【作者】 赵军；

【导师】 董子明；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2005， 硕士

【摘要】 背景与目的: 肿瘤是细胞分化、增殖和死亡机制发生异常的结果。DNA的损伤和基因结构的异常,以及由此造成的所谓癌基因和/或抑癌基因的表达和功能上的改变,是细胞恶性转化的前提。但并不是所有的损伤均导致基因突变,其原因就在于细胞内的DNA修复系统能够清除和修复DNA损伤。其中,碱基切除修复(BER),是清除细胞内DNA损伤的主要途径,而DNA聚合酶β(DNA polymerase β,DNA pol β)又是BER过程的关键酶,主要修复自由基氧化或胞嘧啶和甲基胞嘧啶脱氨基等过程产生的DNA损伤,也能解决外源性因素如亚硝胺类产生的烷基化碱基,在DNA和RNA水平上进行损伤修复,因此能减少损伤导致的基因突变。为此,本研究构建了食管组织野生型DNA pol β与增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP)的真核表达载体,而且将此融合基因导入食管癌细胞EC9706中,使外源野生型的DNA pol β表达,观察转染后的细胞生物学特性的变化,间接的研究DNA pol β的损伤修复作用。 方法: 运用PCR方法,从质粒pcDNA3.1-pol β中扩增出野生型的DNA pol β基因开放阅读框序列,作为目的片段,T-A克隆至pGEM-T载体,再定向克隆至pEGFP-C3真核绿色荧光蛋白表达载体,获得野生型的重组真核表达载体pEGFP-C3-pol β。重组质粒pEGFP-C3-pol β用限制性内切酶酶切鉴定,通用鉴定引物PCR扩增鉴定,并进行DNA序列分析。将重组的野生型的pEGFP-C3-pol β经脂质体介导转更多还原

【Abstract】 Background and objective:Tumor is a consequence that the mechanism of cell differentiation proliferation and death is out of the way .It is the premise of cell malignant transformation that the abnormity of DNA damage, gene structure and the change of expression and function of oncogene and/or cancer suppressive gene ,which is result from the abnormity of DNA damage and gene structure. However ,not all damages lead to gene mutation because DNA repair system of cell can clean DNA damages out and repair them. Base-excision repair(BER) is one of vital ways to clean DNA damages out and that DNA polymerase β(DNA pol β) is a key enzyme in BER process. DNA pol β is involved in repairing DNA damages resulted by ROS oxidation or deamination of cytosine and met-cytosine. It also may be involved in repairing alkyl bases damage resulted by exterior factors such as nitrosamine .Therefore , DNA pol β can decrease gene mutations resulted from DNA damages .On the basis of the peculiar functions of DNA pol β ,we constructed a recombined enhanced green fluorescent protein expression vector carrying wild type DNA pol β gene in esophageal tissue ,and then transfected recombinants into EC9706 cell by lipofectamine method .The fusion protein was expressed in EC9706 cell .We observed the change of biological function of transfected cell to study indirectly the repair function of DNA pol β.Methods:The fragment of wild type DNA pol β gene was amplified by PCR method with plasmid pcDNA3.1-polβ as a template and cloned into pGEM-T vector by T-A clone method. And then, it was inserted directionally into a eukaryotic expression vector pEGFP-C3.The recombinant pEGFP-C3-polβ was obtained .Restricted enzyme analyzing, PCR technique and sequencing were employed to identify the recombined plasmid. The recombinant was transfected into EC9706 cells by lipofectamine method .The stable expressing EC9706-wtpolβ was established through G418 screening. The localization of DNA pol β gene-encoded protein was observed by fluorescent converted microscope. The expression of mRNA level was detected by RT-PCR method. The growth curve was drawn by MTT method and cell cycle was examined by flow cytometer .The effect of biological function of EC9706 cell transfected DNA pol β was analyzed. All data were analyzed statistically.Result:1. It was proved that the sequence of the wide type recombinant was correct and it was transfected into EC9706 cell successfully .2. The localization of wtDNA Pol p protein displayed that pEGFP-C3 and pEGFP-C3-polβ proteins expressed GFP and they were distributed in the whole cell and mostly inside the nuclear at 24 hour, respectively .There was significant difference between these two groups .And the expressing account of wtDNA Pol P protein was significantly higher than untransfected group.3. The proliferous speed of EC9706-wtPolβ was significantly slower than EC9706-neor and EC9706. (P<0.05)。 Furthermore, the cell number of S period was decrease in EC9706-wtPolβ (P>0.05) .Conclusion:1. The recombined expression vector (pEGFP-C3-polβ) is constructed successfully.2. The localization of exogenous wtDNA Pol β is observed and it is mainly distributed inside the nuclear;3. The proliferous speed of EC9706-wtPolβ is significantly slower and the cell number of S period is decrease .It indicates that transfected wtDNA Pol β may be involved in and enhance DNA repair function.更多还原