【作者】 姚峰；

【导师】 王佐；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 动脉粥样硬化是一种动脉血管壁慢性炎症性疾病,其确切机制还未完全阐明,很多因素可促进其发生发展。而趋化因子是炎症的关键调节因子。基质细胞衍生因子SDF-1α(stromal cell derived- factor 1 alpha)是一类具有趋化活性的细胞因子,研究证实它在造血干细胞迁移及归巢、恶性肿瘤尤其血液肿瘤的浸润转移以其HIV感染方面发挥着重要作用。近几年来SDF-1α在动脉硬化性疾病中的作用也开始引起重视。本研究应用RT-PCR法从大鼠血管平滑肌细胞(Vascular smooth muscle cells,Vsmcs;)中克隆出SDF-1α基因,并对其序列进行初步的生物信息学分析;然后构建EGFP-C1- SDF-1α重组质粒进行亚细胞定位分析;最后构建重组质粒pcDNA3.1-SDF-1α,研究其对单核细胞的趋化作用。目的:克隆大鼠SDF-1α基因,并对大鼠SDF-1α基因序列进行分析;分析SDF-1α蛋白亚细胞定位;探讨重组SDF-1α蛋白对单核细胞的趋化作用。方法:从大鼠Vsmcs中提取mRNA,用RT-PCR从中扩增出SDF-1α基因,将扩增片段克隆于pMD-T18载体中,转化DH5α菌;进行蓝白筛选后,运用PCR和酶切鉴定并DNA测序;利用生物信息学方法对SDF-1α基因的序列进行分析。构建EGFP-C1- SDF-1α表达载体,酶切鉴定,脂质体转染法将重组质粒瞬时转染Ecv304内皮细胞株,48h后用荧光显微镜检测绿色荧光蛋白表达;构建重组质粒pcDNA3.1-SDF-1α,脂质体转染法转染Ecv304内皮细胞株,经G418筛选阳性克隆,RT-PCR验证,建立SDF-1α转基因稳定表达细胞系,再用小室迁移实验来研究SDF-1α对THP-1单核细胞的趋化作用。结果: PCR扩增得到378bp的基因片段,经酶切初步证实为SDF-1α基因;阳性克隆经筛选鉴定连接有目的基因;目的基因测序结果与GeneBank上序列比较,结果完全一致,表明靶基因成功插入pMD–T;用脂质体转染法将重组质粒EGFP-C1- SDF-1α瞬时转染内皮细胞,发现绿色荧光蛋白主要在细胞质中表达;用脂质体转染法将重组质粒pcDNA3.1-SDF-1α转染法转染内皮细胞,经G418筛选阳性克隆,RT-PCR验证,表明建立了SDF-1α转基因稳定表达细胞系,小室迁移试验发现SDF-1α对THP-1单核细胞的具有明显趋化作用。结论:成功克隆了SDF-1α基因,SDF-1α蛋白主要在细胞质中表达,重组的SDF-1α对单核细胞具有趋化作用。更多还原

【Abstract】 Atherosclerosis is now viewed as an inflammatory disease of the vascular system, there are so many factors involve in enhancing its development, yet its mechanism remains unclear. Stromal cell–derived factor-1 (SDF-1) is a sort of chemotactic cytokines. Recent publications indicate that the SDF-1αplays a pivotal role during development and in many patho-physiological conditions including hematopoiesis,cancer metastasis,and HIV infection.Latest, SDF-1αis discoveried to be highly expressed in atherosclerosis plaque, so its role in atherosclerosis has been highlighted.In this study, using RT-PCR to amplify SDF-1αgene form rat VSMCs, and analyzing its sequence by bioinformatics. pEGFP-C1-SDF-1αfusion mammalian expression protein was constructed and to observe how did Express in the ECV-304 cells. The fusion mammalian expression protein pcDNA3.1-SDF-1αwere constructed and to to study the effect of SDF-1αon THP-1 cells chemotaxis.Objective To amplify and clone SDF-1αgene and to predict its function by bioinformatics analysis;To observate that pEGFP-SDF-1αwas expressed in the ECV-304;To explore the effect of SDF-1αon THP-1 cells chemotaxis.Methods mRNA was extracted from rat VSMCs, SDF-1αgene was amplified reverse transcriptase polymerase chain reaction (RT-PCR) ;The PCR fragment was inserted into pMD-T18 easy plasmid,and then was transformed into DH5α.After the selection of blue/white screening,the recombinant plasmid was identified by restriction enzyme digest,PCR and sequencing. Function of SDF-1αwas predicted by bioinformatics analysis with Internet and GeneBank databse. pEGFP-SDF-1αfusion mammalian expression protein was constructed and transfected into the ECV-304 cells. Observation under the fluorescent microscopy showed that pEGFP-SDF-1αwas expressed. The fusion mammalian expression protein pcDNA3.1/SDF-1αwere constructed and transfected into the ECV-304 cells, and established stable cell line,then explored the effect of SDF-1αon THP-1 cells chemotaxis with transwell permeable supports.Results The length of the DNA fragment RT-PCR amplified by mRNA was 378 base pairs,which was conformed by enzyme EcoRⅠcutting identification.The correctness of linking between SDF-1αand pMD-T18 easy was verified through selection of blue/white screening,and the sequence of DNA fragment were homology with corresponding published in GenBank,which indicated that the target gene has been inserted into pMD-T18 successfully. pEGFP-SDF-1αfusion protein was transfected into ECV-304 cells using Liposome and showed that pEGFP-SDF-1αwas expressed and existed in the plasma membrane and cytoplasm. pcDNA3.1/SDF-1αplasmid was transfected into ECV-304cells using Liposome ,the positive clones were screened by G418 and the expression of SDF-1αgene in the transfected ECV-304 cells were detected by RT- PCR ,and it showed that it has been established stable cell line. THP-1 chemotaxis was examined with transwell permeable supports and showed that SDF-1αisimplicated in THP-1 cells chemotaxis.Conclusion The SDF-1αgene was cloned successfully.The SDF-1αprotein was expressed mainly in the cytoplasm and effect on THP-1 cells chemotaxis更多还原