靶向人端粒酶逆转录酶（hTERT）RNA干扰的建立及其生物学效应的研究

发布时间：2018-01-20 15:55

本文关键词： RNA 干扰端粒酶逆转录酶 p53 凋亡　出处：《重庆医科大学》2005年博士论文　论文类型：学位论文

【摘要】：端粒酶是一种核蛋白复合物,具有维持和延长真核细胞染色体末端端粒结构、调节细胞增殖潜能和寿命的功能。由于在 90%的人恶性肿瘤细胞中涉及端粒酶的活化而在绝大多数正常体细胞中检测不到端粒酶的活性,故近来端粒酶已成为颇具吸引力的肿瘤治疗的分子靶标。具有活性的端粒酶由一个 RNA 亚基和几个蛋白组份构成。RNA 亚基(human telomerase RNA, hTR)含有端粒 DNA 复制的模板。蛋白组分包括具有催化活性的端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT ) 和端粒酶相关蛋白 1 ( human telomerase-associated protein 1,hTEP1)。hTR 和 hTEP1 在很多正常人组织中都有表达,而 hTERT 则表达于细胞永生化过程中具有端粒酶活性的细胞中,在细胞的癌变过程中起关键作用。近来,越来越多的研究表明,hTERT 的功能除了维持端粒的结构外,还可以通过与其他分子相互作用而发挥不同的生物学功能。为进一步研究 hTERT 的新的生物学功能,发现与之相互作用的相关分子,我们应用 RNA 干扰(RNA interference,RNAi)技术抑制 HepG2细胞 hTERT 的表达,观察 HepG2 细胞的生物学特性如细胞凋亡,生长速度的改变;检测了凋亡通路中几个重要蛋白的表达水平以探讨 hTERT抑制诱导凋亡发生的机制;同时研究了 p53 的表达对 hTERT 的调节作用,本文的主要内容如下: 第一部分靶向 hTERT siRNA 有效序列的筛选采用随机设计方法和全长基因寻靶技术设计针对 hTERT 转录体编码区和非编码区的 5 条双链 DNA 作为合成 siRNA 的模板序列,在体外
[Abstract]:Telomerase is a nuclear protein complex that maintains and extends the telomere structure at the end of the chromosome of eukaryotic cells. The function of regulating cell proliferation potential and longevity. Telomerase activity is not detected in most normal somatic cells because telomerase activation is involved in 90% of human malignant tumor cells. Telomerase has recently become an attractive molecular target for cancer therapy. The active telomerase consists of a RNA subunit and several protein components. HTR) contains a template for telomere DNA replication. Protein components include telomerase reverse transcriptase (telomerase reverse transcriptase) with catalytic activity. Human telomerase reverse transcriptase. HTERT and human telomerase-associated protein 1. HTEP1).hTR and hTEP1 are expressed in many normal human tissues, while hTERT is expressed in cells with telomerase activity during immortalization. Recently, more and more studies have shown that hTERT functions in addition to maintaining the structure of telomere. They can also play different biological functions by interacting with other molecules. In order to further study the new biological function of hTERT and find the interacting molecules, we used RNA interference RNA interference. RNAi technique inhibited the expression of hTERT in HepG2 cells and observed the biological characteristics of HepG2 cells such as apoptosis and the change of growth rate. The expression levels of several important proteins in apoptotic pathway were detected to investigate the mechanism of hTERT inhibition induced apoptosis. At the same time, the regulation of p53 expression on hTERT was studied. The main contents of this paper are as follows: Part one: screening of effective sequences targeting hTERT siRNA A randomized design method and a full-length gene targeting technique were used to design five double-stranded DNA targeting the coding and non-coding regions of hTERT transcripts as template sequences for the synthesis of siRNA. In vitro
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R346

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