【作者】 廖明媚；

【导师】 何剪太；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 目的:研究肝细胞癌、癌旁及正常肝组织中Mcl-1的表达及其临床意义,探讨RNA干扰下调Mcl-1对肝癌细胞生长与药物敏感性的影响,通过研究GCDA对肝癌细胞存活与耐药的作用和可能途径,阐明抗凋亡蛋白Mcl-1在肝细胞癌发生中的作用,分析GCDA调节Mcl-1产生抗凋亡作用的机制,以利于进一步研究新的策略联合靶向Mcl-1,开发肝细胞癌治疗的新方法,开创肿瘤分子靶向治疗的新途径。方法:1.采用免疫组化S-P法检测51例肝细胞癌组织、49例癌旁组织和25例正常肝组织中Mcl-1的表达,并结合临床病理特征进行分析。2.采用免疫荧光法检测GCDA作用前后细胞内蛋白表达强度与细胞内定位的变化。3.用阳离子脂质体Lipofectamine^TM 2000为载体将Mcl-1 siRNA转染至HepG2细胞中,对细胞内Mcl-1基因表达进行干扰。4.采用MTT法检测GCDA±抗肿瘤药物或抗肿瘤药物±Mcl-1siRNA处理前后肝癌细胞的相对活性与增殖性。5.采用流式细胞分析法检测抗肿瘤药、GCDA或Mcl-1 siRNA作用前后细胞周期的变化。6.GCDA±抗肿瘤药物或抗肿瘤药物±Mcl-1 siRNA处理肝癌细胞,Annexin V-FITC和PI合染细胞,荧光显微镜和电镜观察凋亡形态学改变,流式细胞分析法检测细胞凋亡率的变化。7.采用免疫沉淀法检测GCDA作用下Mcl-1与促凋亡蛋白间的相互结合。8.提取各方案的细胞总蛋白,采用BCA法检测蛋白浓度,Western Blotting技术检测细胞内待测蛋白的表达变化,β-Tubulin为内参,蛋白带灰度值采用BandScan软件进行分析。9.采用Comet Assay在单细胞水平上检测GCDA作用后DNA链损伤的情况。结果:1.抗凋亡蛋白Mcl-1在肝细胞癌组织和癌旁组织中均有表达,阳性率分别为68.63%和58.10%,与正常肝组织的8.00%相比,差异具有显著性（p＜0.01）。细胞内定位以胞浆为主,部分出现胞核表达。其表达与临床病理特征如性别、年龄、HBV感染、肿瘤大小、肝硬化及病理分级无相关性（p＞0.05）,高分化肝细胞癌有较高的阳性表达率（67.86%）。2.RNA干扰结果显示Mcl-1 siRNA转染24h、48h、72h后Mcl-1蛋白表达明显减少,与未转染组有明显差异（p＜0.01）,尤以48小时表达最弱,其灰度值仅为未转染时的27.70%。Mcl-1下调后HepG2细胞中抗凋亡蛋白Bcl-xl表达相应减少,而促凋亡蛋白Bak、Bim和Bax表达稍有增多。3.MTT结果显示GCDA在短时间内对肝癌细胞株HepG2细胞有增殖作用,以10～20min最明显（p＜0.05）;其浓度在200μM内对细胞有增殖作用,以50～200μM处最明显（p＜0.05）。RNA干扰下调Mcl-1后HepG2细胞在抗肿瘤药物作用下的存活率明显低于单纯药物组的存活率（P＜0.05）。4.流式细胞仪检测结果显示GCDA与转染载体本身对细胞周期与凋亡无明显影响（p＞0.05）,RNA干扰下调Mcl-1后HepG2细胞在药物作用下的凋亡率（33.17%）高于单纯药物组的凋亡率（17.60%）（P＜0.05）。5.免疫荧光结果显示GCDA能使细胞内Mcl-1和pERK表达明显增强,核内表达逐渐加强。Irinotecan处理后pERK和Mcl-1的表达稍有减弱,尤以核内明显。6.免疫沉淀结果显示GCDA作用后Bak与Bim结合减少,Bim、Bak与Mcl-1结合增加。7.Western Blotting结果显示GCDA能使HepG2细胞中抗凋亡蛋白Mcl-1表达明显增加,促凋亡蛋白Bim、Bak和Bax表达减少。蛋白合成抑制剂使Mcl-1表达在2-3小时开始衰减,在GCDA存在情况下,Mcl-1在6小时的表达仍很强。Irinotecan处理后pERK和Mcl-1的表达被抑制,在GCDA存在情况下两者表达明显回升。8.Comet Assay检测结果显示随着GCDA作用浓度的增加HepG2细胞中DNA链损伤程度加重。Western Blotting显示DNA修复蛋白PARP明显增强。结论:1.抗凋亡蛋白Mcl-1在肝细胞癌组织中呈过表达状态,在肿瘤发生过程中呈逐渐增强的趋势,高分化肝细胞癌就有较高的阳性表达率,提示肝细胞癌的发生与Mcl-1的抗凋亡作用有关。2.RNA干扰能有效降低肝癌细胞中Mcl-1蛋白表达水平,Mcl-1蛋白的特异性下调能增加化疗药物作用后肝癌细胞的凋亡率,增强肝癌细胞的药物敏感性。当Mcl-1蛋白水平下调后,与Mcl-1同源的Bcl-2家族抗凋亡成员的功能受到部分抑制,促凋亡蛋白Bax、Bim和Bak功能增强,诱导细胞凋亡。3.GCDA能诱导肝细胞癌细胞存活与耐药,其作用可能是通过上调抗凋亡蛋白和下调促凋亡蛋白来实现的。4.GCDA能使肝细胞癌细胞中Mcl-1蛋白表达增强,并阻止Mcl-1在细胞内的降解,增加Mcl-1蛋白在细胞内的稳定性和抗凋亡作用。其机制是通过激活MAPK/ERK1/2信号通道,使ERK磷酸化并向细胞核内转运,激活靶蛋白Mcl-1产生抗凋亡作用。GCDA还能促进Mcl-1中和促凋亡蛋白Bim、Bak的作用使其失活,并减少Bim和Bak的相互结合,阻止细胞色素C的释放,抑制细胞凋亡。5.GCDA作用下存在DNA的损伤与修复,为下一步研究打下了基础。更多还原

【Abstract】 ObjectiveTo study expression and clinical significance of Mcl-1 in hepatocellular carcinoma（HCC） tissues,adjacent liver tissues and normal liver tissues.Identify the effects of Mcl-1 down-regulated by RNA interference on survival and drug sensitivity of HCC cells.Investigate the effects and mechanisms of bile salt（GCDA） on apoptosis and chemotherapeutic drug sensitivity of HCC cells.Then on this basis, clarify the role of Mcl-1 in hepatocellular carcinogenesis and its anti-apoptotic mechanism regulated by GCDA.This foundation will provide a combined target to Mcl-1 with a new strategy and explore new method in HCC therapy and create a new way in molecular targeted therapy of tumors.Methods1.Expression of Mcl-1 were detected in 51 HCC tissues,49 adjacent liver tissues and 25 normal liver tissues by Strepavidin-peroxidase immunohistochemistry staining（SP method） and analyzed with clinical pathological characteristics.2.Changes of protein expression and subcellular localization were detected by immunofluorescence method after GCDA treatment in HCC cells.3.Human HCC cell line HepG2 was cultured in vitro and transfected with Mcl-1 siRNA by Lipofectamine^TM 2000 which interfering the expression of Mcl-1.4.HepG2 cells were treated with GCDA±chemotherapeutic drug or chemotherapeutic drug±Mcl-1 siRNA.Cell viability and proliferation were detected by MTT assay.5.Cells were treated with chemotherapeutic drugs,GCDA or Mcl-1 siRNA.Cell cycle was assayed by Flow Cytometry（FCM）.6.HepG2 cells were treated with GCDA±chemotherapeutic drug or chemotherapeutic drug±Mcl-1 siRNA and dyed with Annexin V-FITC and PI.Cell apoptosis morphology was observed by fluorescence microscope or electron microscope.Cell apoptotic rate was assayed by Flow Cytometry（FCM）.7.The interaction of Mcl-1 and pro-apoptotic proteins was assessed by immunoprecipitation（IP） and Western Blotting（WB） after GCDA treatment.8.Total protein of HCC cells was extracted,and the concentration was measured by BCA method.Expressions of apoptosis-regulating proteins were assessed by Western Blotting.The reference gene wasβ-Tubulin.Gray scale values of proteins were analyzed by BandScan software. 9.DNA strand damage after GCDA treatment was assessed by Comet Assay in single cell level.Results1.Mcl-1 was expressed in HCC tissues and adjacent liver tissues with the positive rate of 68.63%and 58.10%,respectively.Compared to 8%of normal liver tissues,they had markedly significance of difference （p＜0.01）.Intracellular location of Mcl- 1 was mainly in cytoplasm,partly in nuclei.The expression of Mcl-1 had no relationship with clinical pathological characteristic such as gender,age,HBV infection,tumor size and pathology grade（p＞0.05）.However,the positive rate in well-differentiated HCC tissues was high to 67.86%.2.The results of RNA interference indicated expression of Mcl-1 protein was down-regulated obviously after Mcl-1 siRNA was transfected to HepG2 cells about 24h,48h,72h.The level of Mcl-1 protein had significance of difference（p＜0.01） to control group,especially after 48h, which the gray scale value was 27.70%of control group.After Mcl-1 was down-regulated in HepG2 cells,expression of anti-apoptotic protein such as Bcl-xl decreased while pro-apoptotic proteins such as Bak,Bim,Bax increased slightly.3.The results of MTT indicated GCDA had proliferation effect to HepG2 cells within short time,especially 10-20rain（p＜0.05）,or with concentration lower than 200μM,especially 50～200μM（p＜0.05）.With the treatment of chemotherapeutic drugs in HepG2 cells,the survival rate after Mcl-1 siRNA was significantly lower than single drug group（P＜0.05）.4.FCM results indicated GCDA or Lipofectamine^TM 2000 had no effect to cell cycle and apoptosis（p＞0.05）.With the treatment of chemotherapeutic drugs in HepG2 cells,cell apoptotic rate after Mcl-1 down-regulation（33.17%） was markedly higher than single drug group （17.60%）（P＜0.05）.5.Immunofluorescence results indicated GCDA strengthened cellular expression of Mcl-1 and pERK,especially in nuclei.Expression of Mcl-1 and pERK weakened slightly after treatment of Irinotecan, especially in nuclei.6.Immunoprecipitation results indicated the binding of Bak/Bim reduced after GCDA treatment while Bak/Mcl-1 and Bim/Mcl-1 bingding increased.7.Western Blotting results indicated GCDA increased the expression of anti-apoptotic protein Mcl-1 and decreased the expression of pro-apoptotic proteins such as Bim,Bak and Bax.Though protein synthesis inhibitor made Mcl-1 degradation in 2 to 3 hours,the time was last to over 6 hours at the co-presence of GCDA.The expressions of pERK and Mcl-1 were inhibited after treatment of Irinotecan and revived at the co-presence of GCDA.8.Comet Assay results indicated DNA strand damage of HepG2 cells was enhanced gradually when the concentration of GCDA increased. Western Blotting results indicated DNA repair protein PARP was increased obviously.Conclusions1.Anti-apoptotic protein Mcl-1 is over-expression in HCC tissues.It has a progressive tendency in tumorigenesis.It has a high positive rate in well-differentiated HCC tissues.This indicates that anti-apoptosis function of Mcl-1 is related to HCC genesis.2.RNA interference can reduce expression level of Mcl-1 protein in HCC efficiently.Specific down-regulation of Mcl-1 protein increases apoptotic rate of HCC cells with the treatment of chemotherapeutic drugs and enhances drug sensitivity of HCC cells.When Mcl-1 protein level is down-regulated,the function of its homologous anti-apoptotic members in Bcl-2 family is inhibited partly and the pro-apoptotic proteins such as Bax,Bim,Bak are activated to induce cell apoptosis.3.GCDA can induce cell survival and chemoresistance of HCC cells through up-regulating expression level of anti-apoptotic proteins and down-regulating expression level of pro-apoptotic proteins.4.GCDA can increase expression of Mcl-1 protein in HCC cells and prevent its degradation,enhance its stability and apoptotic function in cells.The mechanism is that GCDA activate MAPK/ERK1/2 pathway to increase ERK phosphorylation and translocation to nuclei,which activate the target protein Mcl-1 and enhance its anti-apoptotic function.GCDA can promote the binding of Mcl-1/Bim and Mcl-1/Bak,and induce inactivation of pro-apoptotic proteins.It can also reduce the binding of Bim/Bak and prevent cytochrome release and prohibit cell apoptosis.5.GCDA can induce occurrence of DNA damage and repair,which is a good foundation of further study.更多还原