骨髓间充质干细胞在碘酸钠注射大鼠视网膜下的分化的研究

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1、骨髓间充质干细胞在碘酸钠注射大鼠视网膜下的分化研究摘要目的研究体外培养的大鼠骨髓间充质干细胞(MSCs)移植到碘酸钠注射大鼠的视网膜下后的存活、分化情况。方法采用全骨髓培养法体外培养Wistar大鼠的MSCs，并经流式细胞仪鉴定后，用病毒上清液转染EGFP标记MSCs。经尾静脉注射4%NaIO340mg/kg破坏Wistar大鼠的RPE细胞。25只大鼠右眼为实验眼，经玻璃体腔注射MSCs细胞悬液到视网膜下，左眼为对照眼，视网膜下腔注射等量培养液。发生大量视网膜下出血或玻璃体出血的大鼠弃去不用。术

2、后1周、5周分别处死10只大鼠，摘除眼球行冰冻切片后用免疫荧光显微镜直接观察MSCs在视网膜内的分布，并用免疫荧光染色的方法观察术后5周MSCs的分化情况。结果注射过NaIO3的大鼠视网膜发生变性，RPE细胞数量减少，光感受器细胞外节出现空泡样结构，厚度变薄，外核层光感受器细胞数量减少。MSCs的EGFP转染比例可达到76.9%，术后第一周可见大量MSCs整合入视网膜位于光感受器细胞层，五周后可观察到的细胞数量大幅减少，主要迁移至视网膜下区域，并表达角蛋白（pan-CK）、视紫红质（rhodop

3、sin）和胶质纤维酸性蛋白（GFAP）。结论MSCs植入碘酸钠注射大鼠视网膜下腔后可存活，并表达RPE细胞、光感受器细胞和星形胶质细胞的特异性标志。关键词骨髓间充质干细胞，视网膜移植，碘酸钠，大鼠，分化DIFFERENTIATIONOFMSCsINTHESUBRETINALSPACEOFRATSINJECTEDWITHSODIUMIODATEABSTRACTPurpose:Toinvestigatethedifferentiationofratbonemarrowmesenchymalstemc

4、ells(MSCs)inaretinalpigmentosamodelinducedbytheadministrationofsodiumiodate.Methods:WistarratsMSCsculturedinvitrowereselectedbyadherencetotheculturedish,identifiedbyCD90(+)CD45(-)throughflowcytometryandlabeledwithEGFP.28Wistarratsretinitispigmentosam

5、odelswereestablishedby4%Nal03injectionthroughacaudalveinandsubsequentlyconfirmedbypathologicalsection.4daysaftertheadministrationofsodiumiodate,therighteyesof25ratswereinjectedwithMSCsintothesubretinalspace，whilethelefteyeswereinjectedwiththesameamou

6、ntofDMEMascontrol.Aftertheinjection,andeyesshowingmassivesubretinalhemorrhageorvitreoushemorrhagewereexcluded.Every10ratswerekilledandeyeglobeswereenucleatedlor5weeksafterthesurgeryseparately.Frozensectionswereobservedunderfluorescentmicroscopeandana

7、lysedbyimmunofluorescence5weekslater.Results:AfterNaIO3wasinjected,theouter-nuclearlayerwasdisorganizedandthephotoreceptorcellshaddirectcontactwiththeBruch’smembranewithareductioninthenumberofRPEandphotoreceptorcellunclei(outernuclearlayer).OSthinned

8、andvacuoleswerefoundinOS.ThetransductionefficiencyofEGFPwas76.9%.ViableMSCsweredetectedtohaveintegratedintotheneuralretina1weekafterthetransplantation.5weekslaterthenumberoftransplantedMSCsdeclinedsignifcantly,andmostoftheMSCsmigratedintothesubretina