CAJ | 학술논문

背景视网膜色素上皮(RPE) 细胞和Müller细胞在维持视网膜的正常代谢中起重要作用,多种生理、病理过程与二者的相互作用有关.目的利用Transwell小室体外共培养兔Müller细胞与RPE细胞,观察缺氧条件下Müller细胞对RPE细胞增生和迁移的影响.方法应用组织块培养法和酶消化法分别培养原代兔Müller细胞和RPE细胞,免疫组织化学法鉴定2种细胞.取第3代培养良好的细胞进行试验.应用CoCl2培养细胞以建立细胞化学缺氧模型,分为常氧组和缺氧组,每组又分为对照组(RPE细胞单独培养)、共培养组(Müller细胞与RPE细胞共同培养).利用Transwell小室建立Müller细胞和RPE细胞的共培养模型,各组分别培养3、6、24、48h,利用MTT法和结晶紫染色法对培养存活的RPE细胞进行计数,检测常氧及缺氧条件下Müller细胞对RPE细胞增生和迁移的影响.结果原代培养的RPE细胞符合RPE细胞的特征,90%以上呈现CK-18阳性反应.培养的Müller细胞对胶质纤维酸性蛋白(GFAP)和S-100染色呈阳性反应.与常氧培养条件比较,缺氧条件下单独培养的RPE细胞增生数量增加,细胞迁移数量增加,差异有统计学意义(P＜0.01);缺氧条件下与Müller细胞共培养的RPE细胞增生数量和迁移数量明显增加,与常氧组和单细胞培养情况下比较,差异均有统计学意义(P＜0.01).缺氧条件下,与RPE细胞单细胞培养组比较,共培养组RPE细胞的增生和迁移增加,差异均有统计学意义(P＜0.01).结论缺氧可以促进RPE细胞的增生和迁移,Müller细胞可以加重缺氧引起的RPE细胞的增生和迁移.
배경 시망막색소상피(RPE) 세포화Müller세포재유지시망막적정상대사중기중요작용,다충생리、병리과정여이자적상호작용유관.목적 이용Transwell소실체외공배양토Müller세포여RPE세포,관찰결양조건하Müller세포대RPE세포증생화천이적영향.방법 응용조직괴배양법화매소화법분별배양원대토Müller세포화RPE세포,면역조직화학법감정2충세포.취제3대배양량호적세포진행시험.응용CoCl2배양세포이건립세포화학결양모형,분위상양조화결양조,매조우분위대조조(RPE세포단독배양)、공배양조(Müller세포여RPE세포공동배양).이용Transwell소실건립Müller세포화RPE세포적공배양모형,각조분별배양3、6、24、48h,이용MTT법화결정자염색법대배양존활적RPE세포진행계수,검측상양급결양조건하Müller세포대RPE세포증생화천이적영향.결과 원대배양적RPE세포부합RPE세포적특정,90%이상정현CK-18양성반응.배양적Müller세포대효질섬유산성단백(GFAP)화S-100염색정양성반응.여상양배양조건비교,결양조건하단독배양적RPE세포증생수량증가,세포천이수량증가,차이유통계학의의(P＜0.01);결양조건하여Müller세포공배양적RPE세포증생수량화천이수량명현증가,여상양조화단세포배양정황하비교,차이균유통계학의의(P＜0.01).결양조건하,여RPE세포단세포배양조비교,공배양조RPE세포적증생화천이증가,차이균유통계학의의(P＜0.01).결론 결양가이촉진RPE세포적증생화천이,Müller세포가이가중결양인기적RPE세포적증생화천이.
Background More and more evidences suggest that the interaction of retinal progression of retinal diseases.Objective Present study was to investigate the primarily cultured and digested using explant culture method and trypsas acidic protein(GFAP) staining,S-100 staining and cytokine-18 staining co-cultured under the normoxia and hypoxia using transwell chamber,and the proliferation and migration of cultured RPE cells were examined using MTT and compared with only RPE cells cultured group at 3, 6, 24 and 48 hours.Results Over 90% of primarily cultured RPE cells showed the positive response for S-100.The proliferation and migration of RPE cells were significantly increased in hypoxia condition compared with normoxia condition (P＜0.01).In hypoxia group,amount of proliferation and migration of RPE cells in co-culture group were higher than the only RPE cells cultured group(P＜0.01).Conclusion Hypoxia appear to aggravate the proliferation and migration of RPE cells under the hypoxia status.