【作者】 胡沛臻；

【导师】 杨守京；

【作者基本信息】 第四军医大学 ， 病理学， 2001， 硕士

【摘要】 热休克蛋白（Heat-shock protein，HSPs）是普遍存在于生物体细胞中的一个蛋白质家族，是细胞在生理和病理条件下启动一系列特殊基因编码合成的一类高度保守的蛋白质，它们作为细胞内蛋白质的伴侣分子，发挥重要功能，如保护细胞并促进细胞从各种刺激所造成的损伤中自身修复；近来发现，HSPs也是肿瘤的增生、分化和转移所必需的，这已在许多肿瘤研究中得到肯定。我室从1994年就开始进行热休克蛋白的研究，目前主要集中于热休克蛋白在肿瘤和传染病发病机制中的作用研究上，特别是流行性出血热和肝癌发病机制中的作用研究。目前国内外有关hsp70在肝癌细胞中的表达以及与肝癌发生关系的报道均较少见，运用热休克蛋白作为治疗肝肿瘤的手段也未见文献报道。 本实验在以往研究的基础上，采用免疫组化与分子杂交方法结合分子生物学和分子病理学等研究手段，从多种角度探讨hsp70在肝癌的发生机制中的作用及在肝癌基因治疗方面的可行性。（1）运用免疫组化与原位杂交技术检测了HSP70及hsp70 mRNA在原发性肝癌组织中的表达情况。结果发现：HSP70在人原发性肝癌组织中的阳性检出率81.5％（57/70），其中一胞核阳性率57．9O（33／57），而癌旁肝组织中HSP70 阳性检出率54．10 门0门7），未见胞核阳性，两者阳性率有显著性统计学差异（p＜0．05）； HSP70 的表达在Ill、IV级HCC fA织中的阳性检出率分别为 gi．3o（ZI／23）和 gi．70 （11／12），明显高于其在1级HCC组织中的表达54．60（6／11）和癌旁肝组织54．10门0／37）中勺表达（P＜0．05）；在HSP70阳性勺HCC组织及癌旁肝组织中 HBSAg及HBXAg的阳性表达率明显高于 HSP70阴性的 HCC组织及癌旁肝组织，有显著性统计学差异一O刀5）。提示：＊肥刀核阳性表达是肿瘤细胞所特有的；其表达的上调与 HCC的恶性程度正相关，即与其分化程度负相关；HSP70的过度表达有可能与HBV持续性感染、复制有关。 门）咸功构建了逆转录病毒表达载体pLXSN七，并应用脂质体介导的基因转染方法，对肝癌细胞HepGZ进行有效基因转移，采用细胞原位杂交、免疫荧光等方法进行mRNA及蛋白水平的分析，证实在人肝癌细胞系HepGZ中表达了 卜源目的基因hsp70。门）在体卜i田胞培养状 态下，结合流式细胞仪、TmEL和电镜等检测技术，研究了hsp70对肝癌i田胞HepGZ细胞周期的影响，并检测了转染hsp70后HepGZ细胞中多种基因产物的表达情况。结果发现转染hsp70后肝癌细胞HepGZ发生凋亡，6H细胞数量在GI期细胞百分比增加，S＋GZ＋M期细胞的百分比减少。表明转染hsp70的肝癌细胞发生GI期阻滞，影响瘤细胞增殖。同时发现转染hsp70后；转染细月与对照组细月寸上；Bax、P16、PZI、P53、c－Fos和c－Myc白表达增扒未见n7和助表达水平有明显改变。导致这些基因产物表达水平改变 的原因，有待进一步探讨。 综上所述，我们认为，hsp70可能参与了肝癌的发生发展过程，对 hsp70的进一步深入研究，有助于阐明HCC发病机制，对于探讨hsp70在肿瘤基 因治疗中的应用价值具有深远的意义。更多还原

【Abstract】 Heat shock proteins (HSPs) are highly conserved proteins expressed in prokaryotes and eukaryotes under normal and abnormal condition. HSPs play an important role in protecting proteins by assisting in the refolding of damaged proteins or facilitating the degradation of damaged proteins unable to repair. Recent study showed that HSPs also play an important role in carcinogensis of many tumors, however, little is known on the expression and significance of HSP7O and its potential use in treatment of hepatocellular carcinoma (11CC). Our studies have focused on the roles of HSPs in the pathogenesis of tumor and infectious diseases. On the base of our previous findings, (1) the expression of HSP7O protein and mRNA in 70 cases of 11CC tissues had been detected with immunobistochemistry and in situ hybridization. The results showed that about 81 .5%(57170) of 11CC cases and 54.1 %(20/37) peri-carcinomatic tissues were positive for HSP7O with the cytoplasm and/or nucleus localization. The positive rate of HSP7O in 11CC was significantly higher than that in peri-carcinomatic tissues (p<O.OS). The HSP7O positive nucleuses were observed only in 11CC but not in peri-carcinomatic tissues. The HSP7O highly expressed in Grade III (91.3%)~棐IV(91.7%) 11CC than in Grade I (54.6%)HCC and peri-carcinomatic tissues(54. 1 %)(p<O.OS). The results suggested that the high expression of 3 HSP7O in I-ICC might be correlated with the malignancy of the tumor. The correlation of HBV infection with HSP7O expression had been observed in 55 cases of HCC and peri-carcinomatic tissues. In 10 HCC cases negative for I-ISP7O, there were only 3 cases positive for HBsAg(30.0%) and 4 cases positive for HBxAg(40.0%), whereas in 45 HCC cases positive for HSP7O, there were 30 HCC cases positive for I]IBsAg(66.7%) and 36 HCC cases positive for HBxAg(80.0%), which was significantly higher than that in peri-carcinomatic tissues. This results indicated that HSP7O had close relationship with HBsAg I-IBxAg , the overexpression of HSP7O may contribute to the persistent infection of HBV in HCC. (2) The hsp7o expression vector PLXSN-hsp70 was constructed by inserting the hsp70 gene into a retroviral vector PLXSN, and then transferred into the hepatocarcinoma cell line HepG2 using lipofectin system. hsp70 expression was detected by immunofluorescence and in situ hybridization. The results indicated that the transfected HepG2 cells could express LISP7O proteins and hsp70 mRNA. (3) Using the Flow Cytometry, TUNEL, and EM techniques, the effects of the hsp70 gene transfection on hepatocarcinoma cells HepG2 were demonstrated in vitro. The result showed that the hsp70 transfection could lead to increase the apoptosis of HepG2, and the expression of some gene products, such as Bax. Pl6~. P21 P53 and c-Fos, but P27 and Rb. The ratio of G1/S+G2-i-M in HepG2 was also increased, it means the inhibition of cell proliferation, which might be the result of the cell growth delay in G1 phase. In summary, this study may be of value to elucidate the roles of HSP7O in the carcinogenesis of HCC, and its potential use in the gene therapy for HCC.更多还原