简介：AIM:ToinvestigatetheroleofBrn-3bindifferentiationprocessofstemcellsderivedfromretinalMiillercellsintotheganglioncell.METHODS:ThepassageculturemethodofMiillercellsfromretinaofnewbornSpragueDawleyratswascarriedoutbyrepeatedincompletepancreaticenzymedigestionmethod.Thecellsweredetectedbyfluorescenceactivatedcellsorter(FACS),immunohistochemistrytechnologyandreversetranscription-polymerasechainreaction(RT-PCR)todeterminethepurity.Thethirdpassageofcellswasinducedintheserum-freededifferentiationmedium.TheexpressionofthespecificmarkersKi-67andnestinofretinalstemcellswasmeasuredbyRT-PCRandWesternblot.Thecellproliferationofretinalstemcellswasdetectedby5-Ethynyl-2’-deoxyuridine(Edu)staining.Thecellswererandomlydividedinto5groupsasfollows:groupA:Brn-3bsiRNAgroup;groupB:Brn-3bcontrolsiRNAgroup;groupC:pGC-Brn-3b-greenfluorescentprotein(GFP)group;groupD:pGC-GFPgroup;groupE:controlgroup(withoutanyhandling).ThepurifiedMullercellswereculturedfor3-7d,then,thepercentageofganglioncellswascountedbyimmunofluorescencestaining.RESULTS:FACSdemonstratedthepurityofretinalMullercellswasmore97.44%.Afewsphericalcellspheresappeared.Immunofluorescencestainingshowedthatstemcellswithinthesphereswerepositiveforretinalstemcell-specificmarkersnestin(redfluorescence,92.94%±6.48%)andKi-67(greenfluorescence,85.96%±6.04%).Meanwhile,RT-PCRanalysisshowedcellspheresintheculturetohaveexpressedabatteryoftranscriptscharacteristicofstemcellssuchasnestinandKi-67,whichwereabsentintheMullercells.WesternblotanalysisfurtherconfirmedtheexpressionofnestinandKi-67inthecellspheresbutnotintheMullercells.Edustainingshowedmostofthenucleiwithinthecellsphereswerestainedred(82.80%±6.65%),suggestingthenewcellsphereshadthecapacityforeffectiveproliferation.ThestatisticsresultshowedthedifferencebetweenBrn-3bsiRNAgroupand

简介：目的观察1,25（OH）2D3对高糖诱导牛视网膜血管内皮细胞（BRECs）中血管内皮生长因子（VEGF）的表达水平变化及对细胞凋亡水平的影响。方法将分离培养的BRECs分为三组,分别为正常糖组、高糖组和高糖处理组。正常对照组细胞培养液含5mmol/L葡萄糖,高糖组细胞培养液含30mmol/L葡萄糖,高糖处理组细胞培养液含30mmol/L葡萄糖和50nM,1,25（OH）2D3。培养48h后收集细胞蛋白。蛋白免疫印迹法检测细胞VEGF及细胞凋亡相关蛋白Bax和Bcl-2表达水平;PI/Hoechst双染色法检测细胞凋亡。结果相比于正常糖组,高糖组中VEFG水平和Bax/Bcl-2比值显著增加;而在高糖处理组中表达水平远远低于高糖组,差异均有统计学意义（P〈0.05）。高糖的细胞凋亡水平高于正常糖组,而经过1,25（OH）2D3处理后,其细胞凋亡水平则有所下降,差异均有统计学意义（P〈0.05）。结论1,25（OH）2D3可以抑制高糖诱导BRECs中VEGF表达增加及细胞凋亡。