大鼠骨髓间充质干细胞与神经生长因子的体外构建的实验研究  

Construction of rats mesenchymal stem ceils transfected with nerve growth factor in vitro

作　　者：施永彦[1,2] 喻爱喜[1] 张功礼[2] 余永桂[2] 江良波[2] 

机构地区：[1]武汉大学中南医院骨科,武汉市430071 [2]郧阳医学院附属人民医院显微骨科

出　　处：《中华显微外科杂志》2007年第1期32-34,I0002,共4页Chinese Journal of Microsurgery

基　　金：湖北省教育厅重点科研项目B类（B200624016）

摘　　要：目的探讨骨髓间充质干细胞（mesenchymal stem cells，MSCs）的体外构建表达神经生长因子（nerve growth factor，NGF）的能力，同时使MSCs被绿色荧光蛋白（green fluorescent protein，GFP）稳定标记，通过病毒载体将NGF和GFP基因高效转染到MSCs中。方法取2月龄100—150gWistar大鼠，全骨髓法分离培养纯化大鼠MSCs，利用携带人NGFβ基因的修复缺陷性重组腺病毒（AdhNGFβ）和携带GFP基因的复制缺陷性逆转录病毒（Rt-GFP）转染MSCs，荧光显微镜观察GFP阳性的MSCs，免疫细胞化学和Western-blot检测hNGFβ的表达。结果Rt-GFP转染后，80％-90％MSCs可激发出明亮的绿色荧光，且荧光不随培养时间延长和传代次数增加而衰减。Ad-hNGFβ的转染MSCs中hNGFβ阳性率（90．17±2．14）显著高于阴性对照组（2．17±0．75）和空白对照组（1．83±0．98．P〈0．01），转染后MSCs中NGFβ表达量（188．67±8．71）显著高于阴性对照组（25．67±4．08）和空白对照组（27．50±3．33，P〈0．01）。结论大鼠间充质干细胞的体外构建中Ad—hNGFβ可以高效转染MSCs，实现NGF在MSCs中高效表达，Rt—GFP可以使MSCs获得长效标记。Objective The nerve growth factor（NGF） and green fluorescent protein（GFP） gene were transfeeted into mesenchymal stem cells（MSCs） efficiently in vitro, this study transfected the NGF and green fluorescent protein（GFP） genes into MSCs by virus vectors and label MSCs by GFP. Methods In 2-month 100 -150 g Wistar rats, MSCs were isolated and purified. Human NGFβ（ hGNGβ ） and GFP genes were transfected into MSCs by replication-deficient recombinant adenovirus vector（Ad-hNGFβ） and replication-deficient recombinant retrovirus vector（Rt-GFP） respectiveley. GFP positive MSCs were observed by fluorescent microscopy. The hNGFβ expression of MSCs was detected by immunocytochemistry and Westeru-blot. Results The positive rates of MSCs in Ad-hNGFβ transfected group （90. 17 ± 2. 14 ） were significantly higher than that of negative control group（2. 17 ±0. 75） and those in blank control group（ 1.83 ±0. 98, P 〈0. 01 ）. Similarly, the gray scales of NGFβ Westeru-blot in transfected group（ 188.67± 8. 71 ） were also significantly higher than those in negative control group（25. 67 ±4. 08） and those in blank control group（27. 50 ±3.33 ,P 〈 0. 01 ）. Conclusion The constructed vector Ad-hNGFβ can be transfecterd MSCs efficiently and express NGF efficiently in MSCs. Rt-GFP can make MSCs be labeled in long-term.

关 键 词：骨髓间充质干细胞 组织工程 神经生长因子 体外构建 

分 类 号：R686[医药卫生—骨科学]