Researches On The Genes Associated With Centromere Proteins In Embryonic Villi Cells With Numerical Chromosomal Abnormality

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.PARTâ… THE 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.PARTâ…¡INHIBITION 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. PARTâ…¢Clinical 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.