【作者】 宋志；

【导师】 窦科峰； 陶开山；

【作者基本信息】 第四军医大学 ， 外科学， 2010， 博士

【摘要】 肝癌是我国常见恶性肿瘤之一，其死亡率在恶性肿瘤中仅次于胃、食道而居第三位。在我国每年约有11万人死于肝癌，占全世界肝癌死亡人数的45%。研究表明原发性肝癌的预后比较差的主要原因是缺乏特异的早期诊断指标和治疗手段，而研究肝癌的发病机理有助于提高肝癌的早期诊断率，指导肝癌的正确治疗。Tg737基因是近年发现的一种肝癌抑癌基因，最初进行研究时发现该基因与遗传性多囊肾病（autosomal recessive polycystic kidney disease, ARPKD）orpk鼠突变模型研究中的目的基因相同。但到目前为止，对于Tg737基因在肝癌发病过程中的如何发挥作用并不十分清楚。因此，进一步揭示Tg737基因的生物学功能，丰富和发展肝癌发生发展理论，为肝癌早期诊断及治疗提供依据有着十分重要的意义。本研究共分三个部分：第一部分Tg737基因在肝癌中的表达及相关性研究目的观察Tg737基因在肝癌组织和细胞中的表达，并分析其与肝癌的相关性。方法采用RT-PCR和Western blot方法检测人肝癌组织和肝癌细胞系中Tg737 mRNA和蛋白的表达，并通过统计学方法对表达情况与肝癌的相关性进行分析。结果人正常肝细胞系HL-7702中Tg737的mRNA表达和蛋白表达水平均高于肝癌细胞HepG2和MHCC97。65例肝癌组织标本中有30例Tg737mRNA表达呈阳性，且表达水平较相应癌旁组织低；34例检测出Tg737蛋白的阳性表达。而65例癌旁组织中有61例检测出Tg737 mRNA的阳性表达；59例Tg737蛋白表达呈阳性。18例早期（Ⅰ-Ⅱ）肝癌组织中15例Tg737蛋白表达呈阳性，47例中晚期（Ⅲ-Ⅳ）肝癌中19例呈阳性，二者比较有统计学意义。结论Tg737基因作为肝癌的抑癌基因，其表达下调可能是肝癌发生、发展过程中的重要因素，检测Tg737基因的表达可能作为判断肝癌分期的参考指标。第二部分Tg737基因对肝癌细胞活性的影响目的构建Tg737基因的真核表达载体，并将其导入肝癌细胞中，观察该基因对人肝癌细胞增殖活性的影响。方法以临床获取的人正常肝组织的mRNA为模板，通过RT-PCR方法获得Tg737基因全长序列，将其克隆至pEGFP-C1载体，进行序列、酶切鉴定。将HepG2细胞随机分为三组：HepG2-Tg737组（pEGFP-Tg737转染组）、HepG2-vect组（空质粒转染组）和HepG2组（空白对照组），利用脂质体转染法将重组质粒转染至肝癌细胞系HepG2中；通过Real-time PCR及Western blot方法明确转染48h后，各组细胞中Tg737的表达情况，并通过流式细胞仪和MTT比色法观察其对细胞周期和活性的影响。结果我们成功构建了真核表达载体pEGFP-Tg737，将其转染至肝癌细胞系HepG248h后，HepG2-Tg737组细胞中Tg737mRNA和蛋白的表达水平均明显高于HepG2-vect组和HepG2组。与其它两组相比，HepG2-Tg737组细胞生长明显受到抑制，G1期细胞比例高于HepG2-vect组和HepG2组，S期细胞比例明显较低。结论Tg737基因能够过表达于肝癌细胞HepG2中，并可抑制该细胞的增殖能力。第三部分肝癌中Tg737基因杂合性缺失的研究目的检测肝癌中Tg737基因的杂合性缺失情况，并分析微卫星位点杂合性缺失与临床病理特征的关系。方法应用聚合酶链反应-单链构象多态性技术（PCR-SSCP）检测Tg737基因的五个微卫星位点(RH65721、RH118612、SHGC-57879、STS-U20362和G64212)杂合性缺失情况，并通过统计学方法对缺失情况与肝癌临床病理参数的相关性进行分析。结果Tg737基因的五个微卫星位点RH65721、RH118612、SHGC-57879、STS-U20362和G64212的LOH发生频率分别为61.54% (32/65)、52.31%(34/65)、65.45%(36/65)、58.33%(28/65)、74.47%(35/65)；五个位点之间的LOH发生率差异无统计学意义（P>0.05）。微卫星位点SHGC-57879和G64212的LOH发生率与肝癌的转移及TNM分期相关（P<0.05）。结论Tg737基因在肝癌中存在较大比例的杂合性缺失，其微卫星位点表现出较高的不稳定性，影响基因的表达。通过以上的研究，初步证实了Tg737基因在肝癌发生的过程中发挥着抑癌基因的作用，对肝癌细胞的增殖有明显的抑制作用。而且Tg737基因在肝癌中存在杂合性缺失，其微卫星位点表现出较高的不稳定性，进而影响基因的表达。因此，Tg737基因有可能作为肝癌的基因治疗靶点，为肝癌的基因治疗提供新思路。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common malignanttumors in China and its mortality rate ranks the third place after stomach cancerand esophagus cancer. There are over 11 million people who died from HCCevery year, which accounts for 45% of deaths in the world. Studies have shownthat the prognosis of primary liver cancer is still not optimistic, mainly due tothe lack of specific indicators of early diagnosis and treatment. The research ofpathogenesis in HCC is helpful to promote the rate of diagnosis in the earlyperiod.Tg737 gene is a kind of tumor suppressor gene in HCC. The initial studyfound that the gene was the same as the target gene of genetic polycystic kidneydisease (autosomal recessive polycystic kidney disease, ARPKD) orpk mutantmouse model. But so far, the mechanism of Tg737 gene in HCC is not very clear.Therefore, it is very important to reveal the biological function of Tg737 geneand develope the theory of HCC for diagnosis and treatment of HCC in the earlyperiod. This study is divided into three parts.PartⅠStudy of expression of Tg737 and correlation in hepatocellularcarcinomaObjective Expression of Tg737 in HCC was analyzed and relationshipbetween the expression of Tg737 and mechanism of HCC was investigated.Materials and methods RT-PCR and Western blot methods were used toanalyze the expression of Tg737 mRNA and protein.Results The results showed that Tg737 expression of the mRNA andprotein in liver cells were higher than HepG2 and MHCC97 cells. Thirty casesof Tg737 mRNA expression in 65 cases of HCC tissues were lower than thecorresponding adjacent tissues, while 61 cases of Tg737 mRNA expression inthe adjacent tissues were positive. Fifty-nine (90.78％) cases of Tg737 proteinexpression in 65 cases of the adjacent tissues were positive. There were 34(52.31％) cases of protein positive expression in HCC tissues. Fifteen caseswere positive in 18 (55.56％) cases of early (Ⅰ-Ⅱ) HCC tissues. There were 19(27.66％) cases of positive expression in 47 cases of advanced (Ⅲ-Ⅳ) HCCtissues. Statistical analysis showed that the Tg737 protein in tumor tissue wassignificantly lower than the adjacent liver tissue. The Tg737 protein positive rateof early (Ⅰ-Ⅱ) HCC tissue was significantly higher than in advanced (Ⅲ-Ⅳ)HCC tissues.Conclusions It is indicated that Tg737 gene expression in HCC isreduced, which may be important factor in the process of hepatocarcinogenesis.Analysis of Tg737 gene expression may be reference index of HCC stages.PartⅡTg737 affecting activity of HCC cellsObjective Tg737 gene was amplificated from human normal liver tissueand constructed to the eukaryotic expression vector. Then it is studied that Tg737 influences activity of HCC cells.MaterialMaterials and methods Plasmid pEGFP-Tg737 was transfected intoHepG2 cell and increased expression level of Tg737. The experiment wasdivided into three groups: HepG2-Tg737 group (pEGFP-Tg737 transfectiongroup), HepG2-vect group (empty vector transfection group), and HepG2 group(blank control group). Cell cycles were analyzed by flow cytometry andexpression of Tg737 was detected by Real-time PCR and Western blot methods.Cell growth curves were made by MTT test.Results The eukaryotic expression vector pEGFP-Tg737 was constructedsuccessfully and transfected into hepatocellular carcinoma line. Compared withHepG2-vect group and HepG2 group, Tg737 expression of HepG2-Tg737 groupincreased remarkedly after 48 hours of transfection. Cell growth in HepG2-Tg737 group was significantly inhibited and the proportion of G1 phase cells inHepG2-Tg737 group was higher than that of HepG2-vect group and HepG2group and cells of S phase was significantly lower.Conclusions Tg737 gene can inhibit cell proliferation markedly andinduce block of G1 stage.PartⅢStudy of loss of heterozygosity on Tg737 geneObjective Loss of heterozygosity (LOH) of Tg737 in HCC was detectedand relationship between LOH status and clinical pathology characters wasanalyzed.Materials and methods Polymerase chain reaction-single strandconformation polymorphism (PCR-SSCP) method was used to detect LOH offive microsatellite sites on Tg737.Results The results showed that LOH frequences of five microsatellitesites in Tg737 gene were 61.54%(32/65), 52.31%(34/65), 65.45%(36/65), 58.33%(28/65) and 74.47%(35/65). There was no significant difference (P>0.05)between LOH of five microsatellite sites. However, LOH frequences of SHGC-57879 and G64212 related to tumor metastasis and TNM staging (P<0.05).Conclusions High frequences LOH of Tg737 gene exist in HCC andmicrosatellite sites had high instability, which can affect on expression of thegene.In conclusion, this study confirms that tumor suppressor gene Tg737 playsan important role in the period of hepatocarcinogenesis and inhibits theproliferation of HCC cells. Nevertheless, loss of heterozygosity of the Tg737gene exists in hepatocellular carcinoma and high instability is shown on itsmicrosatellite sites which affect the gene expression. So Tg737 can serve astargets for gene therapy of HCC and provide new ideas for gene therapy of HCC.更多还原