兔眼与猪眼视网膜色素上皮细胞分离及培养方法的对比.pdfVIP

眼科研究 2010年 1月第 28卷第 l期 · 实 验 研 究 · 兔眼与猪眼视网膜色素上皮细胞分离及培养 方法的对 比 刘丽娅 马景学 高彦军 安建斌 温晓英 Comparisonofinvitroisolationandcultureofretinalpigmentepithelialcells in rabbiteyeandpigeye LiuLiya，MaJingxue，GaoYanjun，AnJianbin，WenXiaoying．DepartmentofOphthalmology，AffiliatedSecond HospitalofHebeiMedicalUniversity，Shijiazhuang050000，China Abstract Background Cultureofretinalpigmentepithelium (RPE) cellsisvery importantforestablishmentof proliferativeVitre0retinopathy(PVR)model，preventionandtreatmentofPVR aswellasRPEcelltransplantation．Isolationof animalRPEcellsbytrypsinizationisacriticalstep． Objective Thepresentstudyistoestablishthemethodsofisolationand cultureofretinalpigmentepithelium (RPE)cellsinrabbitandeompariedwiththatofpigRPEculture． Methods RPEcells wereisolatedbytrypsinization inpigmentedrabbitandpigandcultured inDMEM containing20％ fetalbovineserum．Cuhured RPE cellswere identified by immun0chemistrv．The fourth generationsofcellswere used in this experiment．Morphology and characteristicsofculturedRPE ceilsfrom rabbitandpigwereexaminedandcomparedunderthe lightmicroscope． Results IsolatedRPE cellsfrom pigwereobtainedby oncetrypsin digestion，buttwotimesoftrypsinizationwereneeded inrabbitRPE cellsisolation．Thedifferentiation in responsetotrypsinizationwasrelated toanatoulicdifferencebetweenthetwotypesofcells． TheadherencetimeofpigRPE cellswas24 hours ，however，therabbitRPE needed48—72hoursafterculture．Proliferationand vitality ofculturedcellsweregraduallyattenuatedandmelanindecreasedafterseveraltimessubculture．Themorphologyofculture RPE cellswasobviously differentbetween rabbitand pig because speciesdifference．Imnmnohistochemistry demonstrated the positiveresponseofRPE cellsforkeratin． Conclusion RPEcellscanbeacquiredfrom bothrabbitandpigbytrypsinization andculture．ThecultureprocessofRPE cellsofpigi