本文關(guān)鍵詞：著絲粒蛋白CENP-E和CENP-I與染色體分離關(guān)系研究　出處：《重慶醫(yī)科大學(xué)》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 免疫血清 著絲粒 CENP-I 流產(chǎn) 自然 細(xì)胞培養(yǎng)技術(shù) 染色體分離 RNA干擾 流式細(xì)胞術(shù) 免疫印跡法

【摘要】： 在胚胎發(fā)育的早期進(jìn)行著旺盛的細(xì)胞有絲分裂,染色體正常分離到兩個(gè)子細(xì)胞是保證有絲分裂正常進(jìn)行的重要基礎(chǔ),而染色體的正常分離與著絲粒結(jié)構(gòu)和功能、紡錘體檢驗(yàn)點(diǎn)監(jiān)控機(jī)制等有關(guān)。著絲粒蛋白質(zhì)(Centromere proteins,CENPs)是著絲粒組裝和功能發(fā)揮的基本組成單位,由位于著絲粒上的一組蛋白質(zhì)(已發(fā)現(xiàn)的將近80余種[1])構(gòu)成,它們?cè)诩?xì)胞分裂間期或分裂期定位在著絲粒上,共同參與著絲粒的組裝并影響其功能的正確行使。其中CENP-E不僅參與著絲粒的組裝,而且還是紡錘體關(guān)卡蛋白檢查點(diǎn)的重要組成部分,是聯(lián)系著絲粒與紡錘體微管間的紐帶,并提供染色體運(yùn)動(dòng)的動(dòng)力[2、3、4、5]。CENP-I是著絲粒組裝過(guò)程中較上游的一個(gè)蛋白質(zhì),在著絲粒裝配和功能發(fā)揮過(guò)程中可能具有重要作用。Nishihashi等[6]研究發(fā)現(xiàn)在雞的DT40細(xì)胞敲除CENP-I基因后,細(xì)胞將長(zhǎng)時(shí)間停滯于分裂的前中期,并出現(xiàn)染色體錯(cuò)排。為了揭示在人類細(xì)胞中,CENP-E、CENP-I是否參與調(diào)控染色體分離和細(xì)胞正常分裂,本課題利用定量PCR、Western-blot和間接免疫熒光技術(shù)對(duì)染色體數(shù)目異常和核型正常的胚胎絨毛細(xì)胞中CENP-E、CENP-I的表達(dá)進(jìn)行檢測(cè)分析,發(fā)現(xiàn)與后者相比,染色體數(shù)目異常的胚胎絨毛細(xì)胞中CENP-E、CENP-I在mRNA水平和蛋白質(zhì)水平上的表達(dá)均明顯降低;并用RNAi技術(shù)構(gòu)建CENP-E、CENP-I缺陷表達(dá)的人胚胎絨毛細(xì)胞模型,發(fā)現(xiàn)與未轉(zhuǎn)染或轉(zhuǎn)染空載體的胚胎絨毛細(xì)胞相比,CENP-E、CENP-I表達(dá)降低的胚胎絨毛細(xì)胞生長(zhǎng)速度明顯減慢,G2/M期細(xì)胞增加,分裂期細(xì)胞比例增大,染色體數(shù)目異常率顯著增加。說(shuō)明CENP-E、CENP-I參與著絲粒對(duì)細(xì)胞染色體分離和細(xì)胞增殖的調(diào)控,二者表達(dá)的降低可能是導(dǎo)致自然流產(chǎn)發(fā)生的重要原因。對(duì)CENP-E、CENP-I的進(jìn)一步研究,將為研究染色體數(shù)目異常胚胎自然流產(chǎn)機(jī)制提供新的參考指標(biāo)。 第一部分著絲粒蛋白質(zhì)CENP-I的原核表達(dá)、純化及抗體制備 目的: 1、構(gòu)建pET32a/CENP-I原核表達(dá)載體。 2、純化重組蛋白rCENP-I。 3、將重組蛋白免疫兔制備抗CENP-I抗體。方法: 1、用RT-PCR的方法從Hela細(xì)胞總RNA中獲得編碼CENP-I蛋白第1～293位氨基酸的CENP-I cDNA序列,采用基因體外重組法將其克隆到原核表達(dá)載體pET32a(+)內(nèi),獲得重組質(zhì)粒pET32a/CENP-I。 2、將測(cè)序鑒定正確的重組質(zhì)粒轉(zhuǎn)化到大腸桿菌BL21(DE3)中,用IPTG誘導(dǎo)表達(dá)、Ni2+-NTA親和層析純化獲得高純度的重組蛋白質(zhì)。 3、用純化的蛋白質(zhì)免疫家兔制備抗體,瓊脂糖免疫雙擴(kuò)法、ELISA法和Western-blot得到高效價(jià)的免疫血清,并用飽和硫酸銨初步純化抗體。 結(jié)果: 1、經(jīng)測(cè)序鑒定,成功構(gòu)建了重組表達(dá)質(zhì)粒pET32a/CENP-I。 2、重組蛋白經(jīng)Ni2+-NTA親和層析柱純化后在SDS-PAGE上呈現(xiàn)單一條帶,灰度掃描后用Bandscan分析重組蛋白純度達(dá)90%以上,成功的獲得了rCENP-I蛋白。 3、將rCENP-I蛋白免疫兔后獲得了高效價(jià)的免疫血清,瓊脂糖雙擴(kuò)顯示免疫血清效價(jià)達(dá)1:16以上;ELISA法測(cè)免疫血清效價(jià)達(dá)1:20000,Western-blot顯示抗體效價(jià)達(dá)1:400,能用于檢測(cè)臨床標(biāo)本中的CENP-I蛋白。 結(jié)論:成功的構(gòu)建了pET32a/CENP-I表達(dá)載體、純化了CENP-I重組蛋白,并制備了效價(jià)較高的抗CENP-I抗體。 第二部分 染色體數(shù)目異常的自然流產(chǎn)胚胎絨毛細(xì)胞中CENP-E和CENP-I基因表達(dá)的臨床研究 目的: 1、通過(guò)T-A克隆獲得FQ-PCR標(biāo)準(zhǔn)品。 2、流產(chǎn)胚胎絨毛組織原代細(xì)胞培養(yǎng),并通過(guò)染色體核型分析結(jié)果劃分對(duì)照組和實(shí)驗(yàn)組。 3、了解染色體數(shù)目異常的自然流產(chǎn)胚胎絨毛細(xì)胞中是否存在CENP-E和CENP-I基因的蛋白和/(或)mRNA水平的異常,探索靶基因的表達(dá)在染色體數(shù)目異常的胚胎發(fā)生中可能具有的作用。 方法: 1、設(shè)計(jì)、合成CENP-E、CENP-I和內(nèi)參GAPDH基因的FQ-PCR引物和探針序列,進(jìn)行普通PCR擴(kuò)增,再將PCR產(chǎn)物與載體pMD18-T載體連接,構(gòu)建靶基因的定量PCR標(biāo)準(zhǔn)品; 2、采用組織塊貼壁法、消化法和組織塊研磨法原代培養(yǎng)流產(chǎn)絨毛組織,通過(guò)直接染色體制備法、傳統(tǒng)染色體制備法和原位染色體制備法對(duì)培養(yǎng)的胚胎絨毛細(xì)胞進(jìn)行染色體核型分析:將取自人工流產(chǎn)絨毛組織的染色體數(shù)目正常的胚胎絨毛細(xì)胞定為對(duì)照組,將來(lái)自自然流產(chǎn)絨毛組織的具有染色體數(shù)目異常核型的胚胎絨毛細(xì)胞定為實(shí)驗(yàn)組。 3、用real-time RT-PCR檢測(cè)CENP-E和CENP-I基因的mRNA水平的表達(dá); 4、用Western-blot和間接免疫熒光技術(shù)檢測(cè)CENP-E和CENP-I蛋白水平的表達(dá); 結(jié)果: 1、成功構(gòu)建CENP-E、CENP-I和內(nèi)參GAPDH基因的FQ-PCR標(biāo)準(zhǔn)品; 2、成功培養(yǎng)胚胎絨毛細(xì)胞,并通過(guò)染色體核型分析,從人工流產(chǎn)絨毛組織中篩選到33例染色體數(shù)目正常的胚胎絨毛細(xì)胞,從自然流產(chǎn)的絨毛組織中篩選到23例染色體數(shù)目異常的胚胎絨毛細(xì)胞。 3、real-time PCR、Western-blot和間接免疫熒光檢測(cè)結(jié)果顯示,與對(duì)照組相比,實(shí)驗(yàn)組中CENP-E和CENP-I的基因水平和蛋白質(zhì)水平的表達(dá)均明顯降低。 結(jié)論:染色體數(shù)目異常的胚胎絨毛細(xì)胞中CENP-E和CENP-I表達(dá)降低可能是引起胚胎染色體數(shù)目異常和胚胎發(fā)育異常的重要原因之一,對(duì)進(jìn)一步探索臨床早期診斷指標(biāo)和檢測(cè)方法具有重要意義。 第三部分RNA干擾技術(shù)抑制內(nèi)源CENP-E或CENP-I基因在胚胎絨毛細(xì)胞中的表達(dá) 目的: 1、構(gòu)建針對(duì)CENP-E和CENP-I基因的短發(fā)夾狀RNA(shRNA)表達(dá)載體; 2、shRNA重組表達(dá)質(zhì)粒載體轉(zhuǎn)染胚胎絨毛細(xì)胞后,觀察靶基因表達(dá)的抑制。 3、了解CENP-E和CENP-I基因的表達(dá)抑制對(duì)胚胎絨毛細(xì)胞增殖和染色體分離的影響,以及對(duì)細(xì)胞周期的調(diào)節(jié)。 方法: 1、設(shè)計(jì)、合成CENP-E和CENP-I基因的shRNA序列,與載體pgenesil-1連接,構(gòu)建靶基因的shRNA重組表達(dá)質(zhì)粒; 2、shRNA重組質(zhì)粒轉(zhuǎn)染細(xì)胞后,用real-time PCR、Western-blot和間接免疫熒光檢測(cè)mRNA水平和蛋白水平的變化,篩選有效的干擾質(zhì)粒和最佳基因抑制時(shí)間。 3、MTT法制備細(xì)胞生長(zhǎng)曲線,檢測(cè)基因抑制后對(duì)細(xì)胞生長(zhǎng)的影響。 4、FCM檢測(cè)細(xì)胞周期的變化。 5、細(xì)胞分裂指數(shù)測(cè)定和染色體核型分析檢測(cè)基因抑制對(duì)染色體分離的影響。 結(jié)果: 1、成功構(gòu)建針對(duì)靶基因的shRNA重組質(zhì)粒。 2、設(shè)計(jì)的干擾序列中pshRNA-CENP-E-3和pshRNA-CENP-I-3是有效的干擾質(zhì)粒,于轉(zhuǎn)染后72h胚胎絨毛細(xì)胞靶基因的內(nèi)源性表達(dá)受到明顯抑制,其蛋白水平和mRNA水平都顯著下降。 3、MTT實(shí)驗(yàn)發(fā)現(xiàn)有效干擾組細(xì)胞生長(zhǎng)較對(duì)照組明顯減慢,細(xì)胞增殖受到抑制。 4、FCM檢測(cè)顯示干擾后G2/M期的細(xì)胞比例增加;細(xì)胞分裂指數(shù)測(cè)定結(jié)果示隨轉(zhuǎn)染時(shí)間增加實(shí)驗(yàn)組中分裂期細(xì)胞所占比例也增加;核型分析顯示實(shí)驗(yàn)組中染色體數(shù)目異常細(xì)胞比例顯著增加。 結(jié)論:設(shè)計(jì)、合成針對(duì)靶基因的shRNA重組質(zhì)粒在胚胎絨毛細(xì)胞中能有效抑制靶基因的表達(dá),細(xì)胞生長(zhǎng)減慢、分裂期細(xì)胞增多、染色體數(shù)目異常細(xì)胞比率增加,說(shuō)明CENP-E和CENP-I對(duì)細(xì)胞增殖和染色體分離可能具有重要調(diào)控作用,成功構(gòu)建CENP-E和CENP-I抑制后觀察染色體數(shù)目異常變化的體外細(xì)胞模型,為后期的臨床研究奠定基礎(chǔ)。
[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, and normal chromosome separation and centromere structure and function, the spindle checkpoint control mechanism and so on. Centromere protein (Centromere, proteins, CENPs) is the basic unit of centromere assembly and function, by a group of proteins located in the centromere (more than 80 kinds of [1] have been found), they in interphase cells or mitotic centromere localization in the exercise of the right of assembly, involved in the centromere and affect its function. The CENP-E is not only involved in centromere assembly. But the spindle checkpoint is an important component, is the link between the centromere and spindle microtubules, and chromosome movement dynamic [2,3,4,5].CENP -I is a protein with upstream centromere assembly process, play may play an important role in.Nishihashi [6] research found in chicken DT40 cells after CENP-I gene knockout in the process of centromere assembly and function, will be a long time before the mid cell arrest in the division, and staggered. In order to reveal the chromosomes in human cells CENP-E, CENP-I, is involved in the regulation of normal chromosome segregation and cell division, the subject of the use of quantitative PCR, Western-blot and indirect immunofluorescence on abnormal chromosome number and karyotype of normal embryonic villi cells in CENP-E were detected and analyzed the expression of CENP-I, found that compared with the latter, the CENP-E chromosome in the embryonic villus cells, the expression of CENP-I at the mRNA level and protein level were significantly decreased; and the use of RNAi technology to build CENP-E human embryonic cashmere hair cell model of CENP-I defect expression, That compared with untransfected or empty vector transfected embryonic villi cells CENP-E, CENP-I reduced expression of embryonic villus cells growth was significantly reduced, G2/M phase cells increased, increasing the percentage of mitotic cells, chromosome abnormality rate increased significantly. The results showed that CENP-E, CENP-I and regulation of cell proliferation and centromere separation cell chromosome, reduce the two expression may be an important cause of spontaneous abortion. On CENP-E, the further study of CENP-I, will provide a new reference index for the study of chromosome number abnormity embryo abortion mechanism.
The first part of the prokaryotic expression, purification and antibody preparation of centromere protein CENP-I
Objective:
1, the prokaryotic expression vector of pET32a/CENP-I was constructed.
2, purified recombinant protein rCENP-I.
3, the recombinant protein was immunized with rabbits to prepare anti CENP-I antibody.
1, the CENP-I cDNA sequence encoding first to 293 amino acids of CENP-I protein was obtained from the total RNA of Hela cells by RT-PCR method. The recombinant plasmid pET32a/CENP-I. was cloned into the prokaryotic expression vector pET32a (+) by in vitro recombination method.
2, the correct recombinant plasmid was transformed into Escherichia coli BL21 (DE3), induced by IPTG, and purified by Ni2+-NTA affinity chromatography to obtain high purity recombinant protein.
3, the purified protein was used to immunize rabbits to prepare antibodies, agarose immune double expansion method, ELISA method and Western-blot were used to obtain highly effective immune serum, and the antibody was purified by saturated ammonium sulfate.
Result:
1, the recombinant expression plasmid pET32a/CENP-I. was successfully constructed by sequencing and identification.
2, the recombinant protein was purified by Ni2+-NTA affinity chromatography and showed a single band on SDS-PAGE. After purification, the purity of recombinant protein was over 90% by Bandscan, and rCENP-I protein was successfully obtained.
3, after the rCENP-I protein was immunized to rabbits, a highly effective immune serum was obtained. The agarose double expansion showed that the titer of immune serum reached more than 1:16. The titer of ELISA was 1:20000 and Western-blot showed that the titer of antibody reached 1:400, which could be used to detect CENP-I protein in clinical specimens.
Conclusion: the expression vector of pET32a/CENP-I was successfully constructed, the recombinant protein of CENP-I was purified and the anti CENP-I antibody with high titer was prepared.
The second part
A clinical study on the expression of CENP-E and CENP-I genes in the villous cells of spontaneous abortion
Objective:
1, FQ-PCR standard was obtained by T-A clone.
2, the primary cell culture of the aborted embryo chorionic tissue was divided into the control group and the experimental group by the karyotype analysis.
3, we need to know whether there are abnormalities in protein and / or mRNA levels of CENP-E and CENP-I genes in natural chorionic villus cells with abnormal chromosome numbers, and explore the possible role of target gene expression in the development of abnormal chromosome numbers.
Method:
1, design and synthesis of CENP-E, FQ-PCR primers and probe sequences CENP-I and reference GAPDH gene, were amplified with PCR, and then PCR products were connected with pMD18-T vector, constructing quantitative PCR standard target gene;
2, by the method of tissue adherence, digestion and tissue grinding cultured abortion, preparation method by direct chromosome system, traditional chromosome preparation in situ chromosome preparation method and preparation method of karyotype analysis of cultured embryonic villus cells: chromosome number from villi of normal embryonic abortion villus cells as the control group, from the chorionic villi of spontaneous abortion with chromosome karyotype of embryonic villus cells as the experiment group.
3, the expression of mRNA levels of CENP-E and CENP-I genes was detected by real-time RT-PCR.
4, the expression of CENP-E and CENP-I protein levels was detected by Western-blot and indirect immunofluorescence.
Result:
1, the successful construction of CENP-E, FQ-PCR standard CENP-I and reference gene GAPDH;
2, we successfully cultured embryonic chorionic villi cells, and through chromosome karyotype analysis, we screened 33 cases of chorionic villi with normal chromosome numbers from the villi of induced abortion. 23 cases of abnormal chorionic villi cells with abnormal chromosome numbers were screened from the villi of spontaneous abortion.
3, real-time PCR, Western-blot and indirect immunofluorescence detection showed that compared with the control group, the expression level of CENP-E and CENP-I gene level and protein level in the experimental group decreased significantly.
Conclusion: the decreased expression of CENP-E and CENP-I in chorionic villi cells with abnormal chromosome numbers may be one of the important reasons for abnormal chromosome number and embryo development. It is of great significance to further explore the early diagnostic indicators and detection methods.
Third RNA interference technique inhibits the expression of endogenous CENP-E or CENP-I gene in embryonic chorionic cells
Objective:
1, a short hairpin RNA (shRNA) expression vector for CENP-E and CENP-I genes was constructed.
2, after transfection of shRNA recombinant plasmid vector to embryo chorionic villus cells, the inhibition of target gene expression was observed.
3, we know the effect of inhibition of CENP-E and CENP-I gene expression on the proliferation and chromosome segregation of embryonic chorionic cells, and the regulation of cell cycle.
Method:
1, design, synthesize shRNA sequence of CENP-E and CENP-I gene, connect with carrier pgenesil-1, construct shRNA recombinant expression plasmid of target gene.
2, after transfection of shRNA recombinant plasmid, real-time PCR, Western-blot and indirect immunofluorescence were used to detect the change of mRNA level and protein level, and effective interfering plasmid and optimal gene suppression time were screened.
3, the cell growth curve was prepared by MTT method, and the effect of gene inhibition on cell growth was detected.
4, FCM detected cell cycle changes.
5, cell division index determination and chromosome karyotype analysis were used to detect the effect of gene inhibition on chromosome segregation.
Result:
1, the recombinant plasmid shRNA for target gene was successfully constructed.
2, pshRNA-CENP-E-3 and pshRNA-CENP-I-3 were effective interference plasmids in the designed interference sequences. After transfection, the endogenous expression of target genes in 72h embryonic chorionic cells was significantly inhibited, and their protein levels and mRNA levels were significantly decreased.
3, the MTT experiment found that the cell growth in the effective interference group was significantly slower than that in the control group, and the cell proliferation was inhibited.
4, FCM detection showed that the proportion of cells in G2/M phase increased after the interference. The cell division index showed that the proportion of mitotic cells increased in the experimental group with increasing transfection time. Karyotype analysis showed that the proportion of abnormal number of chromosomes in the experimental group increased significantly.
Conclusion: design, synthesis of target genes of shRNA recombinant plasmid can inhibit target gene expression in embryonic villus cells, cell growth slowed, mitotic cells increased, the chromosome number of abnormal cell ratio increased, indicating that CENP-E and CENP-I might play an important role in the regulation of cell proliferation and chromosome segregation, successfully constructed a cell model in vitro changes the chromosome number of CENP-E and CENP-I were observed after inhibition, lay the foundation for further clinical researches.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R394;R714.21

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 閆惠娜;探討染色體Cd結(jié)構(gòu)及CENP變化與非整倍體形成的關(guān)系[D];山西醫(yī)科大學(xué);2012年

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本文編號(hào)：1414139