SELEX技术筛选泡沫细胞适配子

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1、·原创性声明本人声明，所呈交的学位论文是本人在导师指导下进行的研究工作及取得的研究成果。尽我所知，除了论文中特别加以标注和致谢的地方外，论文中不包含其他人已经发表或撰写过的研究成果，也不包含为获得南华大学或其他单位的学位或证书而使用过的材料。与我共同工作的同志对本研究所作的贡献均已在论文中作了明确的说明。作者签名：日期：年月日关于学位论文使用授权说明本人同意南华大学有关保留、使用学位论文的规定，即：学校有权保留学位论文，允许学位论文被查阅和借阅；学校可以公布学位论文的全部或部分内容，可以采用复印、缩印或其它手段保留学位论文；学校可根据国家或湖南省有关部门规定送交

2、学位论文。作者签名：导师签名：日期：年月····目录一、主要英文缩略语索引„„„„„„„„„„„„„„„„1二、中文摘要„„„„„„„„„„„„„„„„„„„„„3三、英文摘要„„„„„„„„„„„„„„„„„„„„„5四、论文正文前言„„„„„„„„„„„„„„„„„„„„„„8实验材料„„„„„„„„„„„„„„„„„„„13实验方法„„„„„„„„„„„„„„„„„„„16实验结果„„„„„„„„„„„„„„„„„„„27讨论„„„„„„„„„„„„„„„„„„„„„43结论„„„„„„„„„„„„„„„„„„„„„49参考文献„„„„„„„„„„„„„„

3、„„„„„50五、综述„„„„„„„„„„„„„„„„„„„„„„54六、致谢„„„„„„„„„„„„„„„„„„„„„„66····主要英文缩略语索引Biotin生物素bpbasepair碱基对CEcholesterolester胆固醇酯dNTPdeoxynudeosidetriphosphate脱氧三磷酸核苷酸dsDNAdouble-strandedDNA双链脱氧核糖核酸DPBSDulbeccophosphate-bufferedsaline杜尔贝科磷酸盐缓冲盐水DTTdithiothreitol二硫苏糖醇ECendothelialcell内皮细胞EDTAe

4、thylenediamietetraaceticacid乙二胺四乙酸EBethidiumbromide溴化乙啶FCfreecholesterol游离胆固醇FITCfluoresceinisothiocyanate异硫氰酸酯荧光素IPTGisopropyl-β-D-thiogalactopyranoside异丙基-β-D-硫代半乳糖LBLurib-BertanimediumLB培养基PAGEpolyacrylamidegeleletrophroresis聚丙烯酰胺凝凝胶电泳PBSPhosphate-bufferedsaline磷酸盐缓冲液PCRpolymerase

5、chainreaction聚合酶链反应PMAPhorol12-myristate13-acetate佛波脂rpmrevolutionsperminute每分钟转数SELEXsystemevolutionofligandsby指数富集的配基系统进化exponentialenrichmentssDNAsingle-strandedDNA单链脱氧核糖核酸TCtotalcholesterol总胆固醇TEMEDN,N,N,,N,,-tetramrthylenediamineN,N,N,,N,,-四甲基乙二四甲基联苯胺Tristris(hydroxymethyl)amino

6、methane三羟甲基氨基甲烷VSMCvascularsmoothcells血管平滑肌细胞····X-gal5-bromide-4-chloro-3-indio-β-D-5-溴-4-氯-3-吲哚-β-D-Galactoside半乳糖苷····SELEX技术筛选泡沫细胞适配子硕士研究生汪江波导师严鹏科摘要目的：利用SELEX技术筛选靶向巨噬细胞源性泡沫细胞的寡核苷酸适配子，并初步鉴定其与巨噬细胞源性泡沫细胞及动脉粥样硬化病变结合的特异性。方法：体外培养的THP-1细胞，经100nmol/LPMA孵育72小时,诱导其分化成巨噬细胞，然后用80mg/L氧化性低密度脂蛋

7、白（oxLDL）孵育72小时,建立泡沫细胞模型，通过油红O染色观察细胞内脂滴，HPLC检测细胞内胆固醇含量。体外合成一个含35个核苷酸随机序列、总长81nt、库容量大约为1015-16的ssDNA文库；优化了ssDNA库PCR反应条件，用生物素和`FITC标记的引物扩增为dsDNA文库并以生物素-链霉亲和素磁珠分离方法构建FITC标记的ssDNA文库；以六孔板为筛选介质，ssDNA文库先分别与大鼠血管平滑肌细胞和THP-1巨噬细胞结合，反筛除去与其结合的ssDNA，然后与巨噬细胞源性泡沫细胞结合，洗脱与其结合的ssDNA，PCR扩增构建次级FITC标记的ssDN

8、A文库进行下一次筛选。利