重组腺病毒rad一egf转染hdpscs对其增殖影响的体外研究

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1、中图分类号：R781；R329.28硕士学位论文重组腺病毒rAd一EGF转染hDPSCs对其增殖影响的体外研究院（系）、所口腔医学院研究生姓名付广丽学科、专业口腔临床医学导师姓名刘兴容教授二Ο一二年四月目录重组腺病毒rAd-EGF转染hDPSCs对其增殖影响的体外研究1„„„„„„„„„„„„„„„„„„„„„„„„„11.1中文摘要„„„„„„„„„„„„„„„„„„„„„11.2英文摘要„„„„„„„„„„„„„„„„„„„„„41.3前言„„„„„„„„„„„„„„„„„„„„„„„71.4材料与方法„„„„„„„„„„„„„„„

2、„„„„„141.5结果„„„„„„„„„„„„„„„„„„„„„„„251.6讨论„„„„„„„„„„„„„„„„„„„„„„„301.7结论„„„„„„„„„„„„„„„„„„„„„„„361.8参考文献„„„„„„„„„„„„„„„„„„„„„371.9英汉缩略词对照表„„„„„„„„„„„„„„„„„422致谢„„„„„„„„„„„„„„„„„„„„„„„443成体干细胞与牙再生„„„„„„„„„„„„„„„„45本课题基金资助：四川省卫生厅科研基金资助（060051）重组腺病毒rAd一EGF转染hDPSCs对其增殖影响的体外研

3、究摘要目的：通过体外培养人牙髓干细胞（humandentalpulpstemcells，hDPSCs），观察腺病毒rAd-EGF转染至hDPSCs后对其生长、增殖和凋亡的影响，为EGF对hDPSCs的作用提供实验依据，并为牙齿缺损与牙再生提供可能的新思路和新方法。方法：1.常规收集对数生长期的hDPSCs，将密5度为1×10/ml的细胞悬液接种至6孔板中，每孔细胞悬液量为2ml，根据实验目的分为空白组（只有hDPSCs和培养基）、阴性对照组（转染腺病毒rAd-EGFP的hDPSCs）和实验组（转染腺病毒rAd-EGF的hDPSCs），待

4、24h细胞贴壁后转染腺病毒rAd-EGFP与rAd-EGF，在72h后采用RT-PCR检测腺病毒rAd-EGF转染hDPSCs后对EGFmRNA表达的影响情况。2.体外培养3hDPSCs，常规收集对数生长期的hDPSCs，将密度为4×10/ml的细胞悬液接种于96孔板中，每孔细胞悬液量为200μl，根据实验目的分为空白组、阴性对照组和实验组。待24h细胞贴壁后转染腺病毒rAd-EGFP与rAd-EGF，在1、3、5、7、9、11d的同一时点，采用MTT比色法观察腺病毒rAd-EGF转染hDPSCs后对其体外增殖能力的影响。3.常规收集对

5、数生长期的6hDPSCs，将密度为3×10/ml的细胞悬液接种于6孔板中，每孔细胞贴壁后转染腺病毒rAd-EGFP与rAd-EGF，继续培养48h，用流式细胞仪AnnexinV-FITC/PI法检测腺病毒rAd-EGF转染hDPSCs后的凋亡情况。结果:实验组hDPSCs中EGFmRNA的表达水平相对于阴性对照组与空白组均明显上调(P<0.05)。与阴性对照组与空白组比较，实验组hDPSCs体外增殖能力明显升高，第1-7天升高明显，有显著性差异(P<0.05)，但7天后减弱，无显著性差异(P＞0.05)。流式细胞AnnexinV-FIT

6、C/PI法结果显示在作用时间相同的情况下同阴性对照组与空白组相比，实验组hDPSCs体外凋亡指数明显降低(P<0.05)。结论:腺病毒rAd-EGF转染入hDPSCs，EGFmRNA的表达量增加，在短期内对其增殖能力具有促进作用，对其凋亡具有抑制作用。关键词：hDPSCs；腺病毒rAd-EGF；腺病毒rAd-EGFP；凋亡；细胞增殖；EGF；TheEffectofRecombinantadenovirusofrAd一EGFTransfectionwithhDPSCsonProliferationInvitroAbstract:bject

7、ive:byculturinghumandentalpulpstemcellsonvitro,weobservetheEffectofRecombinantadenovirusofrAd一EGFTransfectionwithhDPSCsonitsgrowth、proliferationandapoptosis,whichcouldnotonlyprovideexperimentalevidencefortheroleofEGFonhDPSCs,butalsocanpossiblenewideasandnewmethodsofdenta

8、ldefectsandtoothregeneration.Methods:1Thecellsexponentialphaseofgrowth,whichwereRoutine5collection,were