【作者】 刘易欣；

【导师】 孙保存；

【作者基本信息】 天津医科大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 目的：通过检测乙肝病毒基因产物、核转录因子-κB、细胞周期素D1、抗凋亡基因Bcl-2、热休克蛋白70在肝炎相关性肝细胞肝癌组、肝炎后肝硬化组及正常对照组的表达差异和相关分析，探讨上述基因编码蛋白的相互影响关系及在肝炎相关性肝细胞肝癌发生发展中作用和意义。方法：对152例肝炎相关性肝细胞肝癌标本、78例肝炎后肝硬化标本经病理复查后制备成4块组织芯片，每份病例穿取4个组织位点，经HE染色光镜下观察确认后对上述基因编码蛋白进行免疫组织化学染色；200倍光镜下对各阳性表达产物的着色强度及阳性细胞百分比进行计数、计量，按Mattern积分法计算各指标的阳性表达指数；应用SPSS11.5医学统计软件包对上述各指标的阳性表达指数进行方差分析及相关分析。结果：1．乙肝基因表达产物中HBsAg、HBcAg、HBx蛋白除彼此相关外HBx蛋白与NF-κB、cyclinD1、HSP70、Bcl-2(p值均＜0.01)；HBcAg除与HSP70无相关关系外其他均与HBx蛋白相关(p值均＜0.01)；HBsAg仅与HSP70相关(p值均＜0.01)。2．NF-κB核内表达与cyclinD1呈显著性正相关(P＜0.01)，与Bcl-2及HSP70无相关关系；3．HSP70与Bcl-2呈显著性负相关(P＜0.01)，与cyclinD1呈显著性正相关(p值均＜0.01)；cyclinD1与Bcl-2无相关关系。4．各指标组间两两比较结果示：HBx蛋白、HBsAg、NF-κB、cyclinD1、HSP70在肝癌组的平均表达指数均显著高于肝硬化组及正常对照组(P均＜0.01)；Bcl-2在肝癌组的平均表达指数则显著低于肝硬化组而高于正常对照组，肝硬化组高于正常对照组(P均＜0.05)；HBsAg在肝癌组的平均表达指数显著低于肝硬化组(P均＜0.05)。结论：1．HVx基因蛋白的表达与NF-κB的表达和激活密切相关；HBx蛋白可能通过其反式激活作用将NF-κB激活，激活后的NF-κB进入细胞核内与其下游细胞增殖调控基因κB位点结合，启动cyclinD1表达的上调，我们推论HBx蛋白的长期持续高表达→NF-κB的异常激活→cyclinD1表达上调是导致肝细胞恶性转化及肝肿瘤细胞持续增殖的重要途径之一。同时也说明HBx蛋白是肝细胞肝癌发生发展的始动因素。2．NF-κB在HCC组的细胞浆及细胞核内平均表达指数均显著高于肝硬化组和正常对照组，表明NF-κB在肝癌细胞内发挥重要的调控作用；NF-κB在肝硬化组胞浆内平均表达指数均显著高于正常对照组，但在肝硬化组胞核内的平均表达指数与正常对照组无显著性差异，表明NF-κB仅在胞浆内表达增高并不具有使细胞发生恶性转化的能力；3．cyclinD1在肝癌组的平均表达指数显著高于肝硬化组和正常对照组，与NF-κB在胞核中的表达呈显著性正相关，且提示NF-κB在肝细胞肝癌的发生中主要通过上调细胞周期调控基因cyclinD1实现的；4．NF-κB与另一下游抗细胞凋亡调控基因Bcl-2的表达无明显相关，表明NF-κB在肝癌的发生过程中缺乏对Bcl-2的调节作用；5．Bcl-2在肝硬化组的平均表达指数显著高于肝癌组及正常对照组Bcl-2与HBx蛋白及HBcAg的表达呈显著性正相关；说明抗细胞凋亡基因Bcl-2在肝组织内的表达上调可能通过乙肝病毒基因产物直接或与其相关的其他途径激活，而Bcl-2在肝癌最主要的癌前病变肝硬化中发挥重要的抑制凋亡作用，延长细胞生存时限，因此，我们推论Bcl-2是肝癌形成及转化中的一个更早的分子事件。6．HSP70在肝癌组的平均表达指数显著高于肝硬化组及正常对照组，而肝硬化组和正常对照组之间无显著性差异，另外HSP70与bcl-2的表达关系在整体上呈负相关，提示肝癌细胞的凋亡调控可能是由HSP70参与的bcl-2以外的其他分子事件。更多还原

【Abstract】 Objectives: To clarify their roles and correlation of HBV genes product, nucleartranscription factor-κB (NF-κB), cyclinD1, Bcl-2 and Heat shock protein70 (HSP70)in the initiation and development of hepatocellular carcinoma (HCC), their proteinexpression level was detected in hepatitis-related HCC group, posthepatitic cirrhosisgroup and control group.Methods: Tissue array was made with 152 cases patients with hepatitis-related HCCand 78 cases patients with posthepatitic cirrhosis after reassessing by pathologists.Four spots in slide were taken out to make the tissue array for each case.Immunohistochemical staining was performed on the slides after HE staining.Staining Index (SI) was used to assess the expression of each factor according toMattem Method. The Statistical Package for Social Sciences (SPSS) version 11.5 wasused for all calculations. The significance level was set at 0.05 for each analysis.Results:1. The expression level of HBcAg, HBx protein, NF-κB, cyclinD1 and HSP70 washigher in HCC group than that in posthepatitic cirrhosis group and control group(P＜0.01), which means they play an important role in the malignant change of HCC.The expression level of Bcl-2 was lower in HCC group than that in posthepatiticcirrhosis group and control group (P＜0.01), which means it can be against apoptosisin precancerous lesions of HCC and maybe an early molecular event. The expressionlevel of HBsAg was lower in HCC group than that in posthepatitic cirrhosis group(P＜0.05), and HBsAg only correlated with the HSP70 (P＜0.01), which means HBsAg has little relationship with the initiation of HCC.2. The expression level of HBx had a positive correlation with the expression ofcyclinD1, NF-κB, cyclinD1, Bcl-2 and HSP70 (P＜0.01), besides correlated with theexpression of HBcAg and HBsAg. HBcAg had the similar results as HBx, excepthad no correlation with the expression of HSP70 (P＜0.01). These data shown thatNF-κB, cyclinD1 , Bcl-2 and HSP70 could be active and over expressed as theincreased expression level of HBx. The increased level of HBcAg could upregulatetranscription and amplification of HBV genes, which can affect the above oncogenesby increasing the copy number of HBx gene.3. The expression level of NF-κB had a positive correlation with the expression ofcyclinD1 (P＜0.01), but had no correlation with the expression of HSP70 and Bcl-2.The activation of NF-κB can increase the expression level of cyclinD1, however hadno effect on the downstream gene Bcl-2.4. There was negative correlation between the expression of HSP70 and Bcl-2(P＜0.01). There was positive correlation between the expression of HSP70 andcyclinD1(P＜0.01), but there was no correlation of the expression of cyclinD1 andBcl-2, which shows that HSP70 level has some effect on the cyclinD1 and Bcl-2.Conclusions:1. NF-κB, activated by HBx, can be transported into nucleus and bind toκB site andthen upregulate the expression of cyclinD1. The axis "High expression of HBx→Activating of NF-κB→Upregulation of cyclinD1" was thought to play an importantrole in the process of malignant transformation of liver cells and continuingproliferation of tumor cells. HBx was regarded as an promoter of HCC.2. High expression of NF-κB in cytoplasm can not make the liver cells aquiring theability of malignant transformation.3. NF-κB can promote the proliferation of tumor cells by upregulating of cyclinD1which let more G1 stage cell become S stage cell.4. NF-κB can not affect the expression of Bcl-2.5. HBV genes product and its relative pathway can affect the expression of Bcl-2which can play an important role in cirrhosis. Activation of Bcl-2 is an earlier process in the process of hepatocarcinogenesis.6. HSP70 can be an alternative molecular pathway which can regulate the apoptosisin the process of hepatocarcinogenesis.更多还原