重组腺病毒rA一EGF转染hDPSCs对其增殖影响的体外研究.pdfVIP

目 录 重组腺病毒rAd-EGF 转染hDPSCs 对其增殖影响的体外研究 1 „„„„„„„„„„„„„„„„„„„„„„„„„1 1.1 中文摘要„„„„„„„„„„„„„„„„„„„„„1 1.2 英文摘要„„„„„„„„„„„„„„„„„„„„„4 1.3 前言„„„„„„„„„„„„„„„„„„„„„„„7 1.4 材料与方法„„„„„„„„„„„„„„„„„„„„14 1.5 结果„„„„„„„„„„„„„„„„„„„„„„„25 1.6 讨论„„„„„„„„„„„„„„„„„„„„„„„30 1.7 结论„„„„„„„„„„„„„„„„„„„„„„„36 1.8 参考文献„„„„„„„„„„„„„„„„„„„„„37 1.9 英汉缩略词对照表„„„„„„„„„„„„„„„„„42 2 致谢„„„„„„„„„„„„„„„„„„„„„„„44 3 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究 摘 要 目的：通过体外培养人牙髓干细胞（human dental pulp stem cells， 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 EGF mRNA 表达的影响情况。2.体外培养 3 hDPSCs，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200 μl，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. 常规收集对数生长期的 6 hDPSCs，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Annexin V-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 EGF mRNA 的表达水平相对于阴性对照组与空白组均明显上调 (P0.05)。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(P0.05)，但7 天后减弱，无显 著性差异(P＞0.05)。流式细胞Annexin V-FITC/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 (P0.05)。结论:腺病毒rAd-EGF 转染入hDPSCs， EGF mRNA 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； The Effect of Recombinant adenovirus of rAd 一EGF Transfection with hDPSCs on Proliferation In vitro A