【作者】 谢毅；

【导师】 钱海鑫；

【作者基本信息】 苏州大学 ， 外科学， 2010， 博士

【摘要】 原发性肝癌是常见的消化系统恶性肿瘤,约占消化系统肿瘤的20%以上,其中肝细胞肝癌约占90%,其生物学特征为易复发、转移、预后差、死亡率高。尽管目前原发性肝癌的治疗方案(手术切除、介入治疗、生物治疗、激光及微波治疗等)日趋完善,但仍难以取得令人满意的疗效,患者生存期极短。因此研究肝癌的发病机理以及探讨新的可行的治疗方案具有极其重要的临庆意义。随着肿瘤学领域中基础研究的不断深入,近年来信号传导通路在肿瘤发生过程中的异常改变及其导致细胞生长失控的机制成为研究的热点。研究发现磷脂酞肌醇3激酶(PI3K)/蛋白激酶B(AKT)信号传导通路在肿瘤的发生、发展、凋亡、血管生成、转移及耐药等方面起着重要的作用。而AKT2作为PI3K/ AKT2信号转导通路的关键分子,在其中更是起着核心的作用。而多种人类恶性肿瘤主要表达AKT家族中的AKT2。细胞内高表达AKT2或激活AKT2的蛋白激酶活性能阻止细胞发生凋亡,增强肿瘤细胞的侵袭能力。目前,国内针对AKT2基因表达与肝细胞癌分化程度、生长方式、临床特点及转移活性的关系尚未进行深入系统的研究。针对AKT2利用小干扰RNA基因治疗肝癌尚无报道。本课题从三个角度进行了系统研究:①肝癌组织AKT2基因表达与肝癌恶性程度、临床特点、转移活性的相关性和在肝癌细胞中的表达。②采用脂质体体外转染针对AKT2的小干扰RNA,并建立稳定株观察转染后肿瘤细胞在体外的生长情况及产生的可能机制。③AKT2小干扰RNA转染后肿瘤细胞体内生长情况观察。结果将为探讨以AKT2为靶点并结合小干扰RNA进行基因治疗,阻止肿瘤的发生和发展,提高生存率,延长生存期奠定理论基础。第一部分AKT2在肝癌组织、肝癌细胞系中的表达及意义本研究对32对肝细胞癌(HCC),4例正常肝组织、4株肝癌细胞株用RT-PCR反应、免疫组化法分别检测AKT2mRNA表达和蛋白表达的情况;分析肝癌AKT2基因表达与肿瘤分期、转移等临床指标之间的关系。1通过RT-PCR方法检测32对人肝细胞癌、4例正常肝脏组织AKT2mRNA表达水平:发现肝癌组织AKT2mRNA的表达明显高于癌旁组织,其中有28对上调,所占比率高达62.5%(P<0.05)。其中4对接近有和无的关系。正常肝中没表达。此外发现:瘤栓组AKT2mRNA表达水平为0.59±0.01,无瘤栓组为0.29±0.02;肝内转移组AKT2mRNA表达水平为0.62±0.03,无肝内转移组为0.30±0.02,相应的两组之间有显著差异。此结果表明AKT2mRNA表达水平增高与临床上肿瘤的侵袭特性有关。2采用免疫组化法检测AKT2蛋白在32例标本中的表达发现在62.5%(28/32例)的肝癌组织内可见到AKT2阳性表达,AKT2阳性表达颗粒主要位于胞膜和胞浆,在同一组织标本中AKT2表达呈现均一性。AKT2的表达在不同分化程度的肝癌有显著差异(P<0.05),高分化肝癌的表达率和表达强度显著低于中分化、低分化肝癌。在肿瘤大小、肝内转移阳性组和阴性组、有和无门静脉癌栓组、HBsAg阳性组和阴性组间有显著性差异(P<0.05)。而与患者的年龄、性别无显著相关性(P>0.05)。3利用Western-blot技术检测AKT2基因在4株肝癌细胞株中的表达,发现肝癌细胞株SMMC-7721AKT2的表达最明显。由此可见,AKT2在肝癌的发生、发展和转移中发挥重要作用,抑制AKT2基因的表达将起到控制肿瘤生长的目的。第二部分封闭人肝癌AKT2的siRNA片段设计、筛选、真核表达载体的构建及SMMC7721细胞转染条件的优化1在哺乳动物细胞的siRNA研究中,有两个关键的步骤,即如何筛选出最有效的针对目标基因的siRNA片段,并使其高效转染入哺乳动物细胞内和表达。本实验通过网上的siRNA设计软件,设计针对AKT2的特异性siRNA。2质粒的扩增、提取及鉴定:采用DH5α大肠杆菌为工作菌,制备感受态细胞,扩增质粒,提取质粒DNA,并经酶切鉴定以备转染使用。3转染效率与脂质体的量和质粒量存在交互作用。在6孔板中以Lipofectamine2000 10μl,质粒量4.0ug时转染效率最高,达到85%以上。Lipofectamine2000转染中可以看到随着转染时间的延长,细胞毒性加大,大于8小时后,转染效率并没有增加。最大的转染效率和最小的细胞毒性的平衡点在8小时。4瞬时转染1,2,3,4,5,6天各组细胞的AKT2的mRNA的变化RT-PCR显示,第3天的时候达到高峰,AKT2的mRNA明显减少,而阴性对照组、空白组AKT2的mRNA没有明显变化。Western-blot检测提示,随着时间的延长,AKT2蛋白在第4天达到被封闭的高峰,而阴性对照组、空白组AKT2的蛋白的变化无显著的差异。研究结果提示:采用脂质体Lipofectamine2000可以成功的将AKT2-siRNA转入SMMC7721细胞中,使后者AKT2mRNA及蛋白的表达受到抑制为进一步观察肿瘤细胞的生物学变化打下基础。第三部分AKT2-siRNA对SMMC7721细胞生物学的影响鉴于第一部分结果显示AKT2在肝癌组织中呈过度表达现象,并且与肿瘤恶性程度、转移能力呈正相关。所以,以AKT2基因为优选靶,采用小干扰RNA技术,抑制AKT2基因表达,将会减少肝癌恶性表型,为肝癌的治疗带来希望。1利用高表AKT2mRNA及蛋白的肝癌细胞系(简称7721细胞)为体外细胞系模型,建立分别含有Supersilencing-AKT2-siRNA、GFP质粒的稳定细胞株。应用MTT法检测细胞生长曲线发现Supersilencing-AKT2-siRNA细胞的生长明显缓慢。应用流式细胞仪检测细胞的细胞周期,发现干扰组S期细胞百分率明显低于对照组(P<0.05),而G0/G1期细胞百分率则明显高于对照组(P<0.05)。Western-blot结果提示干扰组CyclinD1、P27、P21的表达明显低于对照组(P<0.05)。以上说明抑制AKT2基因的表达能够明显降低肝癌细胞的增殖。2利用粘附实验发现干扰组细胞的粘附能力明显下降。通过计算2小时后粘附在平皿上的细胞OD值,我们可以看出,干扰组粘附能力下降约3倍左右。划线实验和Transwell实验都提示干扰组细胞的的侵袭能力明显下降了。明胶酶法检测细胞株MMP-2、MMP-9的结果,可以看到封闭AKT2的表达明显抑制了肝癌细胞MMP-2、MMP-9的表达。从Western-blot可以看到干扰组、LNR、ICAM-Ⅰ在蛋白水平的变化的表达明显低于对照组, E-cadherin在蛋白水平变化明显高于对照组(P<0.05)。研究结果提示:通过靶向阻断AKT2蛋白的表达可以抑制SMMC7721癌细胞株的生长和转移。第四部分封闭AKT2蛋白表达促人肝癌细胞凋亡作用实验研究AKT2的表达与肿瘤的凋亡抑制密切相关,这决定了AKT2在肝癌的靶向治疗上可能具有独特的价值。为了进一步探讨靶向抑制AKT2在肝细胞癌治疗中的意义和价值,我们观察其对肝癌细胞凋亡和化疗药物敏感性的影响。1在CHX(放线菌酮)诱导凋亡后,显微镜下可以观察到干扰组细胞较对照组细胞形态变化明显,表现为细胞间隙逐渐加大,部分细胞边缘变钝;贴壁性能差;易于脱落;死亡细胞较多见。应用DAPI染色显示实验组细胞凋亡明显增加,差异有显著性(P<0.05)。用流式细胞仪检测细胞凋亡,发现CHX处理24小时后可不同程度地诱导三组细胞凋亡,干扰组细胞凋亡数(34.2±4.4%)明显高于各对照组,(16.7±1.8%),(20.4±2.1%)(P<0.05)。而对照组间比较无显著性差异(P>0.05)。本结果表明,AKT2-shRNA可抑制肿瘤细胞生长,增强肿瘤细胞对凋亡药物的敏感性。2在蛋白水平上,CHX诱导凋亡后,干扰组Bax蛋白,Caspase3蛋白表达比对照组明显上调,而Bcl-2蛋白表达明显下降。3用等浓度化疗药物吉西他滨处理发现干扰组细胞生长抑制率明显高于对照组细胞,组间差异有显著性(P<0.05)。由此我可以得出结论AKT2-shRNA可以增加肝癌细胞的凋亡易感性,为进一步的体内实验打下基础。第五部分AKT2小干扰RNA对肝癌细胞体内生长的影响为了进一步揭示AKT2在肝癌的发生、发展中的作用,观察AKT2小干扰RNA转染后的细胞体内生长情况,我们选用BALB-C裸鼠进行了体内实验观察。裸小鼠15只,随机分为3组:A.空白对照组5只:B.空载体组5只;C转染AKT2-siRNA基因组5只。于每只裸鼠腹部的前肢皮下分别接种等量的不同转染细胞(5×106/100ul/只),接种6周后终止实验。①观察肿瘤的生长情况:实验组裸鼠的肿瘤形成与对照组相比潜伏期长,平均为22.2±1.9天,对照组及反义组肿瘤形成的潜伏期分别为13.2±1.5天、10.6±1.5天,干扰组肿瘤形成时间明显被推迟,P<0.05。同时实验组裸鼠肿瘤的生长速度慢于对照组。实验结束时实验组为421.3±30.6mm3而对照组为932.59±48.7 mm3、887.36±38.5 mm3,(P<0.05)。结果说明,AKT2小干扰RNA有效地推迟和抑制了SMMC7721细胞在裸鼠体内肿瘤的形成和生长。②透射电镜观察:干扰组细胞可见到较多的外形变圆,体积缩小,染色深,表面出芽,染色质边集,表现出明显的凋亡细胞的形态。本研究进一步揭示AKT2在肝癌的发生、发展中的作用,表明AKT2小干扰RNA基因治疗能有效地抑制肿瘤细胞在动物体内的生长速度。以此基因为靶基因进行小干扰RNA基因治疗,将有可能为人类肝癌提供有效的治疗手段。更多还原

【Abstract】 Liver cancer is one of the common types of gastrointestinal tumors with poor prognosis metastasis and high mortality in which hepatocellular carcinoma(HCC)accounts for about 90%. Although the different available multi-modalities of therapies,including radical surgical operation、intervention therapy、laser therapy et al,have currently been used in the treatment for liver cancer,the prognosis of liver cancer has not been markedly improved. Therefore, it has an important clinic significance to study on the mechanism of the development and progression of liver cancer and the new strategies for the treatment of liver cancer with the molecular biology. With the development of the basic research on oncological fields, it was found that singal conduction has the focus.It was researched that PI3K/AKT singal conduction takes an important role in the growth、differentiation、angiogenesis、metastasis、proliferation of tumors. AKT2 is the key point in the PI3K/AKT singal conduction.It is an important factor. many tumors have the over expression in the AKT2 gene. The high expression of AKT2 and the action of AKT2 protein kinase can prevent the cell apoptosis and increase the invasion of cancer cell. By now,the systemic researches on the relationship between the expression of AKT2 and histopathologic type, metastasis activity、angiogenesis of HCC have not been reported. And there are also no reports on gene therapy against angiogeneis of liver cancer. This study contains three parts of work: The first, the relationships between the expression of AKT2 in HCC and the tumor grade,tumor metastasis,angiogenesis of liver cancer. The second the inhibitory effect of AKT2 small interfere RNA on the growth of 7721 hepatocellular cell line (7721 cancer hepatocytes)in vivo. The third,the inhibitory effect of AKT2 small interfere RNA on 7721 cell in nude mouse.Part I:Study on the expression of AKT2 and its relationship to metastasis activity and angiogenesis of human HCC1 Expression of AKT2 gene in human HCC and normal liver tissue.The gene expression of AKT2 in 32 pair human HCCs and 4 normal liver were studied by RT-PCR method.The expressive rates of HCCs and normal tissue were 62.5%and 0% respectively.4 paired samples are yes or no.Moreover,The levels of AKT2 mRNA(AKT2 mRNA/GAPDH mRNA)were 0.59±0.01 in embolus, 0.62±0.03 in metastasis respectively.The difference between them are significant.The expression levels of AKT2 mRNA were positively correlated to pathologic grades、embolus and envelops of tumors.2 The protein expression of AKT2 in human HCC and normal liver tissue.The protein expression of AKT2 in 32 paired human HCCs and 4 normal liver were studied by immunohistochemistry.The expression rates of HCCs and normal tissue were62.5% and 0% respectively.The level of AKT2 protein was also significantly correlated to pathologic grades、embolus、capsules and HBsAg of the tumors.3 the expression of AKT2 in 4 cell lines were studied by Western-blot method.4 cell lines were all expressed,especially SMMC-7721.We find AKT2 are expressed in cytoplasm and cytomembrane mainly.It suggested that the AKT2 gene play an important role in the malignant progression of cancers.The overexpression of AKT2 and the correlation between AKT2 expression and tumor grade provided a useful parameter for evaluating the degree of malignancy at molecular level and for selecting the target gene in gene therapy.PartⅡstudy on designing siRNA of blocking AKT2 expression,construction expression vector and optimizing transfection conditionsIn studying siRNA of mammal cells,there are two key steps.One is to select effctive sequence of siRNA,the other is to transfer the sequences into cells effectively.We design the sequence in network.1 there is interaction between Liposome2000 quantity and plasmid quantity in transfection.There is highest efficiency when Lipofectamine2000 volume is 10ul and the palsmid of quantity is 4ug.The efficiency is over 85%. Transfection time is longer,cell cytotoxicity is bigger.The best time is 8 hours after transfection,the biggest transfection,the biggest transfection efficiency and the lest cell cytotoxicity.2 In the siRNA experiment we find the AKT2mRNA is lowest on the thirdth day by RT-PCR,but the AKT2 mRNA is not marked changed in the control teams.We also find the AKT2 protein is lowest on the fourth day by Western-blot but the AKT2 protein is not marked changed in the control teams.The plasmid, Supersilencing- AKT2–siRNA is more,AKT2 protein is lowest.We can draw the conclusion:the plasmid can be transferred into SMMC7721 cell efficiently and AKT2mRNA or AKT2 ptotein is induced.PartⅢThe inhibitory effect of AKT2-siRNA on the growth of HCC cells in vitroBecause there was no effective treatments on liver cancers,it is very urgent and important to find out new methods to solve this problem.According to the results in part I,we believed that AKT2 gene be a target in gene therapy of cancers.So we planed to utilize small interfere RNA technique to observe the inhibitory effects of small interfere RNA AKT2 mRNA on the expression of AKT2.1 We confirmed a/the overexpression of AKT2 in 7721 HCC cells by RT-PCR analysis and immunohistochemistry.We use 7721 HCC cells to Stable cell line which has plasmid Supersilencing-AKT2-siRNA or Supersilencing–AKT2-GFP growth rate among these groups had differences detected by MTT assay.2 The cell cycle is analysizd by FCM assay.The S stage of experimental team is longer than control team(P<0.05)but G0/G1 stage is shorter.The expression of CyclinD1、P27、P21 are reduced magnificently in experimental team by western-blot analysis.3 In adhesion experiment we find the adhesion capability of SMMC7721-siRNA cell is decreased 3 times or so.SMMC7721-siRNA cell invasion is decreased by Scribing test and Transwell test.blocking AKT2 also reduced the decreasing of expression of MMP-2、MMP-9.we find the expression of LNR and ICAM-Ⅰare inhibited marked in SMMC7721-siRNA cell line. but E-cadherin is increased markedly. We can draw the conclusion:blocking of AKT2 expression can inhibit growth and metastasis of SMMC7721-siRNA cell line.PartⅣstudy on blocking AKT2 protein expression to promote SMMMC7721 ApoptosisAKT2 is related to tumor Apoptosis closely.In order to study the value of AKT2 in gene theraphy,we observe the relationship between AKT2 and chemotherapeutics.Induction Apoptosis by CHX,we can see Apoptosis cell is increased in SMMC7721-siRNA cell line.By DAPI analysis,the Apoptosis rate is decreased markedly in in SMMC7721-siRNA cell line.(P<0.05)1 SMMC7721-AKT2-siRNA cell line processed with CHX for 24 hour,we find the apoptotic index (34.2%)is higher than control team(16.7%,20.4%).(P<0.05) by FCM.Outcome make it clear that blocking AKT2 can inhibit cell growth and enhance it sensitivity to chemotherapeutics.2 Processed with CHX,we find the protein Bax,Caspase3 of SMMC7721- AKT2-siRNA cell lines up-regulated(P<0.05),meanwhile Bcl-2 is downregulated by western-blot.3 Using equidensity chemotherapeutics Gem,we see growth inhibiting of SMMC7721-AKT2-siRNA cell line is higher than control team.(P<0.05). From above,we know blocking AKT2 can increase apoptositic sensitivity of hepatoma carcinoma cell to chemotherapeutics.This is foundation to the next in vivo experiment. PartⅤThe therapeutic effect of AKT2 small interfere RNA for HCC 7721 cells in vivoIn order to make further investigation on the role of AKT2 gene in the development of liver cancer and the effect of siRNA gene therapy in vivo,we carried out vivo study with BALB/nu/nu nude mice.15 nude mice were randomly divided into three groups and three prepared cells were subcutaneously injected on the back of mice:(1)control group:10 mice injected with wild-type 7721 HCC cells;(2) positive control group:5 mice injected with 7721 HCC cells transfected with plasmid GFP (3)experimental group:5 mice injected with 7721 HCC cells transfected with AKT2 small interfere RNA.The results showed:the transfected group were significently lower than that of positive control group and control group in volume、growth rate of tumors.In the tumors of experimental group, we knew that AKT2 protein were effectively blocked through analysis of wetern-blot.The present study demonstrated that AKT2 play an important role in the angiogenesis and development of HCC,clarified the therapeutic effect of AKT2 siRNA in vivo and suggested that AKT2 gene be a target for small interfere RNA gene therapy of liver cancer.We get a useful theory basis for gene therapy of liver cance更多还原