中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2012年
11期
1013-1017
,共5页
王业青%李霞%吕勇%王健%张晓梅
王業青%李霞%呂勇%王健%張曉梅
왕업청%리하%려용%왕건%장효매
基质细胞衍生因子-1%整合素连接激酶%视网膜色素上皮细胞%CoCl2%缺氧
基質細胞衍生因子-1%整閤素連接激酶%視網膜色素上皮細胞%CoCl2%缺氧
기질세포연생인자-1%정합소련접격매%시망막색소상피세포%CoCl2%결양
Stromal cell-derived factor-1%Integrin-linked kinase%Retinal pigment epithelium cell%CoCl2%Hypoxia
背景 缺氧是诱导新生血管形成的主要因素,近年来越来越多的研究证实基质细胞衍生因子-1(SDF-1)和整合素连接激酶(ILK)在新生血管性疾病中发挥着重要作用,而缺氧对视网膜色素上皮(RPE)细胞SDF-1和ILK表达的影响尚未阐明. 目的 探讨缺氧对体外培养RPE细胞中SDF-1和ILK表达的影响.方法 从4周龄SPF级健康C57BL/6小鼠眼球中获取RPE组织并进行消化和培养,将达到80％融合的细胞进行传代.取第3代细胞制作细胞爬片后用细胞角蛋白18(CK18)抗体进行免疫组织化学检测和鉴定,以5×104个/ml密度将RPE细胞接种于含胎牛血清的DMEM/F12培养液的培养瓶中,无血清饥饿培养24 h后常氧组在常规培养基中进行培养,缺氧组在CoCl2浓度为200 μmol/L的含胎牛血清的DMEM/F12培养液中进行培养,2个组根据培养时间的不同再亚分为1、3、6、12、24、48、72h组.采用逆转录PCR(RT-PCR)检测RPE细胞中SDF-1 mRNA和ILK mRNA的表达,采用Western blot检测RPE细胞中SDF-1蛋白和ILK蛋白的表达,均以目的基因A值/β-actin A值表示.结果 培养的RPE细胞呈不规则多边形,细胞质内布满黑色素颗粒,90％以上的细胞对CK18呈阳性反应.随着CoCl2培养时间的延长,SDF-1 mRNA和ILK mRNA的表达量逐渐增加,总体比较差异均有统计学意义(SDF-1 mRNA:F=281.875,P=0.000;ILK mRNA:F=187.566,P=0.000),12h达高峰,之后略有降低,但仍高于常氧组,缺氧1h组SDF-1 mRNA及蛋白表达无明显变化;缺氧培养3、6、12、24、48、72 h组SDF-1 mRNA和ILK mRNA的表达量均明显高于常氧培养组,差异均有统计学意义(P＜0.01).随着CoCl2培养时间的延长,SDF-1蛋白和ILK蛋白的表达量逐渐增加,差异均有统计学意义(SDF-1:F=44.719,P=0.000; ILK:F=144.481,P=0.000).缺氧1h组SDF-1及ILK蛋白表达无明显变化;缺氧3、6、12、24、48、72 h组SDF-1蛋白、ILK蛋白表达明显升高,与常氧组比较差异均有统计学意义(P＜0.01). 结论 SDF-1和ILK参与了RPE细胞的缺氧反应,并且在缺血缺氧性视网膜病变的发生发展过程中可能发挥一定的作用.
揹景 缺氧是誘導新生血管形成的主要因素,近年來越來越多的研究證實基質細胞衍生因子-1(SDF-1)和整閤素連接激酶(ILK)在新生血管性疾病中髮揮著重要作用,而缺氧對視網膜色素上皮(RPE)細胞SDF-1和ILK錶達的影響尚未闡明. 目的 探討缺氧對體外培養RPE細胞中SDF-1和ILK錶達的影響.方法 從4週齡SPF級健康C57BL/6小鼠眼毬中穫取RPE組織併進行消化和培養,將達到80％融閤的細胞進行傳代.取第3代細胞製作細胞爬片後用細胞角蛋白18(CK18)抗體進行免疫組織化學檢測和鑒定,以5×104箇/ml密度將RPE細胞接種于含胎牛血清的DMEM/F12培養液的培養瓶中,無血清饑餓培養24 h後常氧組在常規培養基中進行培養,缺氧組在CoCl2濃度為200 μmol/L的含胎牛血清的DMEM/F12培養液中進行培養,2箇組根據培養時間的不同再亞分為1、3、6、12、24、48、72h組.採用逆轉錄PCR(RT-PCR)檢測RPE細胞中SDF-1 mRNA和ILK mRNA的錶達,採用Western blot檢測RPE細胞中SDF-1蛋白和ILK蛋白的錶達,均以目的基因A值/β-actin A值錶示.結果 培養的RPE細胞呈不規則多邊形,細胞質內佈滿黑色素顆粒,90％以上的細胞對CK18呈暘性反應.隨著CoCl2培養時間的延長,SDF-1 mRNA和ILK mRNA的錶達量逐漸增加,總體比較差異均有統計學意義(SDF-1 mRNA:F=281.875,P=0.000;ILK mRNA:F=187.566,P=0.000),12h達高峰,之後略有降低,但仍高于常氧組,缺氧1h組SDF-1 mRNA及蛋白錶達無明顯變化;缺氧培養3、6、12、24、48、72 h組SDF-1 mRNA和ILK mRNA的錶達量均明顯高于常氧培養組,差異均有統計學意義(P＜0.01).隨著CoCl2培養時間的延長,SDF-1蛋白和ILK蛋白的錶達量逐漸增加,差異均有統計學意義(SDF-1:F=44.719,P=0.000; ILK:F=144.481,P=0.000).缺氧1h組SDF-1及ILK蛋白錶達無明顯變化;缺氧3、6、12、24、48、72 h組SDF-1蛋白、ILK蛋白錶達明顯升高,與常氧組比較差異均有統計學意義(P＜0.01). 結論 SDF-1和ILK參與瞭RPE細胞的缺氧反應,併且在缺血缺氧性視網膜病變的髮生髮展過程中可能髮揮一定的作用.
배경 결양시유도신생혈관형성적주요인소,근년래월래월다적연구증실기질세포연생인자-1(SDF-1)화정합소련접격매(ILK)재신생혈관성질병중발휘착중요작용,이결양대시망막색소상피(RPE)세포SDF-1화ILK표체적영향상미천명. 목적 탐토결양대체외배양RPE세포중SDF-1화ILK표체적영향.방법 종4주령SPF급건강C57BL/6소서안구중획취RPE조직병진행소화화배양,장체도80％융합적세포진행전대.취제3대세포제작세포파편후용세포각단백18(CK18)항체진행면역조직화학검측화감정,이5×104개/ml밀도장RPE세포접충우함태우혈청적DMEM/F12배양액적배양병중,무혈청기아배양24 h후상양조재상규배양기중진행배양,결양조재CoCl2농도위200 μmol/L적함태우혈청적DMEM/F12배양액중진행배양,2개조근거배양시간적불동재아분위1、3、6、12、24、48、72h조.채용역전록PCR(RT-PCR)검측RPE세포중SDF-1 mRNA화ILK mRNA적표체,채용Western blot검측RPE세포중SDF-1단백화ILK단백적표체,균이목적기인A치/β-actin A치표시.결과 배양적RPE세포정불규칙다변형,세포질내포만흑색소과립,90％이상적세포대CK18정양성반응.수착CoCl2배양시간적연장,SDF-1 mRNA화ILK mRNA적표체량축점증가,총체비교차이균유통계학의의(SDF-1 mRNA:F=281.875,P=0.000;ILK mRNA:F=187.566,P=0.000),12h체고봉,지후략유강저,단잉고우상양조,결양1h조SDF-1 mRNA급단백표체무명현변화;결양배양3、6、12、24、48、72 h조SDF-1 mRNA화ILK mRNA적표체량균명현고우상양배양조,차이균유통계학의의(P＜0.01).수착CoCl2배양시간적연장,SDF-1단백화ILK단백적표체량축점증가,차이균유통계학의의(SDF-1:F=44.719,P=0.000; ILK:F=144.481,P=0.000).결양1h조SDF-1급ILK단백표체무명현변화;결양3、6、12、24、48、72 h조SDF-1단백、ILK단백표체명현승고,여상양조비교차이균유통계학의의(P＜0.01). 결론 SDF-1화ILK삼여료RPE세포적결양반응,병차재결혈결양성시망막병변적발생발전과정중가능발휘일정적작용.
Background Hypoxia is a crucial factor of neovascularization.Many researches found that stromal cell-derived factor-1 (SDF-1) and integrin-linked kinase (ILK) play an important role in the neovascular disease.However,effect of SDF-1 and ILK in eye neovascular disease is below understood.Objective The aim of this study was to investigate the effect of hypoxia on the expressions of SDF-1 and ILK in cultured retinal pigment epithelium(RPE) cells in vitro.Methods RPE tissue was isolated from 4-week-old C57BL/6 mouse and was digested and cultured in DMEM/F12 with 10％ fetal bovine serum (FBS).The cells with 80％ confluence were collected and passaged.The third generation of cells were identified with cytokeratin 18 (CK18) antibody by immunochemistry.The cells were inoculated at the density of 5×104 cells/ml to free-serum DMEM/F12 for 24 hours and then were cultured in regular medium in the normoxic control group.RPE cells were cultured for 1 hour and 3,6,12,24,48,72 hours with 200 μmol/L CoCl2 in the hypoxia group.Reverse transcription-PCR(RT-PCR) was used to evaluate the expressing change of SDF-1 mRNA and ILK mRNA in RPE cells,and Western blot was used to assay the expressing change of SDF-1 protein and ILK protein in RPE cells in different time points.The detected outcomes were represented as the ratio of target gene A value/β-actin A value.Results Cultured cells showed the polygon in shape with the black pigment granules in cytoplasm.Over 90％ cells were positive response for CK18.Expressions of the SDF-1 mRNA and ILK mRNA were increased in different time points after CoCl2 co-cultured(SDF-1 mRNA:F=281.875,P=0.000 ;ILK mRNA: F=187.566,P=0.000),with the highest expressing value in hypoxia at 12 hours.No significant change in the expression of SDF-1 mRNA and protein was found 1 hour after CoCl2 co-cultured,but expressions of SDF-1 mRNA and ILK mRNA were significantly higher in 3,6,12,24,48 and 72 hours than the normoxic control group(P＜0.01).The expressions of SDF-1 protein and ILK protein were gradually ascended with the time increase of CoCl2 co-culture,showing a significant difference among different time points(SDF-1: F=44.719,P =0.000 ; ILK: F =144.481,P =0.000),and the up-regulation of SDF-1 protein and ILK protein expression was seen mainly in 3,6,12,24,48 and 72 hours after CoCl2 co-cultured in comparison with the normoxic control group (P＜0.01).Conclusions SDF-1 and ILK are involved in the hypoxic response of RPE cells and may play a potential role in ischemic/hypoxic retinopathy.
