【作者】 薛慧琴；

【导师】 罗国容；

【作者基本信息】 广西医科大学 ， 人体组织胚胎学， 2009， 博士

【摘要】 目的:(1)初步了解HSP90和chk1在肝癌组织、配对癌旁组织、正常肝组织中的蛋白表达和mRNA表达的差异,分析HSP90和chk1表达水平与肝癌临床病理的关系及两者的相关性。(2)研究HSP90抑制剂GA对人肝癌细胞株BEL7404的增殖抑制、凋亡及周期阻滞作用,分析HSP90在肝癌细胞凋亡中的作用。(3)研究HSP90抑制剂GA作用前后chk1在mRNA水平和蛋白水平的变化情况,探讨HSP90在肝癌细胞凋亡中的作用机制,为肝癌治疗提供新的靶点。方法:(1)采用免疫组织化学(IHC)技术从蛋白质水平检测41例肝癌组织、41例肝癌旁组织、9例正常肝组织中HSP90和chk1的表达情况。(2)应用逆转录PCR(RT-PCR)方法从mRNA水平检测46例肝癌组织、46例肝癌旁组织、9例正常肝组织中HSP90和chk1的表达情况。(3)体外培养人肝癌细胞株BEL7404,用MTT法研究HSP90抑制剂GA对人肝癌细胞株BEL7404生长的影响;用透射电镜、吖啶橙荧光染色法及Annexin V-EGFP/PI双染法流式细胞仪检测GA对人肝癌细胞株BEL7404细胞凋亡的影响(。4)用流式细胞仪检测HSP90抑制剂GA对人肝癌细胞株BEL7404细胞周期的影响。(5)用RT-PCR方法和Western-blot技术检测HSP90抑制剂GA作用前后,chk1 mRNA含量和蛋白表达的变化情况。结果:(1)免疫组织化学(IHC)结果:HSP90蛋白在肝癌、肝癌旁以及正常肝组织中的阳性表达率分别为85.37 %(35/41)、56.1 %(23/41)、22.22 %(2/9), HSP90在肝癌组织中阳性表达率明显高于癌旁组织和正常肝组织。HSP90阳性表达与肝癌TNM分期、病理分级、有无复发有关(P<0.05),而与性别、年龄、肿瘤大小、肿瘤结节、门脉癌栓、血清AFP含量、有无乙肝、丙肝、及是否合并肝硬化、腹水等无关(P >0.05)。chk1蛋白在上述组织中的阳性表达率分别为80.49 %(33/41)、70.73 %(29/41)、11.13 %(1/9), chk1在肝癌组织中阳性表达率高于癌旁组织,明显高于正常肝组织。chk1阳性表达与肝癌病理分级有关(P<0.05),而与TNM分期、有无复发及性别、年龄、肿瘤大小、肿瘤结节、门脉癌栓、血清AFP含量、有无乙肝、丙肝、及是否合并肝硬化、腹水等无关(P >0.05)。等级相关性分析表明,HSP90和chk1在肝癌组织中的表达呈正相关(P<0.01)。(2)逆转录PCR(RT-PCR)结果:HSP90基因在肝癌组织、肝癌旁组织及正常肝组织中的mRNA相对表达量分别为0.718±0.372、0.437±0.207、0.136±0.104,差异有统计学意义(P<0.01)。HSP90的mRNA表达与肝癌TNM分期、病理分级、有无复发密切相关(P<0.05)。chk1基因在肝癌组织、肝癌旁组织及正常肝组织中的mRNA相对表达量分别为0.320±0.146、0.193±0.071、0.098±0.000,差异有统计学意义(P<0.01)。chk1的mRNA表达与肝癌病理分级密切相关(P<0.05)。(3)增殖抑制实验:MTT法结果显示HSP90抑制剂GA在1～20 umol/L的浓度范围内对人肝癌细胞株BEL7404均有不同程度的增殖抑制作用,生长抑制率在10.1 %～76.7 %之间。GA分别作用24 h、48 h、72 h后,对BEL7404细胞株的生长抑制率逐渐增高,GA对人肝癌细胞株BEL7404的增殖抑制作用呈时间剂量依赖性。(4)流式细胞仪结果显示:GA各剂量组(5 umol/L、10 umol/L、15 umol/L)能引起BEL7404细胞株G0/G1期细胞增加,G2/M期细胞减少,对细胞周期的影响表现为G0/G1期阻滞,并呈时间剂量依赖性。(5)Annexin V-EGFP/PI双染法,用流式细胞仪检测可见BEL7404细胞株在GA各剂量组作用24h后的凋亡率分别为(10.2±1.35)%、(21.3±1.30)%、(38.7±1.43)%,明显高于对照(NS)组(6.31±0.82)%,说明GA可诱导BEL7404细胞株发生凋亡。(6)吖啶橙(AO)荧光染色结果可见随着GA浓度的增加,呈亮绿色的凋亡细胞的比率逐渐增加,GA给药组明显高于对照(NS)组(P < 0.01)。电镜下可见GA给药各剂量组肿瘤细胞核染色质浓缩并边缘化,核碎裂,胞浆空泡化明显,有的细胞可见凋亡小体,表明GA可诱导肿瘤细胞凋亡。以上结果提示GA抑制肿瘤细胞增殖可能与细胞周期阻滞和诱导细胞凋亡有关。(7)10 umol/L GA处理BEL7404细胞不同时间点后chk1的mRNA水平无明显降低。Western blot检测GA作用24h后chk1蛋白表达较GA作用前明显减弱。以上结果表明抑制HSP90的功能可降低chk1蛋白表达量而不影响chk1 mRNA水平的合成。结论:HSP90及其客户蛋白chk1在肝癌组织中的蛋白水平和mRNA水平的表达均高于配对癌旁组织及正常肝组织,并且与肝癌临床病理有关。HSP90抑制剂GA可抑制体外培养的肝癌细胞生长,引起肝癌细胞凋亡和周期阻滞。抑制HSP90的功能可减少chk1蛋白表达量,而不影响chk1 mRNA水平的合成。HSP90抑制剂GA的抗肿瘤作用机制可能与抑制chk1通路有关。HSP90和chk1有望成为肝癌治疗的新靶点。更多还原

【Abstract】 Objective: (1) To investigate the differences of mRNA and protein expressions of HSP90 and chk1 among normal liver, hepatocellular carcinoma and paired adjacent cancerous liver tissues, analyze the correlation of the level of expressions of HSP90 and chk1 and the clinicopathological parameters in liver cancer. (2) To observe the effects of geldanamycin (GA) on cell proliferation inhibition and apoptosis of human hepatoma cell line BEL7404. (3) To observe the changes of the mRNA and protein expression of chk1 before and after treatment with HSP90 inhibitor geldanamycin (GA), discuss the mechanism of HSP90 in apoptosis of human hepatoma cell line BEL7404 and provide new therapeutic targets for hepatocarcinoma.Methods: (1) The levels of protein expression of HSP90 and chk1 were assayed by immunohistochemistry (IHC) in 41 hepatocellular carcinoma, 41 paired adjacent cancerous liver tissues and 9 normal livers. (2) The levels of mRNA expression of HSP90 and chk1 were assayed by reverse transcription PCR (RT-PCR) among 46 hepatocellular carcinoma, 46 paired adjacent cancerous liver tissues and 9 normal livers. (3) Cultured human hepatoma cell line Bel-7404 in vitro and observed the impacts of HSP90 inhibitor geldanamycin (GA) on the growth of human hepatoma cell line Bel-7404 by methyl thiazolyl tetrazolium (MTT) method. Studied the influence of HSP90 inhibitor geldanamycin (GA) on apoptosis of human hepatoma cell line BEL7404 by electron microscope, acridine orange fluorescent staining and Annexin V-EGFP/PI double staining FCM assay. (4) The influence of HSP90 inhibitor geldanamycin (GA) on cell cycle of human hepatoma cell line Bel-7404 was assayed by FCM. (5) The changes of the mRNA and protein expression of chk1 before and after treatment with HSP90 inhibitor geldanamycin (GA) were assayed by RT-PCR and Western-blot.Results: (1) The positive protein expression rates of HSP90 were 85.37% (35/41)、56.1% (23/41)、22.22% (2/9) in hepatocellular carcinoma, paired adjacent cancerous liver tissues and normal liver respectively. The rate in hepatocellular was significantly higher than the other two groups. The positive expression of HSP90 was associated with clinical stages, differentiation degree of cancer cells and recurrence (P<0.05), and was irrelevant with age, gender, tumor size, tumor nodus, serum level of AFP and portal vein embolus(P>0.05). The positive protein expression rates of chk1 were 80.49 % (33/41)、70.73 % (29/41)、11.13 % (1/9) in three tissues respectively. The rate in hepatocellular was higher than that in paired adjacent cancerous liver tissues, and significantly higher than that in normal liver tissues. The positive expression of chk1 was associated with pathological classification (P<0.05), and was irrelevant with age, gender, tumor size, tumor nodus, portal vein embolus, serum level of AFP, hepatic cirrhosis and ascites (P>0.05). Rank correlation analysis showed the expression between HSP90 and chk1 in hepatocellular carcinoma had a positive relationship.(2) The mRNA expression level of HSP90 was 0.718±0.372, 0.437±0.207 and 0.136±0.104 in hepatocellular carcinoma, paired adjacent cancerous liver tissues and normal liver respectively, there are significantly differences among groups (P<0.01). The mRNA expression of HSP90 was associated with TNM stages,differentiation degree of cancer cells and recurrence (P<0.05). The mRNA expression level of chk1 was 0.320±0.146, 0.193±0.071 and 0.098±0.000 in three tissues respectively, and there are significant differences among groups (P<0.01). The mRNA expression of chk1 was associated with pathological classification (P<0.05).(3) HSP90 inhibitor geldanamycin (GA) inhibited BEL7404 cell line at the concentration of 1-20 umol/L with 10.1 %～76.7 % inhibitory ratio. The growth inhibition ratio increased gradually after treatment with GA to BEL7404 cells 24h, 48h, and 72h respectively. And this inhibitory effect showed remarkable time and dose dependence.(4) G0/G1 period cells increased while G2/M period cells decreased either with treatment of 5 umol/L GA , or 10 umol/L GA, or 15 umol/LGA.GA arrested cell cycle at phase G0/G1,which showed time and dose dependency.(5) GA induced apoptosis of BEL7404 cells with the apoptosis rate of 10.2±1.35 %, 21.3±1.30 %, and 38.70±1.43 %, while the rate in the control group was 6.31±0.82 % by Annexin V-EGFP/PI double staining FCM assay. This suggested that GA could induce apoptosis of hepatoma cells.(6) The proportion of apoptosis cells increased gradually along with the different concentration of GA by acridine orange fluorescent staining. Condensation of chromatin at margins of nuclei, disintegration of nucleolus, vacuoles in cytoplasm, even apoptotic body were observed in the GA group under transmission electron microscope (TEM). This suggested that the inhibitory effect of GA on the growth of tumor cells was probably related to its arresting cell cycle at G0/G1 phase and induced apoptosis of tumor cells.(7) After BEL7404 cells were treated with 10 uM GA for various time intervals, chk1 mRNA expression showed no significant difference before and after GA treatment by a semi-quantitative RT-PCR analysis. Chk1 protein showed an obviously decrease after 24h treatment with GA assayed with western blot. These results indicated that the inhibitory effect of GA was due to degradation of chk1 protein but not decreased synthesis.Conclusions: The mRNA and protein expressions of HSP90 and chk1 in hepatocellular carcinoma are higher than paired adjacent cancerous liver tissues and normal liver tissues, and are associated with clinicopathological classification. HSP90 inhibitor geldanamycin (GA) inhibits the growth of hepatoma cells in vitro, induces apoptosis and blocks cell cycle of hepatoma cells. The inhibition of HSP90 can decrease the expression of chk1 in human hepatoma cell line Bel-7404, without affecting the synthesis of mRNA of chk1. The mechanism of anti-tumor activity of HSP90 inhibitor GA may be related to the effect of blocking the chk1 pathway. HSP90 and chk1 are hopeful new therapeutic targets for hepatocarcinoma.更多还原