【作者】 朱娜；

【导师】 孙路虹；

【作者基本信息】 南京医科大学 ， 免疫学， 2008， 硕士

【摘要】 肝癌是我国常见的严重危害人类生命的恶性肿瘤之一,发病率在我国的恶性肿瘤中居第二位。其中,肝细胞性肝癌(hepatocellularcarcinoma,HCc)是最主要的组织类型。探讨HCC组织中异常表达基因的分子生物学机制,对于了解其发生、发展过程以及辅助诊断和治疗,有着重要的理论和实际意义。Gadd45基因家族是在研究肿瘤发生,发展以及转归过程中新发现的相关基因家族,该家族成员主要包括Gadd45a、Gadd45p及Gadd45y。现有资料表明,该基因家族在细胞周期调控过程中发挥重要作用,但其对人肝癌细胞HepG2细胞周期的影响及具体机制尚不清楚,故此,本研究就Gadd45α/γ在HCC组织中的表达及Gadd45α抑制肝癌细胞生长的信号通路进行了初步探讨。第一部分Gadd45α/γ在肝癌组织中的表达及对人肝癌细胞HepG2细胞周期的影响目的:检测Gadd45α/γ蛋白在肝癌组织中的表达情况,并探讨其对人肝癌细胞HepG2细胞周期的影响。方法:随机选取临床HCC标本50例,运用免疫组织化学方法检测Gadd45α/γ蛋白在50例肝细胞性肝癌及癌旁组织中的表达水平并分析相关临床意义;体外培养人肝癌HepG2细胞,分别转染质粒pCMV-tag2B、pCMV-tag2B-Gadd45a和pCMV-tag2B-Gadd45γ,运用流式细胞仪检测Gadd45α/γ对细胞周期分布的影响。结果:(1)Gadd45a蛋白在50例肝细胞性肝癌中阳性率为58.0%(29/50),癌旁组织中阳性率为62.0%(31/50),两者差异没有统计学意义;Gadd45γ蛋白在肝癌组织中的阳性表达率为30.0%(15/50),而在癌旁组织中的阳性表达率为86.0%(43/50),两者比较有显著性差异(p＜0.01)。(2)Gadd45α/γ蛋白表达阳性率与肝癌分化程度有关,它们在高分化癌中阳性率(84.2%,57.9%)显著高于在中低分化癌中的阳性率(41.9%,12.9%);两者有显著性差异(p＜0.05),而Gadd45α/γ蛋白表达阳性率与性别,年龄以及淋巴结转移的相关性不明显(p＞0.05)。(3)将质粒pCMV-tag2B-Gadd45α/γ分别转染至人肝癌细胞HepG2后可诱导细胞周期G2/M期阻滞,且pCMV-tag2B-Gadd45α(G2/M期细胞比值为0.31±0.03)诱导的阻滞程度较pCMV-tag2B-Gadd45γ(G2/M期细胞比值为0.21±0.03)诱导的阻滞程度更为明显。结论:Gadd45α/γ蛋白的表达可能与肝癌的病理分型、发展以及转归过程存在着密切关系,与Gadd45γ相比,Gadd45α在肝癌的转归过程中可能扮演着更为重要的角色,并且为肝癌的基因治疗提供了新的分子靶点。第二部分Gadd45a诱导人肝癌细胞G2/M期阻滞的相关信号转导通路研究目的:探讨Gadd45α抑制肝癌细胞生长可能涉及的信号转导通路,为研究肝癌细胞生长抑制性基因提供初步理论依据。方法:体外培养人肝癌细胞HepG2,分别转染质粒pCMY-tag2B-Gadd45α和pCMV-tag2B至细胞,采用western blot方法检测Gadd45α及其下游基因P38、p-P38、JNK和p-JNK的表达变化;然后,给予信号通路的抑制剂(sB203580和SP600125)分别特异性阻断P38和JNK的磷酸化,再运用流式细胞仪检测并比较各组细胞周期,进而分析Gadd45α诱导肝癌细胞HepG2阻滞在G2/M期可能涉及的信号通路。结果:(1)将质粒pCMV-tag2B-Gadd45α转染至人肝癌细胞HepG2 48h后,Gadd45α蛋白表达峰值为0.68±0.02,其下游基因P38(0.94±0.01)和JNK(0.77±0.04)的磷酸化水平与空白对照组(0.58±0.02,0.50±0.02)相比均明显升高。(2)运用特异性抑制剂使升高的P38和JNK磷酸化表达水平降低至0.56±0.02和0.53±0.03,达到空白对照组磷酸化水平(0.58±0.02,0.50±0.02),但细胞仍表现为G2/M阻滞,阻滞程度较非抑制剂组明显减弱。结论:在HepG2细胞中过度表达Gadd45a蛋白可以激活Gadd45α-P38和Gadd45α-JNK信号转导通路;Gadd45α-P38和Gadd45α-JNK通路参与了Gadd45α诱导的肝癌细胞G2/M期阻滞。更多还原

【Abstract】 Primary hepatoma is the most common and lethal cancer around the world which is very harmful to human health.In china,it is the second most frequent cancer.Hepatocellular carcinoma(HCC)is the most common histological type among primary hepatoma.The identification of the abnormal gene expression profile in HCC will help us to clarify the mechanisms of tumorigenesis and development of HCC and develop new targets for prevention,diagnosis and therapy.The Gadd45 family of proteins,which includesα、βandγisoforms, has recently been demonstrated to play critical role in tumor research, development and the process of turnover.Recently it was documented that Gadd45 could play critical roles in negative growth control. Nevertheless,up to now the mechanism by how Gadd45 exerts its function of negative growth control is not fully understood in hepatoma HepG2 cell lines.In this study,we described the expression of Gadd45α/γin HCC and the related signal transduction pathways that mediated cell cycle G2-M arrest by Gadd45αin HepG2 cells. PartⅠExpression and significance of Gadd45α/γprotein in human hepatocellular carcinomaObjective:To investigate the expression of Gadd45α/γprotein in human hepatocellular carcinoma(HCC)and observe cell cycle arrest inducing by Gadd45αand Gadd45γin hepatocellular carcinoma.Methods:Immunohistochemical method was used to detect the expression of Gadd45α/γfrom the hepatocellular carcinoma tissues and surrounding non-neoplastic liver tissues in 50 cases;then vectors encoding the Gadd45αor Gadd45γgenes were tranisently transfected to HepG2 hepatoma cells and analysed for the cell cycle phase using flow cytometry.Results:(1)The positive expression rates of Gadd45αin the HCC tissues and surrounding non-neoplastic liver tissues in 50 cases was 58.0%(29/50) and 62.0%(31/50)respectively.The rates were no significantly different from each other(p＞0.05).The positive expression rates of Gadd45γin the HCC tissues and surrounding non-neoplastic liver tissues in 50 cases were 30.0%(15/50)and 86.0%(43/50)respectively.The rates were significantly different from each other(p＜0.01).(2)The both expression of Gadd45αand Gadd45γwere close correlatedwith the differentiation degree(P＜0.05):the positive rate of Gadd45α/γin well differentiation carcinoma(84.2%,57.9%)was noticeable higher than poor differentiation carcinoma(41.9%,12.9%).But the expression of Gadd45αand Gadd45γwere not significantly correlated with age,gender and lymphnode metastasis(P＞0.05).(3)After transient transfection of HepG2 cells,the proteins of them were capable of arresting HepG2 cells at the G2/M phase of the cell cycle. Compare to Gadd45γ-induced G2/M arrest,Gadd45α-induced G2/M arrest was more obviously.Conclusions:The Gadd45α/γcould play critical roles in pathological type,development and the process of turnover of hepatocellular carcinoma;The Gadd45αcould play more important role in treatment of hepatocellular carcinoma.PartⅡRelated signal transduction pathways in Gadd45α-induced cell cycle G2-M arrest in hepatoma cellsObjective:To explore the related signal transduction pathways throw Gadd45α-induced cell cycle G2-M arrest in HepG2 cells and provide the primary indications of growth inhibition in hepatoma.Methods:Firstly,HepG2 cells were transfected with pCMV-tag2BGadd45αand pCMV-tag2B respectively and the changes in protein levels of Gadd45α,P38,p-P38,JNK and p-JNK were detected by Western blot. Then,the inhibitors of SB203580 and SP600125 were employed in HepG2 cells to block up the phosphorylation of P38 and JNK.Finally,the cell cycles of HepG2 cells were assayed by flow cytometry.Results: (1)In HepG2 cells transfected with pCMV-tag2B-Gadd45αexpression vector,Gadd45αprotein expression reached the peak level(0.68±0.02)at 48h and the phosphorylation levels of P38(0.94±0.01)and JNK(0.77±0.04)apparently increased.(2)The degree of G2-M arrest was obviously attenuated after applying with the inhibitors in HepG2 cells.Conclusion:Gadd45αand Gadd45γ,utilize P38 and JNK signaling pathways to induce cell cycle G2/M arrest in HepG2 Hepatoma cells更多还原