【作者】 张敏；

【导师】 奚耕思；

【作者基本信息】 陕西师范大学 ， 动物学， 2005， 博士

【摘要】 端粒位于真核细胞染色体末端,由串联排列的DNA重复序列和端粒结合蛋白构成,其作用为保护染色体末端并维护基因组稳定。端粒随细胞分裂次数的增加而不断缩短,当缩短到临界长度时就会导致细胞衰老、死亡。端粒酶是一种特殊的核糖核蛋白复合体,能以其自身RNA(TR)为模板反转录合成端粒重复序列,并将其加到染色体末端,从而恢复端粒功能,使细胞获得无限增殖能力。约90%以上的恶性肿瘤细胞都含有端粒酶活性,而仅有睾丸、卵巢组织及造血细胞系等少数正常组织细胞可检测到端粒酶活性。端粒酶催化亚基TERT是端粒酶活性的限制组分,在原发性肿瘤、癌细胞系和永生化细胞系中,TERT的表达与端粒酶活性的表达水平一致,与端粒酶的活化过程密切相关。端粒酶及TERT在哺乳动物配子发生过程中的表达特点目前尚存在争议,同时其在肿瘤细胞中的表达调控研究也是当今生命科学和医学研究的热点。 为了研究探讨端粒酶催化亚基TERT在正常生殖细胞中的表达特点和生物学意义,以及TERT与细胞周期相关基因的关系及其在肿瘤细胞增殖中的调控作用,本实验首先应用免疫组织化学和原位杂交技术,对不同年龄小鼠睾丸和卵巢不同分化类型的生殖细胞中TERT的表达进行了原位检测,并在此基础上应用RNA干涉和基因芯片等技术,对靶向TERT的RNA干涉后的小鼠EL-4淋巴瘤细胞进行细胞周期相关基因的表达谱分析,研究主要结果如下: 1.10天小鼠睾丸中含有精原细胞和支持细胞两种,未见TERT蛋白及mRNA阳性表达;20天小鼠睾丸中精原细胞有丝分裂和初级精母细胞减数分裂活跃,初级精母细胞尤其是粗线期精母细胞中有TERT阳性表达;30天小鼠睾丸生精上皮分化形成大量精子细胞,初级精母细胞(以粗线期精母细胞为主)和精子细胞均有TERT阳性表达;60天和90天小鼠睾丸曲细精管管腔进一步扩大,初级精母细胞和精子细胞中仍有TERT阳性表达,附睾中的成熟精子无TERT表达,同时精原细胞也始终无表达。TERT在精子发生过程中的表达存在着从弱到强再到弱的动态变化趋势,其表达与精子发生中减数分裂过程存在密切关系,并对各级生精细胞的增殖分化有重要的调节作用,从而保证了精子的端粒长度以及遗传物质的完整性和稳定性。更多还原

【Abstract】 Telomeres are specialized structures at the ends of eukaryotic chromosomes and are composed of conserved sequences of DNA repeats. Telomeric DNA is not completely synthesized by conventional DNA polymerase, but progressively shorten with each cellular division. Cellular aging is characterized by a decrease in telomere length, and this has been implicated as a mitotic clock that signals cells to stop division when telomeres reach a critically short length. Telomerase is a ribonucleoprotein that synthesizes telomere repeats onto chromosome ends and is involved in maintaining telomere length in germline and cancer cells( > 90 % ).The catalytic subunit (TERT) appears to be the limiting component in most telomerase-negative cells. As TERT is expressed constitutively, the presence of telomerase activity is correlated with TERT mRNA expression in extracts from tissue culture cells and normal and cancer tissues.In order to investigate the expression pattern of TERT in reproductive system cells of mouse and address the biological significance of TERT, both immunohistochemistry and in situ hybridization experiments were applied to explore the pattern of TERT expression in different differentiated reproductive cells in the testis and ovaries of mouse. Furthermore, in order to investigate the relationship between TRET and cell cycle related genes and the regulative functions of TERT in proliferation of mouse cancer cells, techniques of RNA interference and gene chip were used to analyze the expression of cell cycle related genes after RNA interference targeted TERT in EL-4 lymphoma cells of mouse. The main results are as following:1. Spermatogonia and sertoli cells are found in the testis of 10-day mice;but there is no positive expression of TERT protein and mRNA. In testis of 20-day mice, active meiosis and mitosis are found, and there are large numbers of primary spermatozoas, in which there are highly positive TERT expression. In testis of 30-day mice, seminiferous epithelium has differentiated into secondary spermatocytes and spermatids cells, and some sperms have been formed. Meanwhile, TERT positive expression can be found in the primary spermatocytes and spermatids cells. In primary spermatocytes, especially inpachytene spermatocytes, the strongest TERT expression level was detected. The seminiferous tubules cavity in testis of 60-day and 90-day mice are expanded and TERT positive expressions can only be detected in the spermatids cells and primary spermatocytes. TERT expression pattern during the process of sperm generation indicates a weak-strong-weak trend of the dynamic character, which may be closely related with the meiosis in spermatogenesis and have important regulative effect on the proliferation and differentiation of different levels of sperm-generation cells. And the role of TERT during the spermatogenesis also ensures the transmission of full-length chromosomes to progeny.2. During the oogenesis, no positive TERT expression has been found in the oocytes of 10-day、 20-day、 30-day 、 60-day and 90-day mice. Strongly TERT positive expression is found in the follicle cells of primordial follicles. This indicates that the follicle cells are main source of telomerase activity in ovaries. During the development of ovary the expression TERT becomes weaker and weaker, which is closely related with the growth of the follicles. The relation between TERT and follicle cells possibly influences not only proliferative capacity of follicle cells, but also communion between follicle cells and oocytes.3. The results of RT-PCR and Western blot show that, after transfection to the EL-4 lymphoma cells of mouse, the TERT specialized shRNA effectively repressed the expressions of endogenous TERT mRNA and protein, which made the targeted genes partially silent. The MTT experiment demonstrates that the amount of EL-4 cells dose not show significant difference between the sample treated with TERT specialized shRNA and the contrast without treatment. This result indicates that the repression of TERT expressions does not essentially lead to apparent death of cells at the time of detection, which may due to the phenotypic lag phenomenon caused by the inhibition of telomerase.4. According to the result of gene chip analysis, among the 96 cell cycle genes of mice, 43 genes show significant expression difference between the sample treated with TERT specialized shRNA and the contrast, and all of 43 genes belong to the down-regulated gene. According to the biological functions of the genes in the cell cycles of mice ,they can be divided into five groups: (1) Genes encoding cyclin, such as CyclinAl, CyclinB2, CyclinE2, CyclinF, CyclinG1, CyclinH etc.. ( 2 ) Genes encoding cyclin-dependent kinase, such as CDK4, CDK5, CDK7 etc.. (3) CDC25B更多还原