【作者】 贺新春；

【导师】 范学工；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 研究背景：原发性肝癌是一种病死率很高的临床危重症,在我国原发性肝癌最常见的病因是病毒性肝炎,其中主要的是乙肝病毒(hepatitis B virus, HBV)感染。而高迁移率族蛋白1 (high mobility group box chromosal protein 1, HMGB1)在细胞外作为一种关键的“晚期”炎症因子,已经成为癌症防治研究中一个重要的新靶标。有研究表明HMGB1可能在某些癌症发生发展中发挥着重要的作用。本研究旨在探讨HMGB1在原发性肝癌发生发展中的潜在作用。目的：(1)研究肝癌患者肝组织、血清及外周血单个核细胞(PBMC)中HMGB1的表达水平。(2)体外观察HMGB1刺激是否能促进人肝细胞株HepG2细胞增殖并初步探讨其可能的机制。(3)探讨小干扰RNA(small interfering RNA, siRNA)抑制HMGB1基因对人肝细胞株HepG2细胞生物学功能的影响。方法：(1)分别收集健康对照者血清及外周血单个核细胞(PBMC),肝癌患者血清、PBMC、肝癌组织及癌旁组织,利用RT-PCR、Western blot和免疫组化技术,分别从mRNA水平和蛋白质水平研究肝癌患者组织、血清及PBMC中HMGB1表达的情况。同时行HE染色,评估肝组织的病理学改变。(2)体外用不同浓度重组HMGB1 (rHMGB1)刺激人肝细胞株HepG2细胞,不同时间点分别收集细胞。台盼蓝拒染法和噻唑蓝(MTT)法测定细胞存活率,TUNEL法检测细胞凋亡率,以明确细胞是否凋亡或坏死。RT-PCR和Westernblot检测细胞中PCNA、cyclin D1表达的变化。(3)设计并化学合成针对HMGB1的3对siRNA分子序列(siRNAH1、siRNAH2、siRNAH3),同时设未转染组(Lipo组)及转染阴性siRNA组(阴性siRNA对照组)。脂质体法瞬时转染HepG2细胞,通过RT-PCR和Western blot检测HMGB1基因在mRNA水平及蛋白质水平的变化以验证HMGB1 siRNA抑制效果；MTT法检测细胞存活率并绘制生长曲线；RT-PCR和Western blot检测PCNA、cyclin D1表达的变化,TUNEL法检测细胞凋亡率,以明确抑制HMGB1表达后能否诱导细胞凋亡,同时测定培养上清中AFP的含量。结果：(1)肝癌患者癌组织中HMGB1无论是mRNA水平(2.02±0.25)还是蛋白水平(0.78±0.13)均明显高于相对应的癌旁组织的mRNA水平(1.08±0.12)和蛋白水平(0.325±0.11)(P<0.01)。同样,免疫组化结果显示癌组织中HMGB1表达明显高于相对应的癌旁组织。而血清中HMGB1蛋白相对浓度及PBMC中HMGB1 mRNA的含量与健康对照组之间无差别(P>0.05)。(2)MTT结果显示不同剂量(10、50、100ng/ml) rHMGB1组HepG2细胞增殖能力均明显高于HepG2细胞空白对照组(P<0.05)。HMGB1抗体(20μg/ml)能阻断HMGB1 (10ng/ml)对HepG2细胞的增殖作用。Western blot和RT-PCR显示,HMGB1能明显上调HepG2细胞中PCNA、cyclin D1蛋白和mRNA的表达(P<0.05), HMGB1抗体能明显抑制HMGB1上调PCNA、cyclinD1蛋白和mRNA表达的作用(P<0.05)。台盼蓝拒染法和TUNEL检测排除了细胞处于凋亡或坏死状态。(3)3对特异性HMGB1-siRNAs转染HepG2细胞后,HMGB1 mRNA相对表达量分别为1.147±0.024,1.014±0.042,0.415±0.055,较单纯脂质体对照组(Lipo组)的1.411±0.065明显减少(t值分别为7.187、9.018和20.689,P值均<0.01)；3对特异性HMGB1-siRNAs转染HepG2细胞后,HMGB1蛋白相对表达量分别为0.369±0.035,0.340±0.028,0.097±0.020,较Lipo组的0.553±0.051明显减少(t值分别为6.678、7.919和17.287,P值均＜0.01),以siRNAH3抑制作用最强,抑制率可达到70-80%。转染siRNAH3后HepG2细胞的增殖能力在72h、96h、120h时受到明显抑制,与Lipo组相比,其F值分别为34.651、540.550、3778.197,P值均<0.01,HepG2细胞中PCNA及cyclin D1 mRNA和蛋白表达下降,AFP含量也下降(P<0.01),而细胞凋亡指数明显增高(P<0.01)。结论：(1)证实HMGB1在肝癌癌组织中是明显升高的,其表达与肝癌的发生、发展密切相关。(2) HMGB1明显促进HepG2细胞增殖,HMGB1抗体能拮抗外源性HMGB1的增殖作用。(3)靶向HMGB1基因的siRNA分子片段可以有效地抑制人肝细胞HepG2的生长,诱导HepG2细胞凋亡,其作用与下调HMGB1的表达有关,HMGB1可能是肝癌治疗的一个潜在靶点。更多还原

【Abstract】 Objectives:(1) To identify the expression levels of HMGB1 in liver tissues, serum and peripheral blood mononuclear cell (PBMC) from hepatocellular carcinoma patients. (2) To investigate the effect of high mobility group box-1 protein (HMGB1) on proliferative activity of human hepatoma cell line HepG2 and its potential regulating mechanism. (3) To investigate the effect of specific inhibiting HMGB1 gene expression by small interfering RNAs (siRNA) on the proliferation and apoptosis of human hepatoma cell line HepG2.Methods:(1)Serum and PBMC from healthy control were collected. Similarly, serum, PBMC and liver tissues from HCC patients were also collected. Then, all samples were subjected to Western blot, RT-PCR or immunohistochemical analysis for the expression of HMGB1, respectively. At the same time HE staining was performed to evaluate the feature of liver tissue damage. (2) The cultured HepG2 cells were treated with recombinant HMGB1 (10、50、100ng/ml) or HMGB1-Ab, or both for 24h. Cell proliferation was observed by MTT and trypan blue exclusive analysis. Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect the expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 protein and mRNA respectively. In situ apoptosis was evaluated by terminal deoxynucleotidyl transferase-deoxyuridine triphosphate nick end labeling (TUNEL) assay. (3) Three specific siRNAs of HMGB1 were designed and synthesized, and transiently transfected into HepG2 cells by Lipofectamine TM 2000.The HMGB1 expression in HepG2 cells was detected by RT-PCR and Western blot after transfection respectively. The proliferation activity in vitro was assessed by MTT assay. Western blot and RT-PCR were used to detect the expression of PCNA and cyclin Dl protein and mRNA respectively. In situ apoptosis was evaluated by TUNEL assay. The concentration of supernatant AFP from the transfected HepG2 cells was detected using chemiluminescence immunoassay (CLIA) method.Results:(1) According to immunohistochemical staining and Western blot analysis, HMGB1 protein levels were significantly overexpressed in carcinoma tissues, as compared to the corresponding non-cancerour tissues from the same HCC patient. Correspondingly, HMGB1 mRNA levels were also elevated in carcinoma tissues, as compared to noncancerous counterparts from the same HCC patient. However, HMGB1 neither protein in serum nor mRNA levels in PBMCs had any difference between HCC patients and healthy controls. (2) Compared with no-treatment, HMGB1 at the concentration of 10ng/ml, 50ng/ml and 100ng/ml obviously increased HepG2 cells proliferation, cyclin Dl and PCNA protein and mRNA expression after treatment for 24 hours respectively (P<0.05). However, when both HMGB1 (10ng/ml) and anti-HMGB1 (20μg/ml) treated HepG2 cells, anti-HMGB1 could significantly inhibit the proliferation effect and cyclin Dl and PCNA mRNA and protein expression of HMGB 1 on HepG2 cells (P<0.05). (3) All of these specific HMGB1-siRNAs (1,2,3) efficiently and specifically inhibited the expression of the HMGB1 gene and the levels of HMGB 1 mRNA were 1.147±0.024,1.014±0.042,0.435±0.055, respectively, in HMGB1-siRNAs transfection group, which were significantly lower than that in LipofectamineTM2000 alone group (1.411±0.065, P<0.01); Correspondingly, all of these specific HMGB1-siRNAs (1,2,3) could efficiently and specifically inhibit the expression of the HMGB 1 protein and the levels of HMGB1 protein were 0.369±0.035,0.340±0.028, 0.097±0.020, respectively, in HMGB1-siRNAs transfection group, which was significantly lower than that in LipofectamineTM2000 alone group (0.553±0.051, P<0.01). Of the 3 specific HMGB1-siRNAs, HMGB1-siRNA-3 (siRNAH3) had the highest inhibition rate (70-80%). The proliferation of HepG2 cells was markedly inhibited by siRNAH3 transfection. Compared to mock-transfection, seventy-two hours after introduction of siRNAH3 into HepG2 cells, siRNAH3 dramatically suppressed not only the proliferation activity of HepG2 cells but also the expression of PCNA and cyclin D1 mRNA and protein, as well as the secretion of AFP in the culture supernatant (P<0.01). Moreover, siRNAH3 can induce apoptosis by inhibiting HMGB1 expression (P<0.01).Conclusions:(1) It is further identified that HMGB1 levels were significantly elevated in hepatocellular carcinoma tissues. Our data indicate a strong correlation between expression of HMGB1 and HCC development. (2) HMGB1 has potent growth-promoting effect on HepG2 cells proliferation, and anti-HMGB1 can reverse this effect. (3) siRNA targeting HMGB1 mRNA can specifically reduce HMGB1 gene and protein expression. siRNAH3 can effectively suppress the proliferation and induce apoptosis of HepG2 cells by inhibiting HMGB1 expression, providing a novel target for hepatocarcinoma therapy.更多还原