载体pAKT2-siRNA转染胰癌细胞株Panc-1，应用RT-PCR、Western blot检测转染siRNA后Panc-1细胞AKT2基因和蛋白的表达变化，应用MTT法检测干扰AKT2后Panc-1细胞对吉西他滨敏感性的变化。结果：转染AKT2 siRNA后Panc-1细胞AKT2基因和蛋白的表达水平明显下降；吉西他滨对Panc-1细胞的半数抑制量从1．96±0．22μg／ml降到0．24±0．03μg／ml，Panc-1细胞对吉西他滨的敏感性明显增加。结论：AKT2 siRNA抑制AKT2表达，能增加人胰癌细胞株Panc-1对吉西他滨的敏感性。第二部分RNA干扰AKT2对人胰腺癌裸鼠移植瘤的吉西他滨敏感性的实验研究目的探讨RNA干扰AKT2对人胰腺癌裸鼠移植瘤的吉西他滨敏感性的影响。方法构建荷胰腺癌裸鼠模型，采用腹腔给药和瘤内注射方式，以吉西他滨配合AKT2siRNA表达载体对瘤鼠进行联合干预治疗对比观测裸鼠肿瘤生长情况，RT-PCR检测肿瘤细胞AKT2 mRNA的表达，免疫组织化学法检测肿瘤AKT2蛋白表达，DNA末端原位标记法（TUNEL法）检测细胞凋亡指数。结果化疗+AKT2-siRNA组移植瘤组织中AKT2 mRNA及AKT2蛋白表达明显低于空白对照组、化疗组、化疗+阴性质粒组。化疗+AKT2-siRNA组瘤重、肿瘤体积显著低于其他各组。化疗+AKT2-siRNA组抑瘤率、凋亡指数显著高于其他各组。结论RNA干扰AKT2明显提高人胰腺癌吉西他滨化疗的敏感性。第三部分胰腺癌吉西他滨耐药细胞系的建立及其特性目的建立胰腺癌吉西他滨耐药细胞系，并对其细胞生物学特性进行研究。方法逐步增加培养基中吉西他滨的浓度，建立了对吉西他滨耐药的胰腺癌细胞系Panc1-Gem。采用四甲基偶氮唑蓝（MTT）法和集落形成实验，计算出Panc1和Panc1-Gem的半数抑制浓度(IC₅₀)和耐药系数（RI）：比较Panc1和Panc1-Gem的生长曲线，并计算出两细胞系的倍增时间。结果吉西他滨对Panc1和Panc1-Gem的IC₅₀分别为1．96±0．22μg／ml和239．82±35．47μg／ml，RI为122．36（P＜0．05）。集落形成实验的RI为118．93。根据生长曲线计算出Panc1和Panc1-Gem的倍增时间为27．1h和35．2h。结论成功建立了胰腺癌吉西他滨耐药细胞系Panc1-Gem，耐药性能明显、稳定，非常适合用于胰腺癌中吉西他滨耐药的研究。第四部分AKT2在胰腺癌细胞吉西他滨化疗耐药中的作用目的探讨AKT2、多药耐药基因（mdr-1）、脱氧胞苷激酶（dCK），在胰腺癌细胞株Panc-1吉西他滨（GEM）化疗耐药中的相互关系及作用。方法通过浓度递增法，诱导建立耐GEM的胰腺癌细胞株。应用RNA干扰耐GEM的胰腺癌细胞株AKT2表达进行逆转耐药实验，采用了Western Blot、RT-PCR法和MTT法，评估AKT2与mdr-1、dCK基因表达的相互关系及逆转耐药的效果。结果Western Blot、RT-PCR结果显示，耐药细胞Panc1-GEM的mdr-1在蛋白表达、mRNA转录水平上比其亲本细胞系Panc1增强，而脱氧胞苷激酶（dCK）基因在蛋白表达、mRNA转录水平上比其亲本细胞系Panc1减弱。经过RNA干扰耐GEM的胰腺癌细胞株AKT2表达作用后，mdr-1在蛋白表达、mRNA转录水平上减弱，而脱氧胞苷激酶（dCK）基因在蛋白表达、mRNA转录水平上增强。耐药细胞Panc1-GEM对吉西他滨的IC₅₀为239．82±35．47μg／ml，耐药系数（RI）为121．94；经过RNA干扰耐GEM的胰腺癌细胞株AKT2表达作用后对吉西他滨的IC₅₀为113．45±21．89μg／ml，RI为57．88。结论胰腺癌细胞对吉西他滨耐药的原因与dCK减少有关，同时也与mdr-1升高相关。AKT2可能参与调控mdr-1、dCK基因表达，从而介导胰腺癌细胞株Panc-1吉西他滨化疗抵抗。更多还原

【Abstract】 PartⅠEffects of the Expression of AKT2 on Sensitivity of Pancreatic Cancer Cell LinePanc-1 to GemcitabineObjectiveTo explore the effects of the expression of AKT2 on sensitivity of pancreatic cancercell line Panc-1 to gemcitabine.MethodsIn vitro the expression vector of pAKT2-siRNA was constructed and transferred intoPanc-1 cell by lipofection. The mRNA and protein expression of AKT2 was detected byusing RT-PCR and Western blot. The changes of gemcitabine sensitivity after siRNA wereexamined by MTT assay.ResultsThe mRNA and protein levels of AKT2 in Panc-1 cells were significantly decreasedafter transfection, and the median inhibition concentration of gemcitabine against Panc-1cells was reduced from 1.96±0.22μg / ml to 0.24±0.03μg / ml. The sensitivity of Panc-1 cells to gemcitabine was increased significantly after AKT2 siRNA.ConclusionThe sensitivity of Panc-1 cells to gemcitabine could be enhanced by AKT2-siRNA.PartⅡThe Effect of Sensitivity of Human Pancreatic Cancer Cell Xenograft in Nude Mice toGemcitabine by RNA Interference Suppressing the Expression of AKT2ObjectiveTo study the effect of sensitivity of human pancreatic cancer cell xenograft in nudemice to gemcitabine by RNA interference suppressing the expression of AKT2.MethodsThe human pancreatic cancer cell implanted tumor model in the nude mice wasestablished. By abdominal cavity administration and intratumoral injection ,the micebearing tumor were treated with gemcitabine in combination with vector ofpAKT2-siRNA. Tumor growth of tumor tissues were observed,the AKT2 mRNAexpression levels by RT-PCR method and, AKT2 protein expression in tumor tissues wasdetected by immunohistochemistry and tumor apoptosis by Tdt-mediated dUTP nick endlabeling（TUNEL）.ResultsIn chemotherapy+AKT2-siRNA group the expression of mRNA and protein wassignificantly lower than in control group, chemotherapy group and chemotherapy+ blankplasmid group. The tumor weight and tumor volume in chemotherapy+AKT2-siRNA groupwere significantly lower than those in other three groups. The inhibition rate and apoptosisin chemotherapy+AKT2-siRNA group were significantly higher than those in other three groups.Conclusionsensitivity of human pancreatic cancer cell to gemcitabine could be significantlyimproved by RNA interference suppressing the expression of AKT2.PartⅢDevelopment and Characterization of Gemcitabine-resistant Pancreatic Cancer CellLine Pane-1ObjectiveTo establish the resistance to gemcitabine in human pancreatic cancer cell line Panc-1and describe the characteristics of its resistant variant.MethodsResistance of gemcitabine was established by exposing Panc-1 cells to increasingconcentration of gemcitabine, which was designated as Panc1-Gem. The IC₅₀ and resistanceindex （RI） were tested by MTT assay and colony formation test. The growth curve and cellcycle of Panc1 and Panc1-Gem were compared.ResultsThe IC₅₀ increased from 1.96±0.22μg/ml in Pancl to 239.82±35.47μg/ml inPancl-Gem as tested by MTT assay at 72h exposure, the RI was 122. 36（P＜0.05）. The RIof colony formation test was 118.93. Double time of Panc1 and Panc1-Gem were 27.1h and35.2h respectively, as evaluated by the growth curve.ConclusionPanc 1-Gem, the gemcitabine resistant phenotype, is stable and suitable for the study ofgemcitabine resistance in pancreatic cancer. PartⅣThe Role of AKT2 in Resistance to Gemcitabine in Pancreatic Cancer CellObjectiveTo investigate the role of AKT2, multidrugresistance-1（mdr-1） and deoxycytidinekinase（dCK） in Gemcitabine （GEM） chemoresistance in the pancreatic cancer cell linePanc-1.MethodsThe GEM-resistant pancreatic cancer cell line model was constructed using a stepwiseincrease in concentration gradient of Gemcitabine. RNA interference of AKT2 was used toreverse the drug resistance in gemcitabine-resistant pancreatic cancer cell line（Panel-GEM）, RT-PCR, Western Blot and MTT assay were employed to evaluate therelationship between AKT2 and mdr-1,dCK and the effect of reversing drug resistance byRNA interference of AKT2.ResultsAs compared to the parental Panc-1 cells, Panc1-GEM cells showed increasedexpression of mdr-1 revealed by Western Blot and RT-PCR, while Panc1-GEM cells alsoshowed decreased expression of dCK revealed by Western Blot and RT-PCR. After theAKT2 expression of Panc1-GEM had been interferenced by siRNA, Panc1-GEM showeddecreased expression of mdr-1 revealed by Western Blot and RT-PCR, while Panc1-GEMcells also showed increased expression of dCK revealed by Western Blot and RT-PCR. TheIC50 of Pancl-GEM to gemcitabine was 239.82±35.47μg/ml and RI of Panc1-GEM was 121. 94. After the AKT2 expression of Panc1-GEM had been interferenced by siRNA, theIC50 of Panc1-GEM to gemcitabine was 113.45±21.89μg/ml and RI of Pancl-GEM was57.88.ConclusionThe resistance to gemcitabine of pancreatic cancer cell line is due to decreasedexpression of dCK and increased expression of mdr-1. The AKT2 participates in theregulation of the expression of mdr-1 and dCK, most likely contributing to the GEMchemoresistance in the Panc-1 pancreatic cancer cell line.更多还原