染色体数目异常胚胎绒毛细胞着丝粒相关蛋白基因研究

发布时间：2018-01-02 06:43

本文关键词：染色体数目异常胚胎绒毛细胞着丝粒相关蛋白基因研究　出处：《重庆医科大学》2007年博士论文　论文类型：学位论文

【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。
[Abstract]:In the early embryonic development of exuberant cell mitosis, normal chromosome separated into two daughter cells is an important basis to ensure the normal mitosis, related proteins should include at least: centromere associated protein (centromere associated proteins) (MAD, BUB), centromere specific proteins (centromere protein) (CENPs) and spindle microtubule associated protein (spindle microtube associated proteins). The normal separation of chromosomes during cell division by mitotic spindle checkpoint (mitotic spindle checkpoint) regulation, with the normal function of the spindle checkpoint can ensure the correct separation of sister chromatids into two daughter cells when there is. Mitotic spindle and chromatid attachment, the checkpoint is activated and the activation of the checkpoint inhibits late start complexes (APC/C) of the Ubiquitin ligase activity, mitotic anaphase delay.MAD2 (mitosis arrest deficiency) and BUB1 (budding uninhibited by benomyl) is the two important components of centromere associated protein, is composed of spindle checkpoint complexes, also known as checkpoint protein (checkpoint proteins, CP), they are highly conserved from yeast to humans. These proteins ensure that only in all chromosomes attached to spindle microtubules when cells can enter anaphase, the chromosomes began to move to opposite poles of the cell, the normal separation of chromosomes. Mammalian animal cells, BUB1 and MAD2 protein in the prophase of mitosis in the centromeric region, they will be with the mid spindle microtubule attachment to centromere and.Li and Ouyang argue that higher concentrations of disappearance of these CP proteins is not connected with the spindle of the centromere on the spindle connected to the centromere The concentration of hsMAD2 and / or hBUB1 may play an important role. We studied the gene expression of hsMAD2 and / or hBUB1 in the chorionic villi of chromosome number abnormality, and found that the expression level of hsMAD2 and / or hBUB1 protein decreased significantly.
RNA interference (RAN interference RNAi) technology is a new technology in recent years, widely used in mammalian cells to inhibit gene expression, mainly used for the research of gene function and gene therapy and so on. This paper discusses the application of RNAi technology, villus cells transfected with target normal chromosome number to the hsMAD2 or hBUB1 gene recombinant RNAi and the expression of target gene transcription and protein. Our data showed that shRNA-MAD2-1 and shRNA-BUB1-2 interference sequence can specifically and effectively inhibit the expression of target genes in embryonic cells, and abnormal chromosome number increased. The proportion of cells in the clinical research of target genes, we directly detected decreased expression of the target protein embryo abnormal chromosome number of spontaneous abortion, obtained the same results with the RNAi interference model.
Therefore, we speculate that the down regulation of hsMAD2 and / or hBUB1 gene expression in embryonic cells may be one of the reasons leading to abnormal chromosome numbers. This study laid a foundation for further exploring the mechanism of chromosome abnormality.
Part one
Study on the culture of villous cells and the strategy of transfection
Objective:
1, the primary cells of the aborted embryo were cultured, and the methods of preparing the chromosomes were obtained.
2, the aborted embryo was cultured in the villi tissue for primary culture, and the villus cells were purified and the species of cells were identified.
3, to understand the growth characteristics of chorionic cells and plasmid transfection conditions.
Method:
1, the karyotype of the villus cells in the abortion tissue was analyzed.
2, the primary culture and purification of the aborted embryos were carried out. The type of cell species was identified by anti -vimentin antibody and anti -cytokeratin antibody.
3, the growth curve was drawn by MTT experiment, and its logarithmic growth phase was found. The optimal pH for villus cell growth was found by cell adherence rate, and the transfection efficiency and cell survival fraction were used to evaluate the optimal dosage of plasmid transfection.
Result:
1, 40 normal embryo villi specimens were screened by chromosome analysis.
2, the primary cell culture of the aborted embryo chorionic villus was 80%, and the content of the villus cells after purification.
3, after cell passage, the 12~36 h cells were in logarithmic growth phase, and entered the plateau stage after 48 h. The most suitable pH for villi cell growth was 6.8~7.0, and the optimal transfection efficiency and cell survival fraction were found to find the best liposome dosage.
Conclusion: embryo chorionic cells were successfully cultured, and their growth characteristics were preliminarily understood. Meanwhile, the necessary conditions of RNA interference plasmid transfection were optimized, so as to prepare for the later RNA interference experiment.
The second part
RNA interference technique inhibits the expression of endogenous hsMAD2 or hBUB1 genes in chorionic cells
Objective:
1, a short hairpin RNA (shRNA) expression vector for hsMAD2 and hBUB1 genes was constructed.
2, shRNA recombinant plasmid vector was transfected into villous cells with normal chromosome number, and the inhibition of target gene expression and the change of chromosome number were observed.
3, we know the effect of inhibition of hsMAD2 and hBUB1 gene expression on the proliferation of chorionic villi and the regulation of cell cycle.
Method:
1, design, synthesize shRNA sequence of hsMAD2 and hBUB1 gene, connect with carrier pTZU6+1, construct shRNA recombinant expression plasmid of target gene.
2, after transfection of shRNA recombinant plasmid to 48 h cells, the protein level of the target gene was observed by immunocytochemistry and Western blot, and the level of mRNA was detected by quantitative real-time PCR.
3, the analysis of the number of chromosomes in the chorionic villus cells was effectively interfered.
4, MTT test was used to detect the cell survival fraction before and after interference.
5, the effect of two gene on the cell cycle of embryonic chorionic villi was evaluated by FCM.
Result:
1, the recombinant plasmid shRNA for target gene was successfully constructed.
2, after transfecting the recombinant plasmid with effective interference shRNA, the endogenous expression of the target gene of the villus cells was obviously inhibited, and the protein and mRNA level were significantly decreased.
3, the cell karyotype analysis of effective interference group also found the number of chromosomes of various types.
4, MTT experiments showed that the survival fraction of the effective interference group was significantly decreased, and the cell proliferation was inhibited. After FCM detection, more chorionic cells were arrested during the split phase (M phase).
Conclusion: design, synthesis of the target gene shRNA recombinant plasmid can inhibit target gene expression in villi cells, abnormal chromosome number of villus cells ratio increased, MAD2 and BUB1 on chromosome segregation may play an important role in regulation, successfully constructed an in vitro model of abnormal changes in chromosome number MAD2 and BUB1 inhibition was observed after. Lay a foundation for further clinical researches.
The third part
A clinical study on the expression of hsMAD2 and hBUB1 genes in the villous tissue of spontaneous abortion embryos with abnormal chromosome number
Objective:
1, we need to know whether there are abnormalities in protein and / or mRNA levels of MAD2 and BUB1 genes in natural chorionic villus tissues with abnormal chromosome numbers, and explore the possible role of target gene expression in the development of abnormal chromosome numbers.
2, to understand whether the protein coding region of hsMAD2 and hBUB1 genes in fetal tissues with abnormal chromosome number of spontaneous abortion has a genetic variation in clinical significance.
Method:
1, the number of chromosomes in the chorionic cells of early spontaneous abortion (12 weeks) was analyzed, and the normal group was the control group, and the abnormal group was the experimental group.
2, the total RNA and protein in the villi tissues of the control group and the experimental group were extracted respectively.
3, the mRNA amount of hsMAD2 and hBUB1 genes was detected by real-time RT-PCR.
4, the level of hsMAD2 and hBUB1 in each protein sample was detected by Western blot.
5, in the experimental group, 8 cases of cDNA with minimal protein expression were selected. The CDS region of the two target gene was amplified by PCR and sequenced, and the sequencing results were compared with genebank database by bioinformatics.
Result:
1, 25 cases of abnormal chromosome number and 31 normal chromosome number were selected from the villi tissue of early spontaneous abortion.
2, Real-time RT-PCR results showed that there was no significant difference in the mRNA level between the hsMAD2 and the hBUB1 genes in the experimental group and the control group.
3, the results of Western Blot detection showed that the expression of hsMAD2 and hBUB1 protein in the experimental group was significantly lower than that in the control group.
4, the results of CDS sequencing were compared with the genebank database, and the two target genes were not displaced and missing, and only 3 mutation of the synonymous point were found.
Conclusion: the expression of hsMAD2 and hBUB1 in natural chorionic villus tissues with chromosome number abnormalities is significantly decreased, suggesting that as an important gene encoding CP, it is of great significance in the occurrence of abnormal chromosome numbers.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R394

【参考文献】