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大鼠骨髓间充质干细胞在脉络膜新生血管微环境中分化的实验研究
【作者】 于伟泓；
【导师】 董方田；
【作者基本信息】 中国协和医科大学 ， 眼科， 2007， 博士
【摘要】 目的研究体外培养的大鼠骨髓间充质干细胞(Mesenchymal StemCells,MSCs)视网膜下移植后在脉络膜新生血管(Chroidal Neovascularization,CNV)微环境中的分化及其对CNV渗漏情况的影响。方法体外培养挪威棕色大鼠(Brown Norway,BN)MSCs并经流式细胞仪鉴定CD90(+)CD45(-)后,用Brdu进行标记。用氪绿激光视网膜光凝的方法建立25只大鼠双眼CNV模型,并经眼底荧光血管造影(FluoresceinceAngiography,FA)证实CNV生成。将25只大鼠随机分为5组,每组5只。每只大鼠右眼为实验眼,经玻璃体腔注入视网膜下腔MSCs,左眼视网膜下腔注射等量PBS做为对照。术后1,2,3,4,5周分别做FA检查后处死一组动物,摘除眼球行冰冻病理切片后分别用小鼠抗人BrdU单抗、FITC标记羊抗小鼠二抗和小鼠抗大鼠CD31单抗、小鼠抗大鼠Rhodopsin单抗、小鼠抗大鼠全角蛋白(Pan-Cytokeratin,PCK)单抗、Cy3标记羊抗小鼠二抗双重荧光标记。观察术后1,2,3,4,5周MSCs在CNV微环境中的分化,计算视网膜下腔注射MSCs/PBS前、后FA面积并比较CNV渗漏情况的变化。结果视网膜下移植术后第1周即可见MSCs位于CNV表面,但CD31标记阴性,第2周至第5周可见位于CNV内的MSCsCD31标记阳性,第5周可见移行于RPE层的MSCs rhodopsin和PCK标记阳性。FA分析结果初步表明,实验眼视网膜下移植MSCs后CNV的渗漏面积较对照眼有减少的趋势。结论MSCs移植入CNV模型大鼠视网膜下腔后第2周至第5周可以向内皮细胞方向分化,第5周可以向光感受器细胞方向和RPE方向分化。视网膜下移植MSCs后有可能使CNV渗漏情况得到一定的改善。
【Abstract】 Objective To access the differentiation of subretinal-transplanted rat mesenchymal stem cells （MSCs） in the microenvironment of choroidal neovascularization （CNV） and changes of CNV exudation after the transplantation .Methods In-vitro cultured Brown Norway （BN） rats MSCs were identified CD90（+） CD45（-）by flow cytometry and labeled with Brdu. 25 CNV rats models were established by Krypton laser photocoagulation and subsequently confirmed by fluorescein angiography（FA）. 25 rats were randomly distributed into 5 groups, 5 rats in every group. The right eyes were injected with MSCs into subretinal space, while the left eyes were injected with the same amount of PBS. The rats were killed and globes enucleated 1,2,3,4 or 5 weeks after the surgery respectively. Frozen sections were stained with Brdu primary monoclonal antibody, FITC- labeled secondary monoclonal antibody and CD31 , rhodopsin or pan- cytokeratin（PCK） primary monoclonal antibody respectively combined with Cy3-labeled secondary monoclonal antibody . The sections were observed under fluorescent microscope. Calculate and compare the exudation areas change pre-and post-transplantatively by FA.Results MSCs distributed on the surface of CNV in the 1st week after surgery with CD31 negative.However, from the 2nd to 5th week, CD31 positive MSCs were found in CNV. Rhodopsin positive and PCKpositive MSCs were found near CNV in the 5th week. FA analysis demonstrated a mild decrease tendency of CNV exudation after the transplantation of MSCs.Conclusions The results demonstrates that MSCs have the potential to differentiate into endothelium from 2th to 5th weeks, into photoreceptor cells and RPE in 5th weeks after subretinal transplantation in the microenvironment of NCV.CNV exudation may be mildly improved after subretinal transplantation of MSCs.