【作者】 张沁宏；

【导师】 王东；

【作者基本信息】 第三军医大学 ， 病理学， 2007， 硕士

【摘要】 肝细胞性肝癌(hepatocellular carcinoma,HCC)是我国最常见的恶性肿瘤之一,由于难于早期发现,发病后患者的生存期很短、病死率高,目前临床采用的多种传统治疗手段都不能收到满意的效果。基因治疗是当代医学和生物学的一个新的的研究领域,随着分子生物学理论和技术的发展,基因治疗研究的重点逐渐由单基因缺陷所致的遗传病转向肿瘤这种多基因参与、多步骤形成的疾病。肿瘤发生、发展过程复杂,涉及多种基因异常,联合基因治疗成为当前肿瘤基因治疗研究的发展方向。脱嘌呤/脱嘧啶核酸内切酶(apurinic/apyrimidinic endonuclease 1,APE1)具有DNA损伤修复及氧化还原双重功能,是一种多功能蛋白,对维持DNA的稳定和调节细胞因子的表达具有重要作用。APE1蛋白的不正常表达、分布及功能改变与抗细胞凋亡、肿瘤发生密切相关。野生型p53基因是细胞凋亡的关键基因,p53基因突变与HCC发生密切相关。HCC细胞凋亡能力下降推测与APE1过表达及p53突变有关。本研究应用免疫组化检测肝癌组织中APE1和突变型p53的表达,分析其与肝癌演进及p53基因突变的关系,并在体内外应用重组腺病毒Ad5/F35-APE1 siRNA联合Ad-p53感染肝癌细胞,通过观察体内外肝癌细胞生长的抑制效果,探讨二者联合促进细胞凋亡的作用机制,为联合基因治疗提供新的思路和实验依据。研究目的1.探讨DNA损伤修复基因APE1在肝细胞癌组织中的表达与肿瘤演进及p53基因突变的关系;2.体外联合感染细胞实验研究对肝癌细胞生长的抑制协同效应;3.体内联合治疗实验研究对荷瘤裸鼠肿瘤生长的抑制协同作用。研究内容和方法1.肝癌组织APE1和p53蛋白表达及其临床意义:采用免疫组化方法检测肝癌组织APE1和p53蛋白表达,分析APE1表达与肝癌临床病理因素及p53突变之间的关系,为进一步以APE1和p53为靶点的基因治疗提供临床怖砘　?2.重组腺病毒Ad5/F35-APE1 siRNA联合Ad-p53体外抑制肝癌细胞生长的实验研究:采用流式细胞术和免疫荧光检测Ad5/F35-APE1 siRNA和Ad-p53对肝癌细胞感染效率;Western blot检测Ad5/F35-APE1 siRNA对肝癌细胞APE1基因的沉默作用,及Ad-p53对肝癌细胞p53基因的增强作用;免疫组化检测Ad5/F35-APE1 siRNA和Ad-p53在肝癌细胞内转染目的基因与相应蛋白表达的时效关系;MTT法检测腺病毒感染滴度与细胞存活的关系;台盼蓝染色法绘制细胞生长曲线检测腺病毒联合抑制细胞生长的作用;TUNEL法检测SMMC-7721细胞凋亡。3.重组腺病毒Ad5/F35-APE1 siRNA联合Ad-p53体内抑制荷瘤裸鼠肿瘤生长的实验研究:建立肝癌裸鼠动物模型,观察Ad5/F35-APE1 siRNA联合Ad-p53对肝癌生长抑制的协同作用,测量肿瘤的生长,绘制肿瘤生长曲线,HE染色,光镜观察肿瘤的组织学变化,免疫组化和TUNEL法检测肿瘤细胞APE1和p53表达、细胞凋亡。研究结果1.肝癌组织APE1和p53蛋白表达及其临床意义:正常肝脏组织APE1呈胞核阳性,肝硬化和HCC组织APE1表达特征发生改变,呈胞核表达、单纯胞浆表达或核浆共同表达。APE1胞核表达在HCC、肝硬化及正常肝相互间比较均无显著差异;APE1核浆共表达在三组间比较均具有显著差异(p＜0.01);APE1胞浆表达仅在4例HCC发现。APE1胞核阳性分度和胞浆阳性分度在正常肝、肝硬化、HCC中比较有显著差异(p＜0.01),且与HCC组织学分级有关。HCC组织中p53阳性率60.2%,与APE1表达方式无相关性;APE1/p53不同表达状态的HCC组织学分级有明显差异(p＜0.01),APE1异位表达/p53~+的HCC恶性程度高。2.重组腺病毒Ad5/F35-APE1 siRNA联合Ad-p53体外抑制肝癌细胞生长的实验研究:重组腺病毒Ad5/F35-APE1 siRNA和Ad-p53的最佳感染效率分别为20MOI、50MOI。Ad5/F35-APE1 siRNA能有效抑制肝癌细胞APE1的表达,48～72h达到最大抑制;Ad-p53能有效增强肝癌细胞p53的表达,48～72h达到最高。重组腺病毒Ad5/F35-APE1 siRNA和Ad-p53均能诱导细胞凋亡、抑制细胞生长,联合感染时对肝癌细胞的诱导细胞凋亡作用及抑制作用均明显增强(p＜0.01)。3.重组腺病毒Ad5/F35-APE1 siRNA联合Ad-p53体内抑制荷瘤裸鼠肿瘤生长的实验研究:Ad5/F35-APE1 siRNA联合Ad-p53注射后肿瘤的生长抑制率为54.23%,显著高于单药组(p＜0.01)。与对照组肿瘤细胞相比,两种腺病毒分别有效抑制了APE1或提高了p53蛋白表达水平。TUNEL法检测细胞凋亡情况显示,联合组肿瘤细胞凋亡率为(15.48±2.95)%,显著高于Ad5/F35-APE1 siRNA组(6.85±1.11)%、Ad-p53组(8.23±1.60)%以及对照组(3.06±1.35)%(p＜0.01)。结论1.APE1蛋白在肝细胞中的表达异位及增强与HCC进展密切相关,当细胞浆内出现APE1表达时可作肝细胞早期癌变的指标之一。2.p53突变是HCC发生的高概率事件,在HCC组织中APE1异位表达与p53突变没有相关性。APE1异位表达和p53突变可能在肿瘤的发生、发展过程中具有协同促进作用,APE1和p53是肝癌治疗潜在的分子靶点,具有重要的临床应用价值。3.Ad5/F35-APE1 siRNA可特异性沉默肝癌细胞APE1的表达,沉默效率达到94.24%;Ad-p53能够提高细胞内野生型p53的表达,增强效率达到82.31%。4.Ad5/F35-APE1 siRNA联合Ad-p53能使肝癌细胞凋亡显著增多,抑制肿瘤生长作用显著增强,以APE1和p53为靶点的联合基因治疗可能成为今后肝癌治疗的新方法。更多还原

【Abstract】 Hepatocellular carcinoma(HCC) is one of the most frequent and devastating malignancies in China,and early diagnosis is very difficult,therefore with poor prognosis and high mortality.Even with the combined use of the three classic treatment modalities: surgery,radiation,chemotherapy,little has changed over the last five years to alter the deathly outcome.The rapid development of molecular biology makes the gene therapy a kind of new treatment mode in clinical application.Because the carcinogenesis and progression of HCC is a complicate process involved many genes and several transforming steps,the combination gene therapy against HCC has emerged as a promising approach.The human apurinic/apyrimidinic endonuclease(APE1) is a ubiquitous multifunctional protein which has both DNA repair activity and redox regulatory activity.APE1 plays an important role in maintaining genomic integrity and regulating gene expression,and its abnormal expression is associated with anti-apoptosis and tumor progression.Wild-type p53 plays a key role in cell cycle control and apoptosis,and the mutation of p53 gene is correlated with the carcinogenesis and progression of HCC.So we hypothesis that the APE1 and mutant p53(mtp53) may contribute to the resistance to apoptosis in HCC.In this study,we first detected the expression of APE1 and investigated its correlation with the expression of mtp53 and clinic-pathological parameters in HCC.Then,we examined the effect of chimeric adenoviral vector Ad5/F35 carrying human APE1 siRNA combined with recombinant adenovirus carrying wild-type p53 on cell proliferation inhibition and apoptosis induction in human hepatocellular carcinoma cells in vitro and in vivo.The present study will present a new strategy for combined gene therapy against cancer.Objective1.To investigate the expression of APE1 and explore its correlation with the expression of mtp53 and clinic-pathological parameters in HCC. 2.To investigate the inhibitory effects of Ad5/F35-APE1 siRNA combined with Ad-p53 on human HCC cells in vitro.3.To investigate the inhibitory effects of Ad5/F35-APE1 siRNA combined with Ad-p53 on human HCC cells in a tumor-bearing nude mice model.Materials and Methods1.Expression feature of APE1 and mtp53 and its significance in HCC:Expression of APE1 and mtp53 was detected by S-P immunohistochemical method in 10 cases of normal liver tissue,40 cases of liver cirrhosis and 103 cases of HCC.2.Study of inhibitory effects of Ad5/F35-APE1 siRNA combined with Ad-p53 on human HCC cells in vitro:The infection efficiency of Ad5/35-EGFP and Ad-EGFP to human HCC SMMC-7721 cell line was measured by flow cytometry and observed by fluorescence microscopy.The expression of human APE1 and p53 protein was detected after Ad5/F35-APE1 siRNA and Ad-p53 were transfered into SMMC-7721 cells by western blot analysis and immunohistochemical technique.The cellular proliferation capacity was observed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. Cell growth curve was detected by trypanblau,and cell apoptosis was determined by TUNEL.3.Study of inhibitory effects of Ad5/F35-APE1 siRNA combined with Ad-p53 on human HCC cells in a tumor-bearing nude mice model:Nude mice hepatocarcinoma xenograft model was established by using human HCC SMMC-7721 cell line.When the tumor volumes reached 40～50 mm~3 on the sixth day after inoculation of SMMC-7721 cells, sixteen mice were randomly divided into four groups:control group,Ad5/F35-APE1 siRNA group,Ad-p53 group and Ad5/F35-APE1 siRNA combined with Ad-p53 group.The tumor volumes were measured and the tumor growth curves were plotted.The APE1 and p53 expression were observed by immunohistochemistry.Apoptosis index was detected by TUNEL technique.Results1.Expression feature of APE1 and mtp53 and its significance in HCC:In normal liver tissue,APE1 was detected in nucleus of cells,and the shifts of APE1 from nucleus to cytoplasm was observed in liver cirrhosis and HCC tissues.There was no significant difference in the positive nuclear expression rate of APE1,while there was a significant difference(p＜0.01) in the positive cytoplasmic expression rate of APE1 among normal liver tissue,liver cirrhosis and HCC tissues.There was a significant difference(p＜0.01) in the cytoplasmic and nuclear staining intensity of APE1 among normal liver tissue,liver cirrhosis and HCC tissues,and the cytoplasmic and nuclear staining intensity of APE1 was correlated with histological grade of HCC(p＜0.01).The location of APE1 combination with the p53 status was significantly correlated with tumor grade,and the cytoplasmic APE1 expression/p53~+ status implied a higher tumor grade(p＜0.01).2.Study of inhibitory effects of Ad5/F35-APE1 siRNA combined with Ad-p53 on human HCC cells in vitro:The results showed that the infection efficiency of Ad5/35-APE1 siRNA at 20 multiplicity of infection(MOI) and Ad-p53 at 50 MOI to SMMC-7721 cells was 99%and 85%,respectively.Infection of SMMC-7721 cells with Ad5/F35-APE1 siRNA resulted in a dose-dependent suppression of APE1 protein,and the APE1 protein expression lowest on 48～72h.Infection of SMMC-7721 cells with Ad-p53 resulted in a dose-dependent enhancement of p53 protein,and the p53 protein expression peaked on 48～72h.Apoptosis and inhibition of cell growth were induced by infection with Ad5/F35-APE1 siRNA or Ad-p53.Apoptosis index(AI) and inhibition effect of cell growth in Ad5/F35-APE1 siRNA combined Ad-p53 group was significantly higher,compared with Ad5/F35-APE1 siRNA or Ad-p53 alone(p＜0.01).3.Study of inhibitory effects of Ad5/F35-APE1 siRNA combined with Ad-p53 on human HCC cells in a tumor-bearing nude mice model:Ad5/F35-APE1 siRNA suppressed significantly the expression of APE1 protein,and Ad-p53 enhanced significantly the expression of p53 protein in vivo.The tumor growth inhibition ratio and AI in Ad5/F35-APE1 siRNA combined with Ad-p53 group were significantly higher,compared with the control group,Ad5/F35-APE1 APE1 group and Ad-p53 group(p＜0.01).Conclusion1.The shifts of APE1 from nucleus to cytoplasm and overexpression might play a pivotal role in the carcinogenesis and progression of HCC.Cytoplasmic expression of APE1 might be a useful marker for predicting carcinogenesis of hepatocyte.2.APE1 combination with the p53 status may be risk factors for HCC.It suggests that APE1 and p53 are potential molecular therapeutic targets of HCC.3.Ad5/F35-APE1 siRNA can specifically knock down the APE1 protein expression in HCC cells,and the inhibition rate of expression of APE1 protein reached in 95%.Ad-p53 can specifically raise the p53 protein expression in HCC cells,and the enhancement rate of expression of p53 protein reached in 95%.4.Ad5/F35-APE1 siRNA combined with Ad-p53 transfer can significantly inhibit the growth of HCC,promote tumor cells apoptosis.Therefore,the gene therapy with Ad5/F35-APE1 siRNA combined Ad-p53 may be a promising approach to treat HCC in the future.更多还原