重组腺病毒介导的人表皮生长因子基因体外转染人牙髓干细胞的研究

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1、中图分类号：R781.05；R329.28硕士学位论文重组腺病毒介导的人表皮生长因子基因体外转染人牙髓干细胞的研究院（系）、所口腔医学院研究生姓名鲍艳学科、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1.重组腺病毒介导的人表皮生长因子基因体外转染人牙髓干细胞的研究„„„„„„„„„„„„„„„„„„„„„1.1中文摘要„„„„„„„„„„„„„„„„„„„„„1.2英文摘要„„„„„„„„„„„„„„„„„„„„„1.3前言„„„„„„„„„„„„„„„„„„„„„„„1.3材料与方法„„„„„„„„„„„„„„„„„„„„1.5结果„„„„„„„„„„„„„„„„„„„„„„

2、„1.6讨论„„„„„„„„„„„„„„„„„„„„„„„1.7结论„„„„„„„„„„„„„„„„„„„„„„„1.8参考文献„„„„„„„„„„„„„„„„„„„„„1.9英汉缩略词对照表„„„„„„„„„„„„„„„„„2致谢„„„„„„„„„„„„„„„„„„„„„„„3矿化诱导剂与牙髓干细胞体外增殖及骨向分化能力（综述）„„„„„„„„„„„„„„„„„„„„„„„„„四川省卫生厅科研基金资助项目:编号(060051)重组腺病毒介导的人表皮生长因子基因体外转染人牙髓干细胞的研究摘要目的：本实验将EGF基因CDS片段亚克隆至腺病毒穿梭载体pYr-adshuttle-1，在体外重

3、组至腺病毒载体pAd/BL-DEST，构建重组腺病毒载体，并转染HEK293细胞进行腺病毒包装，获得重组腺病毒rAd-EGF。将重组腺病毒rAd-EGF在体外感染指数生长期人牙髓干细胞，分析人牙髓干细胞被转染后EGF蛋白的表达情况，为后续实验提供材料及数据。方法：在体外用含有10%胎牛血清的DEME培养基传代培养人牙髓干细胞至第三代，镜下观察细胞贴壁及生长情况。双酶切pCR4-TOPO-EGF及腺病毒穿梭载体pYr-adshuttle-1，1%琼脂糖凝胶电泳检测后分别回收EGF片段的带和线状pYr-adshuttle-1载体带，连接转化感受态大肠杆菌DH5α，并进行扩增，提取重组质粒p

4、Yr-adshuttle-1-EGF，进行酶切鉴定。将鉴定正确的重组质粒采用体外同源重组将EGF表达框亚克隆至腺病毒表达载体pAd/PL-DEST，XbaI酶切重组腺病毒载体pAd-EGF，进行酶切鉴定。用PacI酶切pAd-EGF使重组腺病毒载体线性化后转染包装细胞HEK293，包装成重组腺病毒rAd-EGF，PCR鉴定并测定病毒滴度。感染前24h取指数生长期的人牙髓干细胞根据实验目的将其分为3组：重组腺病毒rAd-EGF感染组、对照腺病毒rAd-NC阴性对照组、空白对照组，当细胞密度达到70%时，感染组和阴性对照组加入100MOI重组腺病毒或对照腺病毒，空白组加等量DEME培养液，

5、置于37℃、5%C02培养箱中培养4小时，更换培养基为含10%胎牛血清的DMEM，培养48h后胰酶消化收集各组细胞进行Western-blot，检测基因转染后各组EGF的蛋白表达差异。结果：人牙髓干细胞在镜下成梭形或多角形，2~3个不等突起，生长情况良好。pYr-ads-1-EGF经KpnI、XhoI酶切鉴定分析表明腺病毒穿梭载体pYr-adshuttle-1-EGF构建成功。pAd-EGF进行XbaI酶切鉴定和测序鉴定，确定和目的序列一致，腺病毒载体pAd-EGF构建成功。经PCR鉴定，重组腺病毒rAd-EGF包装成功。重组腺病毒rAd-EGF感染DPSC细胞48h后，Western

6、-blot检测各组EGF的蛋白表达，结果显示重组腺病毒rAd-EGF感染组的EGF的蛋白表达水平均显著高于其他两对照组(p<0.05)。结论：体外成功培养人牙髓干细胞至第三代，细胞生长良好，增殖情况正常。酶切鉴定表明成功构建了重组腺病毒穿梭载体pYr-adshuttle-1-EGF和重组腺病毒载体pAd-EGF后，采用HEK293细胞进行腺病毒包装，最终获得重组腺病毒rAd-EGF。采用重组腺病毒rAd-EGF成功转人牙髓干细胞后，高表达EGF蛋白。说明腺病毒是一种有效的转染载体，人牙髓干细胞是一种理想的基因载体细胞，腺病毒介导的EGF基因可有效转染人牙髓干细胞。关键词：人表皮生长因子

7、人牙髓干细胞腺病毒转染ResearchonAdenovirusmediatedhumanepidermalgrowthfactorgenetransfecthumandentalpulpstemcellinvitroAbstractObjective:Thisexperimentistoculturethehumandentalpulpstemcellstothethirdgenerationoftheexponentialgrowthphas