【作者】 刘子杰；

【导师】 翁亚光；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2009， 博士

【摘要】 细胞分裂时染色体平均分配到子代细胞中,这是保持亲代与子代遗传物质的稳定性的基础,纺锤体检查点是保证染色体正确分离的一个重要机制,如果纺锤体检查点功能异常就会导致非整倍体细胞形成,甚至与肿瘤发生相关,因此研究纺锤体检查点作用机制对进一步探明肿瘤发生的机制具有重要意义。近年来纺锤体检查点机制的关闭/灭活得到越来越多的关注,因为纺锤体检查点机制提前关闭会引起染色体错误分离从而形成非整倍体细胞。CENP-E、Mps1蛋白与纺锤体检查点机制关闭/灭活关系密切。本课题运用分子克隆方法从Hela细胞中扩增CENP-E的cDNA,构建含CENP-E蛋白结构域的六个真核表达载体;利用流式细胞术等方法分析CENP-E蛋白结构域对纺锤体检查点功能的影响,以此分析CENP-E蛋白与微管连接状态与纺锤体检查点关闭/灭活的关系;同时利用原核表达载体将CENP-E具有磷酸化位点的截短体,Mps1全长蛋白进行表达纯化;运行荧光共振能量转移（FRET）方法和免疫共沉淀方法研究与Mps1蛋白有相互作用的CENP-E蛋白结构域,为进一步研究CENP-E蛋白和Mps1间作用与纺锤体检查点机制关闭/灭活的关系打下基础。第一部分CENP-E蛋白结构域重组载体构建目的:1.克隆CENP-E基因cDNA;2.构建含CENP-E蛋白结构域的真核表达载体。3.构建含CENP-E蛋白磷酸化位点的截短体及Mps1全长蛋白的原核表达载体并纯化蛋白。方法:1.用RT-PCR的方法从Hela细胞总RNA中获得编码CENP-E蛋白的CENP-E cDNA序列。2.将CENP-E蛋白结构域cDNA插入pEGFP-C1真核表达载体;3.将测序鉴定正确的重组质粒转染HEK293细胞,在倒置荧光显微镜下观察重组质粒表达情况。4.收集转染重组质粒的HEK293细胞提取总蛋白,western blot鉴定重组蛋白（抗GFP小鼠单克隆抗体）。5.原核表达载体构建及用亲和层析纯化重组蛋白结果:1.RT-PCR扩增的CENP-E cDNA与T J.Yen提交Genebank（ACCESSION:NM₀01813）序列比较,RT-PCR产物缺失CENP-E基因第38个外显子。与CENP-E突变体比对同源性达99%,结果表明RT-PCR得到了正确的产物。2.真核重组质粒转染HEK293细胞后在细胞内定位与预期结果相似。3.重组质粒表达的蛋白与预期分子量相同。4.原核表达出正确分子量大小的蛋白且得到较纯的蛋白。结论:1.得到正确的CENP-E基因cDNA但与最先提交的序列有差异。2.成功的构建了含CENP-E蛋白结构域的真核表达载体。3.成功构建含CENP-E磷酸化位点的及Mps1全长蛋白的原核表达载体并纯化成功。第二部分初步探讨CENP-E基因在癌组织及癌细胞株中的变异情况目的:1.分析Hela细胞中CENP-E基因mRNA表达情况。2.分析几种肿瘤细胞株（Hela、T24、A549、U251、HepG₂）和两种肿瘤（肝细胞癌和肾癌）及癌旁组织中的CENP-E mRNA表达情况。3.研究基因组DNA是否存在CENP-E第38个外显子缺失。方法:1.采用RT-PCR方法,以Hela细胞总RNA为模板,分析Hela细胞中CENP-E mRNA表达情况;2.采用PCR方法扩增几种不同肿瘤组织、癌旁组织和肿瘤细胞株基因组DNA,分析在基因组DNA是否存在第38个外显子缺失。3.采用RT-PCR方法同时扩增缺失型和野生型CENP-E mRNA,分析缺失型和野生型CENP-E mRNA的表达量。结果:1.Hela细胞中同时存在缺失型和野生型CENP-E mRNA。2.肝癌、肾癌及相对应的癌旁组织和几种肿瘤细胞株的基因组DNA没有发现CENP-E缺失突变现象。3.肝癌与肾癌组织及几种肿瘤细胞株中突变型CENP-E表达量较野生型高,癌旁组织中野生型CENP-E表达量较突变体高。结论:CENP-E基因mRNA在同一细胞内存在二种或两种以上的剪切体,CENP-E突变体可能与肿瘤有关。第三部分CENP-E蛋白结构域与纺锤体检查点机制关闭的关系目的:1.分析CENP-E蛋白结构域与纺锤体检查点机制关闭的关系。2.CENP-E蛋白在纺锤体检查点中发挥功能是否依赖于着丝点。方法:1.HEK293细胞转至六孔板培养,分别转入含CENP-E蛋白结构域的重组质粒和pEGFP-C1空载质粒。2.采用胸腺嘧啶核苷对HEK293细胞进行双处理,大部分细胞同步于G₁/S期。去除胸腺嘧啶核苷后,用新鲜培养基继续培养7h加诺考达唑处理2h。3.收集第7h组和第9h组细胞经流式细胞术进行细胞周期分析,分析第7h和第9h各组细胞的G₂/M期细胞比例变化情况,同时进行细胞分裂指数测定和细胞死亡率测定。4.收集第9h的各组细胞进行染色体染色体计数分析,了解CENP-E蛋白结构域对染色体分离的影响。结果:1.HEK293细胞经双胸腺嘧啶核苷处理后,去除药物释放7小时大部分细胞进入G₂/M期。2.流式细胞术结果表明转染含CENP-E蛋白动力结构域（pEGFP-E1组和pEGFP-ET1）组第9h G₂/M期细胞比例低于第7h,与对照组比较差异具有统计学意义（P＜0.05）,而其它组细胞第9h G₂/M期细胞比例高于第7h,与对照组比较无统计学意义（P＞0.05）。细胞分裂指数测定也符合流式细胞术结果。3.细胞死亡率测定发现转染pEGFP-E1组和pEGFP-ET1组第9h细胞死亡率高于第7h,与对照组比较差异具有统计学意义（P＜0.05）,其它转染CENP-E结构域重组质粒组细胞死亡率与对照组比较差异无统计学意义（P＞0.05）。4.转染pEGFP-E1组和pEGFP-ET1组细胞经染色体计数分析,经诺考达唑同步处理后非整倍体细胞数目增多,与对照组比较有统计学意义（P＜0.05）;其它转染CENP-E结构域重组质粒非整倍体细胞数目与对照组比较差异无统计学意义（P＞0.05）。结论:CENP-E蛋白动力结构域与纺锤体检查点机制关闭有关;过度只含CENP-E蛋白动力结构域的蛋白能使纺锤体检查点机制提前关闭。第四部分与Mps1蛋白有相互作用的CENP-E蛋白结构域的确定目的:探索与Mps1有相互作用的CENP-E蛋白结构域方法:1.用荧光共振能量转移方法研究CENP-E结构域与Mps1蛋白间的相互作用。2.免疫共沉淀实验对荧光共振能量转移实验结果进行验证。结果:1.荧光共振能量转移实验结果表明CENP-E3结构域与Mps1蛋白间有相互作用。2.免疫共沉淀结果与荧光共振能量转移实验结果一致。结论:与Mps1蛋白有相互作用的CENP-E蛋白结构域位于1200-2134位氨基酸;荧光共振能量转移对蛋白质相互作用的实验结果可信度较高。更多还原

【Abstract】 It is very important that chromosomes segregation ensures only when all the chromosomes have congressed to the metaphase plate to ensure the even partition of the genetic material into the two daughter nuclei.This is accomplished by a surveillance mechanism known as the spindle assemble checkpoint.The aneuploidy emerge even to induce tumorigenesis when the spindle assemble checkpoint dysfunction,so that it is very important to investigate spindle assemble checkpoint in order to explore the mechanism of tumorigenesis.Recently,more and more researcher pay attention to the mechanism of inactivation of spindle checkpoint,because the incorrect inactivation of spindle assemble checkpoint can lead to segregation of chromosome and induce anuepoliy present.There is close relationship between the mechanism of inactivation of spindle assemble checkpoint and Mps1 and CENP-E protein.In order to further understand the function of CENP-E in the spindle assemble checkpoint,we amplified the cDNA of CENP-E gene from the Hela cell line,and constructed six recombinant vectors containing the structural domain of CENP-E protein.Analysis the effect of structural domain of CENP-E protein on the spindle checkpoint was carried out by flow cytometrics,and we also measured the MI and ratio of cell death,and the ratio of aneuploid cells and Explored the interaction between the Mps1 protein and the structural domain of CENP-E protein by fluorescence resonance energy transfer（FRET） and co-immunoprecipetation method,we want to analyze the role of the structural domain of CENP-E protein in the spindle assemble checkpoint,these research will lay a foundation for further studying the mechanism of inactivation of spindle assemble checkpoint.PARTⅠCONSTRUCTING THE RECOMBINANT VECTORS OF THE STRUCTURAL DOMAIN OF CENP-E PROTEINObjective:1.Amplifying the cDNA of CENP-E.2.Constructing the recombinant vectors of the structural domain of CENP-E protein3.Constructing the prokaryotic expression vector containing the phosphorylation sites of CENP-E and full length of Mpsl protein,and purification of the recombinant proptein.Methods: 1.Amplifying the eDNA of CENP-E gene from the total RNA of Hela cells by RT-PCR method.2.Constructing recombinant vectors containing the structural domain of CENP-E.3.Analyzing the expression of recombinant vectors in the HEK293 cells using inverted fluorescence microscope.4.Identifying the recombinant protein collecting the HEK293 cells transfected with recombinant vectors respectively using western blot method（the anti-GFP monoclonal antibody）.5.Constructing prokaryotic expression vectors and purification of recombinant protein.Results:1.The sequence of RT-PCR products was different from the sequence submitted by T J.Yen（ACCESSION:NM₀01813）,which amplified from Hela cells,however sharing 99%homology with the sequence of the variant CENP-E protein.2.The location of recombinant protein expressed in the HEK293 cells was consistent with expected results.3.The molecular weight of recombinant proteins detected with western blot was consistent with the anticipated.4.The molecular weight of fusion protein purified by Ni2+-NTA agarose column was correct.Purity of recombinant protein was above 92%. Conclusion:1.We successfully amplified the cDNA of CENP-E gene,which was consistent with the sequence of the variant CENP-E protein and had distinct diversity with the sequence submitted by T J.Yen,which amplified from Hela cells too.2.We successfully constructed the recombinant vectors containing the structural domain of CENP-E protein.3.Prokaryotic expression vectors were constructed correctly and we get the high purity fusion protein.PARTⅡPRELIMINARY STUDY THE EXPRESSION OF CENP-E GENE IN THE TUMOR TISSUES AND CANCER CELL LINESObjective:1.Analyzing the expression of the mRNA of CENP-E gene in the Hela cells2.Analyzing the expression of the mRNA of CENP-E gene in two kinds of carcinoma tissues（hepatocellular carcinoma and renal carcinoma） and peri-cancerous tissues,and several kinds of cancer cell lines （Hela,T24,A549,U251,HepG₂）。3.Determining whether there is deletion of the 38^th extron in genomic DNA or not.Methods:1.Analyzing the expression of CENP-E mRNA by RT-PCR method in the Hela cells.2.The deletion mutation of CENP-E gene were Studied by PCR method using DNA as template.3.Analyzing the deletion and wild type CENP-E mRNA in carcinoma tissues and peri-cancerous tissues,and cancer cell linesResults:1.Both deletion and wild type mRNA of CENP-E existed in the Hela cells2.There was not deletion mutation of CENP-E gene in carcinoma tissues’ and pare-cancerous tissues’ DNA,and cancer cell lines’ DNA.3.The expression of deletion mutation type of CENP-E mRNA was higher than that of wild type in carcinoma tissues and cancerous cell lines,the expression in peri-cancerous tissues was just the reverse.Conclusion:Two even more type mRNA of CENP-E gene co-existed in the same cell,CENP-E variant was associated with tumorigenesis. PARTⅢEFFECT OF THE STRUCTURAL DOMEIN OF CENP-E PROTEIN ON THE SPINDLE ASSEMBLE CHECKPOINTObjective:1.Examined the relationship between the structural domain of CENP-E protein and inactivation of the spindle assemble checkpoint;2.To observe whether kinetochore dependent of CENP-E play its role on the spindle assemble checkpoint or notMethods:1.HEK93 cells were cultured in the 6 cells plate;cells were transfected with recombinant vectors and p EGFP-C1 vector respectively.2.HEK293 cells were synchronized by double thymidine,the majority of cells were arrested in G1/S phase,cells cultured 7 hours using fresh medium after removed thymidine,then HEK293 cells treated by nocodazole 2 hours.3.Flow cytometry was used to analysis the percentage of G2/M phase of the 7^th and 9^th HEK293 cells.MI assay and trypan blue exclusion was employed.4.The influence of the structural domain of CENP-E protein on chromosome segregation was evaluated by chromosome numbers and counts of the chromosomes. Results:1.The majority of HEK293 cells arrested in G1/S phase treated double thymidine,after 7 hours removed thymidine,the majority of cells enter G2/M phase.2.Results of FCM showed the percentage of G2/M phase was decreased after treated by nocodazole in the cells transfected with pEGFP-E1 and pEGFP-ET1 vector.In contrast,the percentage of G2/M phase of other group was increased in the situation.The results of MI were consistent with the results of FCM.3.Results of trypan blue exclusion assay showed the ratio of cell death was increased after being treated by nocodazole in the cells transfected with pEGFP-E1 and pEGFP-ET1 vector.The ratio of cell death of other group has no difference compared with control group.4.Counts of the chromosomes showed that ratio of the cells with abnormal chromosomal numbers were significantly increased in cells transfected with pEGFP-E1 and pEGFP-ET1 vector.Conclusion:The motor domain of CENP-E protein is related with inactivation of spindle assemble checkpoint;the role of CENP-E in the spindle assemble checkpoint is kinetochore independent. PartⅣEXPLORING THE STRUCTRAL DOMAIN OF CENP-E PROTEIN INTERACTED WITH MPS1 PROTEINObjective:Study the structural domain of CENP-E interacted with Mps1 proteinMethods:1.The protein-protein interaction between the structural domain of CENP-E protein and Mps1 protein was studied.2.Results of FRET were confirmed by CO-IP method.Results:1.The CENP-E3 domain of CENP-E（aa:1200-2134） can interacted with Mps1 protein.2.Results were consistent with results of FRET.Conclusion:the Mps1 protein binding domain of CENP-E resided between 1,200 and 2134.Result of FRET can be faithfully confirmed in the studying protein-protein interaction.更多还原