【作者】 雒喜忠；

【导师】 王阁；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2009， 硕士

【摘要】 背景和目的:肿瘤是一种多基因、多步骤、多因素、多网络的病理机制复杂的疾病,细胞分化障碍是肿瘤形成的本质,利用某些化合物诱导肿瘤细胞分化为正常细胞或近似正常细胞的肿瘤的诱导分化治疗是继手术、放疗、化疗后的新的治疗模式,已成为研究的“热点”。Tec(tyrosine kinase expressed in hepatocellular carcinoma)是一种存在于胞质内的非受体型蛋白酪氨酸激酶,是肝干细胞和肝细胞增殖、分化、凋亡信号转导途径中关键的蛋白激酶连接分子。我们在前期工作中以Tec激酶结构域(KD)作为“钓饵”蛋白,利用酵母双杂交技术筛选构建于转录激活结构域(AD)载体的人胎肝cDNA文库,经营养缺陷选择、诱导筛选及初步鉴定,发现并确证了一个新的阳性克隆。筛库所得基因片段经测序和BLAST比对分析,无同源性序列,确定为RAI16(Homo sapiens retinoic acid induced 16)基因,已被GeneBank收录(编号为BC052237),组织来源为脑Ⅳ型星状细胞瘤,其编码蛋白为人视黄酸诱导蛋白16。进而以RAI16蛋白为切入点,利用相关的生物信息学站点及计算机软件,对RAI16进行生物信息学分析,从中获得其编码蛋白质结构方面的重要信息,从而根据结构预测其功能。生物信息学分析提示,RAI16蛋白含6个PKC磷酸化结合位点,8个CK2磷酸化结合位点,3个酪氨酸(Tyr)磷酸化结合位点, 5个豆蔻酰化位点,2个酰胺化位点,1个ATP/GTP-结合位点、无跨膜区结构域,具有一段由31个氨基酸组成的信号肽,且其切割位点位于30～31aa之间,因此推测RAI16蛋白可能为RA信号通路中一类胞浆内重要的“新”信号蛋白分子。关于RAI16文献报道很少,功能研究报告更是空白,从其命名知晓,为一种RA诱导蛋白。由于诱导分化剂相关的信号转导过程是揭示其作用机理的关键,结合RA在肿瘤细胞分化中的突出作用和RAI16蛋白的功能的“未知性”,我们选择肝癌细胞为对象,将课题切入点定位于“Tec作用蛋白RAI16的新功能和分化调控机理研究”上,以酵母双杂交系统筛选中Tec酪氨酸激酶与RAI16蛋白相互作用的现象为线索,从肝癌细胞诱导分化和胞内信号转导途径的角度,通过抗体制备、真核表达载体和腺病毒载体的构建及表达检测、免疫组化、激光共聚焦分析、Northern blotting、Western blotting、MTT、流式细胞仪(FCM)检测、划痕实验、Transwell细胞迁移和Metrigel细胞侵袭实验等多种方法明确RAI16的表达谱和亚细胞定位,分析RAI16在肝再生、肝癌形成和ATRA对肝癌细胞诱导分化过程中的作用,探讨RAI16对肝癌细胞诱导分化、细胞周期、细胞增殖与凋亡、迁移与侵袭力等多种生物学行为的影响,探索RAI16蛋白“新”功能,并进一步认识和阐明RAI16在RA诱导肝癌细胞分化的信号转导途径中的关键作用,本研究将为肿瘤细胞诱导分化信号转导分子机理的研究提供新思路和理论依据,为肝癌分子治疗提供新的作用靶点,为进一步研究其作用机制奠定基础。研究内容和方法1.用纯化的RAI16蛋白合成肽免疫新西兰大白兔,采用一种快速免疫法制备RAI16多克隆抗体,ELISA追踪检测抗体效价,Protein A亲和层析法纯化特异性IgG抗体,Western blot和免疫组化检测抗体的特性,初步检测RAI16在大鼠再生肝组织和肝癌组织中的表达,为进一步研究RAI16的功能提供抗体。2.以ATRA诱导人肝癌HepG2细胞后,流式细胞仪检测其对细胞周期、细胞增殖与凋亡的影响;提取总RNA和总蛋白,采用RT-PCR和Western blot检测ATRA诱导后RAI16的表达情况,分析ATRA与RAI16在细胞诱导分化中的关系,为进一步探讨RAI16在诱导分化中的作用机理提供理论基础。3.以真核表达质粒pEGFP-C1为载体,从含有全长RAI16基因片段的质粒克隆模板中,利用PCR方法钓取目的基因,将目的基因与目的载体用EcoRI和XhoI分别进行酶切。纯化酶切产物后进行定向连接,构建pEGFP-C1-RAI16,经酶切及测序鉴定。采用激光共聚焦显微镜观察转染pEGFP-C1-RAI16后的亚细胞定位;Western blot分析检测其对HepG2细胞RAI16蛋白水平和诱导分化作用;利用MTT实验检测pEGFP-C1-RAI16质粒转染后对细胞增殖的影响。4.以真核表达质粒pDC315-EGFP为载体,从含有全长RAI16基因片段的质粒克隆模板中,利用PCR方法钓取RAI16基因,将其与真核表达载体pDC315-EGFP分别以EcoR I进行酶切。纯化酶切产物后进行定向连接,构建穿梭质粒pDC315-EGFP-RAI16。经测序鉴定后,利用AdMax腺病毒包装系统构建重组腺病毒Ad-RAI16,并进行包装、扩增、纯化和滴度测定;采用激光共聚焦显微镜观察感染Ad-RAI16后的亚细胞定位;通过Western blotting、MTT、流式细胞仪(FCM)检测、划痕实验、Transwell细胞迁移和Metrigel细胞侵袭实验等多种方法分析RAI16对肝癌细胞诱导分化、细胞周期、细胞增殖与凋亡、迁移与侵袭力等多种生物学行为的影响,进一步认识和阐明RAI16在RA诱导肝癌细胞分化的信号转导途径中的关键作用。研究结果1.成功制备了兔抗人RAI16多克隆抗体。ELISA检测显示该抗体的效价达到1:125 000,相对亲和力常数为10-5~10-6 M-1。Western blot和免疫组化提示该抗体能与RAI16蛋白特异性结合。RAI16在肝、心脏、食管、胃、脑、胰腺等多种组织中均有表达,而且在肝脏再生和肝癌形成过程中其表达呈上调趋势。2. ATRA诱导后RAI16的表达明显上调,并且呈时效性关系;MTT检测结果显示:ATRA能够抑制HepG2在体外的增殖,与对照组比较,诱导组细胞生长均2d开始明显受抑(P<0.05),组内不同处理天数之间有显著性差异(P<0.05)。流式细胞仪检测显示:ATRA能够促进肝癌细胞分化和凋亡,抑制细胞DNA合成,使其停滞于G1期。3. RAI16真核表达载体的构建及其生物学效应:经测序分析表明成功构建重组表达载体pEGFP-C1-RAI16。转染HepG2细胞发现RAI16主要定位于胞质内内质网等细胞器中,且能显著抑制细胞增殖和分化分子AFP、γ-GT的表达。4. RAI16腺病毒表达载体的构建及其生物学效应:经测序分析质粒表达检测表明成功构建重组表达腺病毒Ad-RAI16。感染HepG2细胞发现RAI16主要定位于胞质内内质网等细胞器中;能显著抑制细胞增殖、抑制分化分子AFP、γ-GT的表达和Wnt通路信号传导分子β-Catenin的表达;抑制细胞迁移,削弱细胞侵袭能力。结论1.成功制备兔抗人RAI16多克隆抗体并进行了特异性亲和纯化,抗体效价和纯度良好;Western blot和免疫组化显示制备抗体特异性良好。RAI16在多种组织中表达,并参与了肝再生和肝癌形成过程的调控。2. ATRA能够抑制肝癌细胞增殖,促进其分化和凋亡;能显著增强RAI16的表达。RAI16可能在ATRA的诱导分化过程发挥重要作用。3.成功构建了真核表达载体pEGFP-C1-RAI16,RAI16蛋白定位于胞质内内质网等细胞器中;并具有抑制肝癌细胞增殖和促进分化的作用。4.成功构建重组腺病毒表达载体Ad-RAI16。进一步研究表明RAI16能够抑制肝癌细胞增殖、侵袭和迁移,促进其分化和凋亡;可直接通过调控Wnt信号转导通路参与肝癌细胞的诱导分化。更多还原

【Abstract】 Background and objectiveTumor is a diseas with polygene, multistep, multifactor, many networks and complex pathomechanism. Cell dysdifferentiation is the nature of tumorigeness. The induced differentiation therapy for tumor, which can make tumor cells become mormal cells or similar to the normal cells by using some compounds, is a new therapy pattern following the surgery therapy, radiotherapy and chemotherapy and become the focus of research.Tec is a kind of non-receptor tyrosine kinase which is a crucial joint molecule in signal transduction pathway of liver stem cells and liver cell proliferation, differentiation and apoptosis. We use Tec kinase domain as the bait protein in our preliminary work and take advantage of the technique of yeast two-hybrid system to filter the human foetus liver cDNA library that was built from transcription activation domain vector, and through auxotrophic selecting , inducing filtering and preliminary identification, we found and confirmed a new positive clone. After sequencing and making BLAST comparison analysis on the gene fragment got from the filtering result of gene library in this study, we were sure that it was the RAI16 (Homo sapiens retinoic acid induced 16 gene), which has no homology sequence and has been embodied by Genebank and the number is BC052237, and the source of tissue is Brain astrocytoma (grade IV). Its coding protein is the Homo sapiens Retinoic Acid induced protein 16. Afterwards we looked on RAI16 as the cut through point. We mainly used related computer softwares and some bioinformatics websites to analyze RAI16,from which we obtained the significant information of RAI16 protein structure and thus we could predict its unknown new functions according to its structure. The analyses of bioinformatics shows that RAI16 protein contains six protein kinase C phosphorylation sites, nine casein kinase II phosphorylation sites, three tyrosine kinase phosphorylation sites, six N-myristoylation sites, two amidation sites, three tyrosine sulfation sites and one ATP/GTP-binding site. Furthermore, this protein has no transmembrane domain, which contains a section of signal peptide consisted of 31 amino acid and the cleavage site is located between 30-31aa. So we predict it to be a kind of important new decapentaplegic molecule of RA signal path in intracytoplasm.At present there is a few of documents reported about RAI16 and its function study is vacuity. While learning from its name it is a kind of inducing protein of RA. Because signal transduction process is the key to reveal its mechanism of action and thinking of its unknowning function and the outstanding effection of RA for cancer cells in induced differentiation, we choose HCC cells which were induced to differentiate by RA or RAI16 to act as our study object and we looked on‘Preliminary Research of function and differentiation regulating mechanism of RAI16 Protein Interacting withTec Tyrosine Kinase’as the breakthrough point. Herein, from the induced differentiation of HCC cells and intracellular signal transduction path, we looked on phenonmenon that RAI16 inacted with Tec in yeast two-hybrid system as clue to identify expression spectrum and subcellular localization of RAI16, to analyze its function in the process of liver regeneration, hepatocarcinogenesis and HCC differentiation induced by ATRA, to investigate RAI16 effect for many kind of biological behavior of HCC cells, such as cell differentiation, cell cycle, cell proliferation or apoptosis , cell migration or invasiveness and etc, and explored the new function by antibody preparation, the construction of eukaryotic expression vector and adenovirus vector, immunohistochemisty, laser confocal microscop, immunoblotting, MTT, FCM, and so forth, which can make us to learn and interpret its crucial role in signal transduction pathway that RA induced HCC cells to differentiate. This study will provide new idea and theoretical evidences for the molecule mechanism of tumorous cell induced differentiation and signal transduction, and provide a new target for molecule targeted therapy of HCC, which can make the found base for the next step of the function mechanism study of RAI16.Materials and Methods1. Rabbit anti-human RAI16 polyclonal antibody was prepared by using its protein synthetic peptide and a modified rapid immune procedure followed by purification of specific avidity column. The efficacy of this polyclonal antibody was certificated by ELISA, Western blot and immunohistochemistry. The expression of RAI16 was preliminarily detected in regenerating liver tissue and cancer tissue of liver. This can provide antibody for the next step of the function study of RAI16.2. To detect the influence of RAI16 for cell cycle, cell proliferation and apoptosis by flow cytometer after HepG2 cells were induced by ATRA, then extracted total RNA and total protein to detect the expression of RAI16 by RT-PCR and Western blot and analyze the relation of ATRA with RAI16 in inducing differentiation, which can provide the theoretical base for the next step of the function mechanism study of RAI16 during the induced differentiation process.3. First,the RAI16 genes was amplified by PCR from plasmid cloning templates which contains total length gene fragment of RAI16 then inserted into pEGFP-C1 between EcoRI and XhoI sites. Then, the plasmid vector was sequenced and named as pEGFP-C1-RAI16. Laser confocal microscopy was used to observe the subcellular localization of recombinant RAI16. Western blot was applied to measure the increase level of RAI16 protein and induced differentiation function. Subsequently, cellular survival and proliferation was assayed by MTT.4. First,the RAI16 genes was amplified by PCR from plasmid cloning templates which contains total length gene fragment of RAI16 then inserted into pDC315-EGFP at the side of EcoRI sites. The plasmid vector was sequenced and named as pDC315-EGFP-RAI16. Second, AdMax system was used to construct the Ad-RAI16 and to enclose, amplify, depurate and detect titer.Then laser confocal microscopy was used to observe the subcellular localization of recombinant RAI16. Western blot was applied to measure the increase level of RAI16 protein and induced differentiation function. Subsequently, cellular survival and proliferation was assayed by MTT, cellular cycle and apoptosis was analyzed by FCM and the effect for cell biological behaviour was investigated by Transwell cell migration experiment and Metrigel invasion experiment.Results1. Rabbit anti-human RAI16 polyclonal antibody has been preparated successfully: The titer of the antibody obtained in this experiment determined by ELISA was up to 1∶125 000.The kaff value of the antibody was 10-5～10-6 M-1. Western blot results showed the antibody has high specificity to RAI16 protein. RAI16 can express in many tissues, such as liver, heart, esophagus, stomach, brain, pancreatic and so forth, and its expression was up-regulated during the liver regeneration and hepatocarcinogenesis process. 2. The induced effect of ATRA: That the expression of RAI16 was up-regulated after induced by ATRA was showed by Western blot. MTT results show that ATRA can inhibition HepG2 cells to proliferate in vitro, cell growth in induced group begin to be restrained obviously after two days comparing with control(P<0.05)and there are significant difference(P<0.05)in different phase after treating. FCM shows that ATRA can promote HCC cells to differentiate and apoptosis, suppress DNA synthesis and make them stagnate in G1 period.3. Construction and biological effects of plasmid vector overexpressing RAI16 specifically in eukaryotic: Sequencing analysis show that pEGFP-C1-RAI16 was constructed successfully. After transfected into HepG2 RAI16 was found to mainly localizate in endoplasmic reticulum and intracytoplasm and to significantly suppress cell proliferation and the expression of AFP andγ-GT, which are differentiation molecules.4. Construction and biological effects of adenovirus plasmid vector overexpressing RAI16: Sequencing analysis show that Ad-RAI16 was constructed successfully. After infected HepG2 RAI16 was found to mainly localizate in endoplasmic reticulum and intracytoplasm and to significantly suppress cell proliferation and the expression of AFP andγ-GT, which are differentiation molecules, to inhibite cell migration and to weaken invasing ability of tumorous cells.Conclusion1. Rabbit anti-human RAI16 polyclonal antibody, with high titer and specificity, was successfully produced by a modified rapid immune procedure. This polyclonal antibody can be further applied in ELISA, Western blot and immunohistochemistry to elucidate the roles of RAI16 protein played in many important cellular procedures. The result of initiatory experiment shows that RAI16 genes express in many tissues and regulate liver regeneration and hepatocarcinogenesis.2. The proliferation of HepG2 was significantly inhibited,the differentiation and apoptosis was induced and the expression of RAI16 was obviously up-regulated by ATRA. RAI16 may play important role in the process of ATRA induced differentiation.3. The eukaryotic overexpressing vector pEGFP-C1-RAI16 was successfully constructed, which may satisfy experiment demand. RAI16 proteins localizate in endoplasmic reticulum and intracytoplasm,which can inhibit the proliferation of HCC cells and make them differentiate.4. The recombinant adenovirus overexpressing vectorwas successfully constructed. It was proved that RAI16 can inhibit proliferation, invasion and migration of HCC cells, promote cancer cells to differentiate and apoptosis and play induced differentiating function in HCC through regulating Wnt signal transduction path.更多还原