【作者】 徐力善；

【导师】 董家鸿；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学， 2009， 博士

【摘要】 肿瘤发生机制的研究一直是肿瘤分子生物学中的一个重要研究领域,其中肿瘤相关基因的克隆和鉴定,不仅对肿瘤发生机制的研究具有重要的理论价值,而且对肿瘤的诊断和治疗具有潜在的应用价值。肝癌是常见的恶性肿瘤,发生机制尚不完全清楚,现被认为是多步骤多因素作用的结果,认为与癌基因的激活和抑癌基因的失活等基因背景有关系。鉴定癌周肝组织与肝癌组织表达有差异的基因,可为肝癌的预防、诊断和治疗提供有价值的候选基因。基于基因敲除后的表型分析及其研究,发现内胚层转录因子HNF6调控中胚层转录因子Foxfl的表达,进而通过调控Foxfl的下游基因Notch2的表达而调控胆管的发育和生长。由于发现肝部分切除术后再生肝脏的肝细胞内HNF6表达上调,并且肝癌的发生,表现为顺序出现的增生前和增生细胞群体,与正常细胞相比,其增殖行为发生明显改变,同时伴有大量肝脏特异性蛋白质的表达和活性改变。因此我们推测,上述转录因子在肝癌中也可能存在表达异常,基于上述考虑,本研究初步探索了上述3种转录因子与肝癌发生,发展的相关性。一、Forkhead box(FOX)蛋白质是一组进化上保守的转录因子超家族,以Forkhead DNA结合结构域为共同结构特征,调控广泛的生物学过程。本研究通过收集手术切除的人类癌周肝组织、肝癌组织新鲜标本,用Trizol试剂法提取各组织总RNA,通过OD260吸光度测定RNA浓度,各组织取等量RNA,通过随机引物引导,在M-MLV逆转录酶作用下进行逆转录反应,获得cDNA产物,以cDNA为模板,通过PCR法扩增FOXF1的基因片段。发现肝癌组织中转录因子FOXF1的表达水平显著低于癌周肝组织。上述PCR扩增产物经过测序,证实与MEDLINE序列一致;将各样本用含去垢剂NP-40的裂解液匀浆后提取组织总蛋白质,用考马斯亮蓝G-250染色法测量蛋白浓度后,取等量蛋白质上样进行SDS聚丙烯酰胺凝胶电泳,电泳分离后的蛋白质转移至硝酸纤维素膜上,用含牛血清白蛋白的封闭液进行封闭后,与稀释的FOXF1多克隆抗体进行杂交,再与辣根过氧化物酶标记的二抗孵育。经化学发光、显影和定影,发现肝癌组织中转录因子FOXF1的表达水平显著低于癌周肝组织。免疫组织化学检测,FOXF1在蛋白质水平上的表达与上述结果一致。二、Notch转录因子其家族成员的结构具有高度保守性,在细胞分化、发育中起着关键作用。本研究采用RT-PCR法在各样品中均观察到Notch2在基因水平的表达,但在癌周肝组织与肝癌组织之间,未观察到Notch2基因表达水平上的显著差异。Western杂交法未能在蛋白质水平上检测到各组织中Notch2的表达。免疫组织化学检测,肝癌组织与癌周肝组织之间Notch2蛋白质的表达水平没有差异。三、HNF6是肝细胞丰富转录因子家族中较新的成员,可调控肝细胞特异性基因的表达,在肝脏增殖过程中HNF6表达水平明显增高,在肝细胞的分化及功能维持上起重要作用。本研究通过RT-PCR法可在基因水平上检测到各组织中HNF6的表达。Western杂交法可在蛋白质水平上检测到各组织中HNF6的表达。结果表明,癌周肝组织与肝癌组织之间HNF6基因和蛋白质表达水平无显著差异。本研究结合免疫组织化学、逆转录PCR和Western杂交技术,分别在基因和蛋白质水平上初步探索了转录因子FOXF1、Notch2和HNF6在人类肝癌中的表达规律。在基因和蛋白质水平上均发现转录因子FOXF1在肝癌组织中的表达水平显著下调,提示该因子可能在肿瘤抑制中发挥作用,与抑癌基因可能存在某种程度的相互作用,有可能成为肿瘤基因诊断新的筛选标志和肿瘤基因治疗新的靶标,为进一步研究该转录因子在肝癌发生和发展中的作用奠定了基础。本研究同时初步探索了各转录因子在肝癌发生中的相互关系以及他们之间的上下游关系,为寻找可能存在的新的上下游因子关系提供了线索。通过基因水平表达和蛋白质水平表达差异上的相互弥补,克服各种检测技术的缺陷,发挥各种检测手段的优势,以期了解上述各基因表达规律的全貌。更多还原

【Abstract】 Mechanism of carcinogenesis has always been an important research area in tumor molecular biology.Cloning and identification of tumor related gene not only has theoretical value in tumor mechanism investigation,but also has potential practical value in tumor diagnosis and therapy.Liver cancer are the most common malignant tumors. Mechanism of carcinogenesis of liver cancer has not been clearly defined.Multi step and multi factor action may result in their occurrence.Oncogene activation and tumor suppressive gene inactivation may be associated with their development.Identification of differently expressed genes between normal liver tissue and liver cancer,can provide valuable candidate genes for prevention,diagnosis and therapy of liver cancerBased on phenotype analysis after gene knockout,endoblast transcription factor HNF6 may regulate mesoblast transcription factor Foxfl.Notch2 is a downstream gene of Foxf1.HNF6 may regulate Notch2 through Foxfl and regulate cystic development and growth.Since HNF6 expression was upregulated in surgical resected hepatocytes and bile duct epithelial cells of obstructive jaundice models,and liver cancer and cholangiocarcinoma are all proliferative diseases,we speculate that abnormal expression of above transcription factors may exist.Based on this concern,correlation study between transcription factor FOXF1,Notch2,HNF6 and human liver cancer was conducted.Forkhead box(FOX) proteins are evolutionary conserved transcription factor superfamily characterized by Forkhead DNA binding domain.It can regulate multiple biological processes.Operative dissected normal human hepatic tissue,hepatic carcinoma tissue,normal bile duct tissue and cholangiocarcinoma fresh samples were collected.Tissue total RNA was extracted by Trizole method.RNA concentration was measured by OD260 obsorbance.The same amount RNA was reverse transcribed into cDNA by M-MLV reverse transcriptase primed by random primer.Partial sequence of FOXF1 were amplified by PCR from cDNA template,mRNA level of transcription factor FOXF1 in hepatic carcinoma was found significantly lower than that in normal liver tissue.Homogenization with tissue lysis buffer containing detergent NP-40 obtained total protein.After protein concentration measurement with Coomassie brilliant blue G-250,the same amount proteins were loaded onto SDS-PAGE. Electrophoresis separated proteins were transferred onto Nitrocellulose membranes. After blocking with blocking solution containing bovine serum albamin(BSA), membranes were hybridized with diluted rabbit polyclonal antibody to human FOXF1. Membranes were incubated with HRP labeled second antibody.After chemical illuminance,develop and fixation,protein level of transcription factor FOXF1 in hepatic carcinoma was found significantly lower than that in normal liver tissue. Immunohistochemical detection confirmed that above results.Notch was found expressed in multiple species from non vertebral to vertebral animals.There is high conservation among Notch family members which play key roles in cell differentiation and development.Notch2 can be amplified from normal liver tissue, hepatic carcinoma,normal bile duct tissue and cholangiocarcinoma fresh samples.PCR products were confirmed by sequencing.No significant difference of Notch2 expression was observed between normal liver tissue and hepatic carcinoma.Western blot failed to detect Notch2 protein expression in all tissues.HNF6 is a new member of liver enriched transcription factor family.It can regulate liver specific gene expression.By action on HNF3β,HNF6 play some role in endoblast differentiation.Reverse transcription PCR can detect HNF6 gene expression in all tissues. Western blot can detect HNF6 protein expression in all tissues.No significant difference of HNF6 gene and protein expression was observed between normal liver tissue and hepatic carcinoma.Immunohistochemistry,reverse transcription PCR and Western blot were combined in this research.Expression of transcription factor FOXF1,Notch2 and HNF6 in operative dissected normal liver tissue and liver cancer were investigated.FOXF1 mRNA and protein expression was found significantly downregulated in hepatic carcinoma.This result laid foundations for further research concerning its roles in occurrence and development of liver cancer.The interrelationship of these transcription factors in carcinogenesis of liver cancer was explored.Clues were given to search new upstream and downstream factors.Detection methods of gene level and protein level were combined overcoming their limitations and exerting their advantages.Overall perspective ofgene expression were investigated.更多还原