着丝粒蛋白CENP-E和CENP-I与染色体分离关系研究

发布时间：2018-01-12 12:00

本文关键词：着丝粒蛋白CENP-E和CENP-I与染色体分离关系研究　出处：《重庆医科大学》2008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,而染色体的正常分离与着丝粒结构和功能、纺锤体检验点监控机制等有关。着丝粒蛋白质(Centromere proteins,CENPs)是着丝粒组装和功能发挥的基本组成单位,由位于着丝粒上的一组蛋白质(已发现的将近80余种[1])构成,它们在细胞分裂间期或分裂期定位在着丝粒上,共同参与着丝粒的组装并影响其功能的正确行使。其中CENP-E不仅参与着丝粒的组装,而且还是纺锤体关卡蛋白检查点的重要组成部分,是联系着丝粒与纺锤体微管间的纽带,并提供染色体运动的动力[2、3、4、5]。CENP-I是着丝粒组装过程中较上游的一个蛋白质,在着丝粒装配和功能发挥过程中可能具有重要作用。Nishihashi等[6]研究发现在鸡的DT40细胞敲除CENP-I基因后,细胞将长时间停滞于分裂的前中期,并出现染色体错排。为了揭示在人类细胞中,CENP-E、CENP-I是否参与调控染色体分离和细胞正常分裂,本课题利用定量PCR、Western-blot和间接免疫荧光技术对染色体数目异常和核型正常的胚胎绒毛细胞中CENP-E、CENP-I的表达进行检测分析,发现与后者相比,染色体数目异常的胚胎绒毛细胞中CENP-E、CENP-I在mRNA水平和蛋白质水平上的表达均明显降低;并用RNAi技术构建CENP-E、CENP-I缺陷表达的人胚胎绒毛细胞模型,发现与未转染或转染空载体的胚胎绒毛细胞相比,CENP-E、CENP-I表达降低的胚胎绒毛细胞生长速度明显减慢,G2/M期细胞增加,分裂期细胞比例增大,染色体数目异常率显著增加。说明CENP-E、CENP-I参与着丝粒对细胞染色体分离和细胞增殖的调控,二者表达的降低可能是导致自然流产发生的重要原因。对CENP-E、CENP-I的进一步研究,将为研究染色体数目异常胚胎自然流产机制提供新的参考指标。第一部分着丝粒蛋白质CENP-I的原核表达、纯化及抗体制备目的: 1、构建pET32a/CENP-I原核表达载体。 2、纯化重组蛋白rCENP-I。 3、将重组蛋白免疫兔制备抗CENP-I抗体。方法: 1、用RT-PCR的方法从Hela细胞总RNA中获得编码CENP-I蛋白第1～293位氨基酸的CENP-I cDNA序列,采用基因体外重组法将其克隆到原核表达载体pET32a(+)内,获得重组质粒pET32a/CENP-I。 2、将测序鉴定正确的重组质粒转化到大肠杆菌BL21(DE3)中,用IPTG诱导表达、Ni2+-NTA亲和层析纯化获得高纯度的重组蛋白质。 3、用纯化的蛋白质免疫家兔制备抗体,琼脂糖免疫双扩法、ELISA法和Western-blot得到高效价的免疫血清,并用饱和硫酸铵初步纯化抗体。结果: 1、经测序鉴定,成功构建了重组表达质粒pET32a/CENP-I。 2、重组蛋白经Ni2+-NTA亲和层析柱纯化后在SDS-PAGE上呈现单一条带,灰度扫描后用Bandscan分析重组蛋白纯度达90%以上,成功的获得了rCENP-I蛋白。 3、将rCENP-I蛋白免疫兔后获得了高效价的免疫血清,琼脂糖双扩显示免疫血清效价达1:16以上;ELISA法测免疫血清效价达1:20000,Western-blot显示抗体效价达1:400,能用于检测临床标本中的CENP-I蛋白。结论:成功的构建了pET32a/CENP-I表达载体、纯化了CENP-I重组蛋白,并制备了效价较高的抗CENP-I抗体。第二部分染色体数目异常的自然流产胚胎绒毛细胞中CENP-E和CENP-I基因表达的临床研究目的: 1、通过T-A克隆获得FQ-PCR标准品。 2、流产胚胎绒毛组织原代细胞培养,并通过染色体核型分析结果划分对照组和实验组。 3、了解染色体数目异常的自然流产胚胎绒毛细胞中是否存在CENP-E和CENP-I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。方法: 1、设计、合成CENP-E、CENP-I和内参GAPDH基因的FQ-PCR引物和探针序列,进行普通PCR扩增,再将PCR产物与载体pMD18-T载体连接,构建靶基因的定量PCR标准品; 2、采用组织块贴壁法、消化法和组织块研磨法原代培养流产绒毛组织,通过直接染色体制备法、传统染色体制备法和原位染色体制备法对培养的胚胎绒毛细胞进行染色体核型分析:将取自人工流产绒毛组织的染色体数目正常的胚胎绒毛细胞定为对照组,将来自自然流产绒毛组织的具有染色体数目异常核型的胚胎绒毛细胞定为实验组。 3、用real-time RT-PCR检测CENP-E和CENP-I基因的mRNA水平的表达; 4、用Western-blot和间接免疫荧光技术检测CENP-E和CENP-I蛋白水平的表达; 结果: 1、成功构建CENP-E、CENP-I和内参GAPDH基因的FQ-PCR标准品; 2、成功培养胚胎绒毛细胞,并通过染色体核型分析,从人工流产绒毛组织中筛选到33例染色体数目正常的胚胎绒毛细胞,从自然流产的绒毛组织中筛选到23例染色体数目异常的胚胎绒毛细胞。 3、real-time PCR、Western-blot和间接免疫荧光检测结果显示,与对照组相比,实验组中CENP-E和CENP-I的基因水平和蛋白质水平的表达均明显降低。结论:染色体数目异常的胚胎绒毛细胞中CENP-E和CENP-I表达降低可能是引起胚胎染色体数目异常和胚胎发育异常的重要原因之一,对进一步探索临床早期诊断指标和检测方法具有重要意义。第三部分RNA干扰技术抑制内源CENP-E或CENP-I基因在胚胎绒毛细胞中的表达目的: 1、构建针对CENP-E和CENP-I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胚胎绒毛细胞后,观察靶基因表达的抑制。 3、了解CENP-E和CENP-I基因的表达抑制对胚胎绒毛细胞增殖和染色体分离的影响,以及对细胞周期的调节。方法: 1、设计、合成CENP-E和CENP-I基因的shRNA序列,与载体pgenesil-1连接,构建靶基因的shRNA重组表达质粒; 2、shRNA重组质粒转染细胞后,用real-time PCR、Western-blot和间接免疫荧光检测mRNA水平和蛋白水平的变化,筛选有效的干扰质粒和最佳基因抑制时间。 3、MTT法制备细胞生长曲线,检测基因抑制后对细胞生长的影响。 4、FCM检测细胞周期的变化。 5、细胞分裂指数测定和染色体核型分析检测基因抑制对染色体分离的影响。结果: 1、成功构建针对靶基因的shRNA重组质粒。 2、设计的干扰序列中pshRNA-CENP-E-3和pshRNA-CENP-I-3是有效的干扰质粒,于转染后72h胚胎绒毛细胞靶基因的内源性表达受到明显抑制,其蛋白水平和mRNA水平都显著下降。 3、MTT实验发现有效干扰组细胞生长较对照组明显减慢,细胞增殖受到抑制。 4、FCM检测显示干扰后G2/M期的细胞比例增加;细胞分裂指数测定结果示随转染时间增加实验组中分裂期细胞所占比例也增加;核型分析显示实验组中染色体数目异常细胞比例显著增加。结论:设计、合成针对靶基因的shRNA重组质粒在胚胎绒毛细胞中能有效抑制靶基因的表达,细胞生长减慢、分裂期细胞增多、染色体数目异常细胞比率增加,说明CENP-E和CENP-I对细胞增殖和染色体分离可能具有重要调控作用,成功构建CENP-E和CENP-I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基础。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R394;R714.21

【引证文献】