Researches On The Relationship Between The Centromere Proteins CENP-E Or CENP-I And The Chromosome Segregation

At the early stage of embryo development, cells are going on vigorous mitosis. Proper mitotic process of a cell is ensured by the correct separation of chromosomes to two daughter cells,which is associated with the centromere's structure and function and the spindle surveillance system etc. The centromere proteins(CENPS) are the fundamental elements in the period of centromere assembling.Some researches revealed that the abnormal expression of CENPS may cause the chromosome segregation or the cell division abnormality.CENPS are constituted by a series of proteins (now have already discoverded more than 80 proteins)which located at the centromere during the mitotic phase or interphase, they all influence the centromere assembly or function.CENP-E not only participates the assembling of centromere,but also joins the spindle checkpoint's construction,it can offer the power for the chromosomes movement.As an upper stream protein in the process of centromere constructing, CENP-I may have an important effect in the process of centromere assembly. Nishihashi[1] etc found that when conditional knockout of CENP-I in the DT40 cells, the cells will be staying at the prometaphase with misaligned chromosomes for a long time .In order to reveal whether CENP-E or CENP-I participates in adjusting the human cells'chromosome separation or cell division, quantitative real-time PCR,Western-blot and indirect immunofuorescence were used to evaluate the endogenous expression level of CENP-E or CENP-I mRNA and protein in human embryonic villus cells with normal karyotype or numerical chromosomal aberration.The results revealed that comparing with normal embryonic villus cells the expression levels of CENP-E or CENP-I mRNA and protein were significantly decreased in embryonic villus cells with numerical chromosomal aberration; A human embryonic villus cell model which specially suppresses the CENP-E's or CENP-I's endogenous expression was constructed by the RNAi technique targeted to CENP-E or CENP-I.In embryonic villus cells with low CENP-E or CENP-I expression level ,the rate of cell growth decreased markly , cells of G2/M phase,the mitotic index and the rate of abnormal chromosome numbers were increased conspicuous compared with the cells transfected with the mock vector or untransfected. Comparing with untransfected cells or cells transfected with empty plasmids, down regulation of CENP-E or CENP-I expression of embryonic villus cells by sequence specific siRNA could delay the cell growth and postpone the cell division.So a deduction was acquired that CENP-E or CENP-I participates in the centromere's adjusting the human cells'chromosome separation or cell division. The further research of CENP-E or CENP-I will provide a new reference index for studing mechanism of spontaneous abortion with numerical chromosomal abnormality and the clinical treatment of tumors with abnormal chromosomes.PART I THE PROKARYOTIC EXPRESSION AND PURIFICATION OF CENTOMERE PROTEIN CENP-I AND PREPARION OF IT'S ANTIBODYObjective:1.Construct a prokaryotic expression vector pET32a/CENP-I.2.Purify the recombinant protein of CENP-I.3.Prepare antibody by immuning rabbit with rCENP-I.Methods:1. Template cDNA was obtained by RT-PCR from cultured HeLa cells'whole RNA. By gene recombination technology in vitro, the sequence encoding the amino acid 1~293 of CENP-I was cloned into pET-32a(+) expression vector to construct pET32a/CENP-I recombinant plasmid.2. The pET32a/CENP-I recombinant plasmid being confirmed by sequencing was transformed into E.coli BL21(DE3) and was induced by IPTG. The Ni2+-NTA affinity column was used to purify rCENP-I.3. Rabbits immuned with recombinant proteins produced high levels of CENP-I-specific antibodies which were detected by the agarose double Immunodiffusion,ELISA assay and Western-blot.And the antibodies were rough purifid by saturated ammonium sulfate sediment.Results:1. The expression vector pET32a/CENP-I was constructed successfully confirmed by sequencing.2.The recombinant proteins were purified by Ni2+-NTA affinity column .SDS-PAGE and Bandscan analysis showed that the target proteins were highly purified and their purity was more than 90%.3. High levels of CENP-I-specific antibodies were acquired,the valence of antibody is more than 1:16 ditected by the agarose double Immunodiffusion or 1:20000 confirmed by ELISA assay or 1:400 tested by Western-blot.The antibody can be used to identify the CENP-I protein in clinical specimen. Conclusion: We successfully constructed the expression vector pET32a/CENP-I,purified the recombinant proteins and acquired the high valence antibody target to CENP-I. PARTâ…¡THE CLINICAL RESEARCH ON THE EXPRESSION OF CENP-E AND CENP-I GENE IN THE SPONTANEOUS ABORTION EMBRYONIC VILLUS CELLS WITH NUMERICAL CHROMOSOMAL ABNORMALITYObjective:1.The standard plasmds of FQ-PCR were aquired by T-A clone.2.Primary culture of embryonic villus cells from induced or spontaneous abortion embryonic villi tissues,and divided to two groups by chromosomes karyotype analysis.3. To determine whether there is an abnormal expression of CENP-E and CENP-I gene in proteins and/or mRNA level in embryonic villus cells of spontaneous abortion embryos with numerical chromosomal abnormalities.Methods:1.The FQ-PCR primers and probes of CENP-E,CENP-I and GAPDH were designed and synthesized.After PCR amplification , the products were ligated with vector pMD18-T vector to construct the standard plasmids.2. The tissue adherence method,tisse triturate method or tissue digesting method were used to primary culture the embryonic villus cells;The direct preparation of chromosomes,the traditional preparation of chromosomes and the the situ preparation of chromosomes were applied to analyze the cultured embryonic villus cells'karyotype ; Cells with normal chromosome numbers from induced abortion were designated as the control group and cells with numerical chromosomal abnormality from spontaneous abortion were designated as the experimental group according to the karyotype.3.The expression of mRNA level of CENP-E and CENP-I were detected by FQ- PCR.4. The expression of protein level of CENP-E and CENP-I were detected by Western-blot and indirect immunuofuorescence.Results:1.The standard plasmids of CENP-E,CENP-I and GAPDH gene were constructed successfully.2.The embryonic villus cells were cultured successfully,33 cases with normal chromosome numbers from induced abortion and 23 cases with numerical chromosomal abnormality from spontaneous abortion were obtained.3.The results of FQ-PCR,Western-blot and indirect immunuofuorescence showed that comparing with the control group the expressions in mRNA level and protein level of CENP-E and CENP-I were significantly decreased in experimental group.Conclusion: The decreased expression of CENP-E and CENP-I in the embryonic villus cells with abnormal chromosome numbers may be one of the important reasons for numerical abnormality of embryo chromosomes and fetal abnormalities, and have great significance for further exploration of early clinical diadynamic criteria and detection techniques.PART III INHIBITION OF RNAI TECHNOLOGY ON THE ENDOGENOUS EXPRESSION OF CENP-E OR CENP-I IN EMBRYONIC VILLUS CELLSObjective:1. To construct a recombinant plasmids vector expressing short hairpin RNA (shRNA) targeting at CENP-E and CENP-I genes respectively;2. To observe the down-regulation of CENP-E or CENP-I genes expression after transfection of the recombinant shRNA plasmids in embryonic villus cells.3. To explore the influence of down-regulation of CENP-E or CENP-I genes expression on the cell proliferation and chromosome separation of embryonic villus cells and the regulation to cell cycle.Methods:1. ShRNA sequences targeting at CENP-E or CENP-I gene were designed and synthesized, and then ligated with vector pgenesil-1 to construct shRNA recombinant plasmids;2. After cells were transfected with the shRNA recombinant plasmids,the FQ-PCR and Western-blot or indirect immunofluorescence were applied to detect the expression of mRNA and protein level of target genes, and the effective interfering plasimids and the best gene suppression time were selected.3. MTT experiment was used to prepared the cell growth curve of cells before and after RNAi,which could be used to evaluate the influence of cell growth by gene inhibition.4. The influences of the two genes on cell cycle were evaluated by FCM.5.Analysis of mitotic index and chromosome karyotype of cells before and after RNAi was applied to detect the influence of intergenic suppression to chromosomal separation.Results:1.ShRNA recombinant plasmids of target genes were successfully constructed .2.The pshRNA-CENP-E-3 and pshRNA-CENP-I-3 were the effective interference plasmids. At 72h after transfection, endogenous expressions of target genes in embryonic villus cells were obviously inhibited and the mRNA and protein levels were significantly decreased.3.MTT showed cell growth of the effective interference group was significantly slower than that of the control group, and cell proliferation was inhibited.4.FCM showed the ratio of the cells during G2/M was increased after interference; Mitotic index showed the ratio of the cells during mitotic phase in the experimental group was increased following the prolong of transfection time; Karyotype analysis showed that ratio of the cells with abnormal chromosomal numbers were significantly increased in the experimental group.Conclusion: The shRNA recombinant plasmids designed and synthesised of target genes could effectively inhibit the expression of target genes in embryonic villus cells. Cells grew slowly, mitotic cells increased and ratio of the cells with abnormal chromosomal number was increased which demonstrated CENP-E and/or CENP-I might play an important regulatory role in cell proliferation and chromosome separation. We successfully constructed a cell model in vitro to observe abnormal changes in chromosome number after CENP-E or CENP-I gene was inhibited which established the foundation for the later stage of clinical research.