【作者】 施琼；

【导师】 翁亚光；

【作者基本信息】 重庆医科大学 ， 生物医学工程， 2007， 博士

【摘要】 在胚胎发育的早期进行着旺盛的细胞有丝分裂,染色体正常分离到两个子细胞是保证有丝分裂正常进行的重要基础,与之相关的蛋白质种类至少应包括:着丝粒相关蛋白(centromere associated proteins)(MAD,BUB等),着丝粒特异蛋白(centromere specific proteins)(CENPs等)和纺锤体微管相关蛋白(spindle microtube associated proteins)。在细胞分裂中染色体的正常分离受到有丝分裂纺锤体检查点(mitotic spindle checkpoint)的调控,具有正常功能的纺锤体检查点可保证姐妹染色单体正确地分离进入两个子细胞。当有丝分裂纺锤体未与染色单体附着时,检查点处于激活状态,激活的检查点会抑制后期启动复合物(APC/C)的泛素连接酶活性,有丝分裂后期延迟。MAD2(mitosis arrest deficiency)和BUB1(budding uninhibited by benomyl)既是两种着丝粒相关蛋白,也是组成纺锤体检查点复合物的重要成分,因此又称为检查点蛋白(checkpoint proteins,CP),它们从酵母到人类都高度保守。这些蛋白确保只有在所有染色体着丝粒连接到纺锤体微管上时,细胞才能进入后期,染色体开始向细胞两极移动,完成染色体的正常分离。哺乳动物细胞中,BUB1和MAD2蛋白在有丝分裂前期存在于着丝粒区域,中期它们将随着纺锤体微管附着于着丝粒上而消失。Li和Ouyang认为这些CP蛋白在未与纺锤丝连接的着丝粒点上的浓度较高,在纺锤丝连接的着丝粒点上的浓度较低。鉴于hsMAD2和/或hBUB1可能具有重要的作用,我们对染色体数目异常胚胎绒毛组织hsMAD2和/或hBUB1基因表达进行了研究并发现:hsMAD2和/或hBUB1基因蛋白水平的表达显著降低。RNA干扰(RAN interference,RNAi)技术是近年来广泛应用于哺乳动物细胞抑制基因表达的新技术,主要用于基因功能学和基因治疗学等方面的研究。本课题应用RNAi技术,探讨染色体数目正常的绒毛细胞转染靶向hsMAD2或hBUB1基因的RNAi重组体后,靶基因转录水平和蛋白表达水平的变化。我们的实验数据显示:shRNA-MAD2-1和shRNA-BUB1-2干扰序列能特异地、有效地抑制胚胎细胞中靶基因的表达,并发现染色体数目的异常细胞比例增加。在靶基因的临床研究中,我们直接检测到了染色体数目异常的自然流产的胚胎组织中靶蛋白表达的下降,得到了与RNAi干扰模型相同的实验结果。为此,我们推测在胚胎细胞中hsMAD2和/或hBUB1基因表达下调可能是导致胚胎细胞染色体数目的异常的原因之一。该研究为进一步探讨染色体数目异常发生机理奠定了基础。第一部分绒毛细胞的培养方法及转染策略研究目的:1、流产胚胎绒毛组织原代细胞培养,获得染色体标本制备方法;2、流产胚胎绒毛组织原代培养、纯化绒毛细胞并鉴定细胞种类;3、了解绒毛细胞的生长特性和质粒转染条件。方法:1、制备流产组织绒毛细胞染色体标本进行核型分析;2、对流产胚胎进行细胞原代培养和纯化。用抗-vimentin抗体和抗-cytokeratin抗体进行细胞种类鉴定;3、采用MTT实验绘制生长曲线,找到其对数生长期;用细胞贴壁率找出绒毛细胞生长所需的最适pH;同时采用转染效率和细胞存活分数来评价绒毛细胞质粒转染的最佳脂质体用量。结果:1、通过染色体分析筛选出40例染色体数目正常胚胎绒毛标本;2、人工流产胚胎绒毛组织的原代细胞培养、纯化后绒毛细胞含量>80%;3、细胞传代后12~36 h细胞处于对数生长期,48 h后进入平台期;绒毛细胞生长最适的pH为6.8~7.0;综合转染效率和细胞存活分数找到最佳脂质体用量。结论:成功培养出胚胎绒毛细胞并初步了解其生长特性,同时对RNA干扰质粒转染所需的条件进行优化,为后期的RNA干扰实验作好准备。第二部分RNA干扰技术抑制内源hsMAD2或hBUB1基因在绒毛细胞中的表达目的:1、构建针对hsMAD2和hBUB1基因的短发夹状RNA(shRNA)表达载体;2、shRNA重组表达质粒载体转染染色体数目正常的绒毛细胞后,观察靶基因表达的抑制,以及染色体数目异常的变化情况;3、了解hsMAD2和hBUB1基因的表达抑制对绒毛细胞增殖的影响以及对细胞周期的调节。方法:1、设计、合成hsMAD2和hBUB1基因的shRNA序列,与载体pTZU6+1连接,构建靶基因的shRNA重组表达质粒;2、shRNA重组质粒转染细胞48 h后,用免疫细胞化学和western blot观察靶基因的蛋白水平,并用定量real-time PCR检测mRNA水平;3、有效干扰组胚胎绒毛细胞的染色体数目分析;4、MTT试验检测干扰前后的细胞存活分数;5、用FCM评价两基因对胚胎绒毛细胞周期的影响。结果:1、成功构建针对靶基因的shRNA重组质粒;2、转染有效干扰shRNA重组质粒后,绒毛细胞靶基因的内源性表达受到明显抑制,其蛋白和mRNA水平都显著下降;3、有效干扰组细胞核型分析也发现多种类型的染色体数目异常;4、MTT实验发现有效干扰组细胞存活分数明显下降,细胞增殖受到抑制;且经FCM检测知道有效干扰后使较多绒毛细胞停滞在分裂期(M期)。结论:设计、合成的针对靶基因的shRNA重组质粒在绒毛细胞中能有效抑制靶基因的表达,染色体数目异常绒毛细胞比率增加,说明MAD2和BUB1对染色体分离可能具有重要调控作用,成功构建了MAD2和BUB1抑制后观察染色体数目异常变化的体外细胞模型,为后期的临床研究奠定了基础。第三部分染色体数目异常的自然流产胚胎绒毛组织中hsMAD2和hBUB1基因表达的临床研究目的:1、了解染色体数目异常的自然流产胚胎绒毛组织中是否存在MAD2和BUB1基因的蛋白和/(或)mRNA水平的异常,探索靶基因的表达在染色体数目异常的胚胎发生中可能具有的作用;2、了解在自然流产染色体数目异常的胚胎组织中hsMAD2和hBUB1基因的蛋白编码区域是否存在具有临床意义的基因变异。方法:1、分别对早期自然流产的胚胎(<12周)标本绒毛细胞进行染色体数目进行分析;数目正常组为对照组,异常组为实验组;2、分别提取对照组和实验组样品绒毛组织中的总RNA和蛋白质;3、用real-time RT-PCR检测hsMAD2和hBUB1基因的mRNA量;4、用western blot检测每一蛋白样品中hsMAD2和hBUB1水平;5、实验组中选择8例蛋白表达量最低的标本组织的cDNA,对两靶基因的CDS区域用PCR分段扩增并测序,再用生物信息学方法将测序结果与genebank数据库进行比对。结果:1、早期自然流产胚胎绒毛组织中筛选出25例染色体数目异常和31例染色体数目正常的组织标本;2、Real-time RT-PCR结果显示,实验组和对照组的hsMAD2和hBUB1基因的mRNA水平无显著性差异;3、Western Blot检测结果显示,hsMAD2和hBUB1蛋白在实验组中的表达显著低于对照组;4、CDS测序结果与genebank数据库比对,两靶基因均无移位、缺失,仅发现3个同义点突变。结论:在染色体数目异常的自然流产胚胎绒毛组织中hsMAD2和hBUB1表达显著下降,提示其作为编码CP的重要基因在染色体数目异常胚胎的发生中可能具有重要意义。更多还原

【Abstract】 At the early stage of embryonic development, cells are going on vigorous mitosis. Proper mitotic process of a cell is ensured by the correct separation of chromosomes into its two daughter cells. At least three classes of proteins are involved in this progression: centeomere-associated proteins (including hsMAD2 and hBUB1), centeomere specific proteins (CENPs) and spindle microtube associated proteins. The inheritance of a normal assortment of chromosomes during each cell division relies on a cell-cycle surveillance system called the mitotic spindle checkpoint, which can ensure the sister-chromatid separation into its daughter cells properly. The existence of MAD2 and BUB1 on the sister chromatids that do not achieve proper bipolar attachment to the mitotic spindle in a cell will activate this checkpoint, which inhbits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C) and delays the onset of anaphase. Recent studies have revealed that the mitotic arrest deficiency2 (MAD2) and budding uninhibited by benomyl1 (BUB1) protein are key conserved components of the spindle checkpoint in organisms ranging from yeast to man. These checkpoint proteins (CP) ensure that chromatids would not separate till chromosomes are properly aligned along the mitotic spindle. In mammalian cells, the BUB1 and MAD2 proteins are detected at the centromere region in the prophase of mitosis, but they will disappear while it was attached to centromere in accompany with spindle microtubule at metaphase. When one of these genes is damaged, the defective cells will complete mitosis in the presence of a lagging chromosome, resulting in an abnormal chromosomal segregation. Li and Ouyang thought when expression of human MAD2 gene encoding CP or BUB1 gene is down-regulated, it would cause mis-segregation of chromosomes which resulted in the numerical chromosomal abnormality in cells. Thus, considering our study as well, we may presume that the down-regulation of hsMAD2 or/and hBUB1 gene expression in embryonic cells is one of the causes leading to numerical chromosomal abnormality, and finally resulting in the spontaneous abortion.RNA interference (RNAi) is the phenomenon in which double-stranded RNA (dsRNA) specifically suppresses the post-transcriptional expression of a gene target. RNAi, a new and rapidly evolving technology too, is already regarded not only as a technique that has great promise for mammalian functional genomics, but also as a therapeutic agent. In this study, we have developed potent shRNA plasmids targeting at hsMAD 2 or hBUB1 gene, the two plasmids were transfected into human villi cells respectively, specificity of shRNA was assessed by examining the protein and mRNA levels of hsMAD 2 or hBUB1 gene in embryonic villi cells with normal chromosome. Our data shows that pshRNA-MAD2-1 or pshRNA-BUB1-2 can specifically and efficiently inhibit target gene expression and cause numeric chromosomal abnormality, including aneuploidy and polyploidy. Then in the clinical research on target proteins, we found out that the protein expression levels of target genes were down-regulated in some villi tissues of spontaneous abortion embryos with numerical chromosomal abnormality, as it was in the model of RNA interference. The experimental results were consistent with that abnormal expression of hsMAD2 or hBUB1 protein contributed to the first trimester spontaneous abortion with numerical chromosomal abnormality. The researches will become an important foundation for studing mechanism of spontaneous abortion with numerical chromosomal abnormality.PARTⅠTHE STUDY ON CULTURAL METHODS AND PLASMIDS TRANSFECTION STRATEGY FOR VILLI CELLObjective:1. Primary culture of cells with numerical chromosomal normality by numerical chromosomal analysis from induced abortion embryonic villi tissues;2. Purification and identification of villi cells after primary culture of the above cells; 3. Investigate on the growth characteristics of villi cells andtransfection conditions of plasmids into the villi cells.Methods:1. Chromosomal samples of villi cells were prepared for numerical chromosomal analysis in induced abortion villi tissues;2. Villi cells with numerical chromosomal normality were primary cultivated and purified, and the cells were identified with anti-vimentin and anti-cytokeratin antibody;3. The villi cells growth curve was detected by MTT experiment and the optimal pH conditions for culture villi cells were determined based on the cells stick rate. And the ideal amount lipofectamine2000 was valued by Survival fraction of cells and transfiction effection.Results:1. Forty cases of embryonic villi tissues with numerical chromosomal normality were selected by karyotype analysis in villi cells;2. The efficient methods of primary culture and purification of villi cells were successfully obtained, the pure rate of villi cells was no less than 80%; 3. Logarithmic growth phase of villi cells is 12~36 hour, the most optimal condition for the growth of the cells is pH 6.8~7.0; the ideal amount lipofectamine2000 for villi cells transfected with plasmids was also determined.Conclusion: We successfully performed the primary culture and characteristics investigation of the villi cells with numerical chromosomal normality. Simultaneously, we also optimized the transfection conditions of plasmids into the villi cells, followed by RNA interference experiment.PARTⅡINHIBITION OF RNAI TECHNOLOGY ON ENDOGENOUS HSMAD2 OR HBUB1 GENE EXPRESSION IN VILLI CELLSObjective:1. To construct a recombinant plasmids vector expressing short hairpin RNA (shRNA) targeting at hsMAD2 and hBUB1 genes respectively;2. To observe the down-regulation of hsMAD2 or hBUB1 genes expression and numerical chromosomal alteration after transfection of the recombinant shRNA plasmids into villi cells with numerical chromosomal normality;3. To explore the influence of down-regulation of hsMAD2 or hBUB1 genes expression on the cell proliferation and cell cycle in villi cells.Methods:1. ShRNA sequences targeting at hsMAD2 or hBUB1 gene were designed and synthesized, and then ligated with vector pTZU6+1 to construct shRNA recombinant plasmids;2. 48 hours after the shRNA plasmids were transfected into villi cells, immuno-cytochemistry and western blot methods were adopted to detect the protein expression of target genes, FQ-PCR was taken to detect their mRNA levels;3. Numerical chromosomal analysis of villi cells demonstrated that RNAi targeting at hsMAD2 or hBUB1 gene was efficient and specific;4. Cell growth-fraction, shown by MTT experiment before and after RNAi, was used to evaluate cell proliferation;5. The influences of the two genes on cell cycle were evaluated by FCM.Results:1. The shRNA recombinant plasmids targeting at hsMAD2 or hBUB1 genes were successfully constructed;2. After efficient transfection of shRNA recombinant plasmids into embryonic villi cells, the expression of endogenous target genes were inhibited dramatically, the proteins and mRNA level were both significantly decreased;3. Besides, many types of aneuploid and polyploidy cells were identified by numerical chromosomal analysis in the groups with efficient transfection of shRNA plasmids;4. MTT experiment showed that cell survival-fraction was also decreased after efficient transfection with shRNA plasmids, proliferation of cells was inhibited, many cells stagnated at the mitositic phase (M phase).Conclusion: The designed and synthesized short hairpin RNA (shRNA) recombinant plasmids targeting at hsMAD2 or hBUB1 gene can efficiently and specifically inhibit the expression of target genes in villi cells, leading to the mis-segregation of chromosomes, which suggests that MAD2 and BUB1 proteins play an important role during the segregation of chromosomes. We successfully constructed an in vitro model of numerical chromosomal abnormality by the inhibition of the checkpoint proteins, which lays a foundation for further clinical researches. PARTⅢClinical Research on the Expression of hsMAD2 and hBUB1 Genes in Embryonic Villi tissues of Spontaneous Abortion Embryos with Numerical Chromosomal AbnormalityObjective:1. To determine whether there is an abnormal expression of hsMAD2 and hBUB1 gene proteins and/or mRNA level in embryonic villi tissues of spontaneous abortion embryos with numerical chromosomal abnormalities;2. To observe whether or not significant genetic mutations in the villi tissues of spontaneous abortion embryos with numerical chromosomal abnormality.Methods:1. Numerical chromosomal analysis was performed in the villi cells of spontaneous abortions embryonic villi tissues, numerical chromosomal normality group was taken as control group, while numerical chromosomal abnormality group was taken as experimental group;2. Cellular total proteins and RNA were extracted from each villi tissues sample in both control and experimental groups;3. The quantities of mRNA of hsMAD2 and hBUB1 genes were detected by quantitative real-time RT-PCR method;4. The protein expressions of hsMAD2 and hBUB1 genes are tested by western blot;5. CDS regions of target genes were amplified and sequenced in eight embryonic villi tissues samples in which proteins expression were down- regulated dramatically, results were compared with Genebank database by bioinformatics.Results:1. Twenty-five spontaneous abortion embryonic tissues with numerical chromosomal abnormality and thirty-one with numerical chromosomal normality were selected in all of the analyzed samples;2. The results of quantitative real-time RT-PCR displayed that mRNA level of both of hsMAD2 and hBUB1 genes had no significant difference between the control group and experimental group;3. Western blot analysis showed that comparing with the control group, the protein levels of hsMad2 and hBub1 in the experimental group decreased significantly;4. There were three synonmous mutations in all of CDS sequence when we compared CDS sequencing with the corresponding Genebank data.Conclusion: In the vill tissues of spontaneous abortion embryos with numerical chromosomal abnormality, the expressions of hsMad2 and hBub1 proteins were decreased significantly, suggesting that hsMAD2 and hBUB1 genes involved in encoding checkpoint proteins that played an important roles in the development of spontaneous abortion embryos with numerical chromosomal abnormality.更多还原