糖基化终末产物诱导大鼠视网膜微血管内皮细胞表型和功能改变
당기화종말산물유도대서시망막미혈관내피세포표형화공능개변
Phenotypic and functional changes of rat retinal microvascular endothelial cells induced by advanced glycation end products
  • 万方数据
    中华糖尿病杂志 중화당뇨병잡지
    CHINES JOURNAL OF DLABETES MELLITUS
    2010年 5期 362-366 ,共5页

    许世清%姜永玮%王慧%李鸿%娄晋宁%张文健

    허세청%강영위%왕혜%리홍%루진저%장문건

    糖基化终末产物%视网膜内皮细胞%细胞凋亡%表型%功能 糖基化終末產物%視網膜內皮細胞%細胞凋亡%錶型%功能
    당기화종말산물%시망막내피세포%세포조망%표형%공능
    Advanced glycation end products%Retinal endothelial cell%Apoptosis%Phenotype%Function
    目的 研究糖基化终末产物(AGEs)诱导大鼠视网膜微血管内皮细胞表型和功能的变化及相关机制.方法 SPF级雄性Wistar大鼠20只,7～8周龄,体重260～280 g,分离培养大鼠视网膜内皮细胞,采用免疫荧光染色、流式细胞术和体外形成毛细血管样网络结构的方法进行鉴定.视网膜内皮细胞在AGEs刺激后,用噻唑蓝(MTT)法分析细胞的增殖能力;用膜连蛋白V/碘化丙啶(Annexin V/PI)双染法检测细胞凋亡;采用逆转录聚合酶链反应(RT-PCR)和流式细胞术检测细胞AGEs受体、人蛋白激酶C(PKC)、细胞间黏附分子1(ICAM-1)和诱导型一氧化氮合酶(iNOS)的表达变化.采用t检验进行统计学分析.结果 分离纯化的大鼠视网膜内皮细胞表达血管性血友病因子(vWF)并在人工基膜上形成毛细血管网络结构.AGEs以时间和剂量依赖的方式抑制大鼠视网膜内皮细胞增殖能力,在200 mg/L的AGEs组培养至第5天时细胞增殖能力低于对照组(t=8.9,P＜0.05),7 d和9 d时抑制作用更明显(t值分别为15.7和46.1,均P＜0.01).400 mg/L AGEs组在第3天开始出现细胞增殖减慢(t=12.5,P＜0.05),从第5天开始增殖速度明显低于对照组(t值分别为22.4、41.5和77.7,均P＜0.01).并且AGEs诱导视网膜内皮细胞凋亡.进一步分析发现AGEs上调了大鼠视网膜内皮细胞AGEs受体、PKC、ICAM-1和iNOS的mRNA表达(t值分别为91.8、9.22、16和42,均P＜0.01)和蛋白水平的表达(t值分别为20.2、12.3、7.7和13.9,均P＜0.01).结论 AGEs可能通过上调AGEs受体诱导大鼠视网膜微血管内皮细胞表型和功能的改变.
    목적 연구당기화종말산물(AGEs)유도대서시망막미혈관내피세포표형화공능적변화급상관궤제.방법 SPF급웅성Wistar대서20지,7～8주령,체중260～280 g,분리배양대서시망막내피세포,채용면역형광염색、류식세포술화체외형성모세혈관양망락결구적방법진행감정.시망막내피세포재AGEs자격후,용새서람(MTT)법분석세포적증식능력;용막련단백V/전화병정(Annexin V/PI)쌍염법검측세포조망;채용역전록취합매련반응(RT-PCR)화류식세포술검측세포AGEs수체、인단백격매C(PKC)、세포간점부분자1(ICAM-1)화유도형일양화담합매(iNOS)적표체변화.채용t검험진행통계학분석.결과 분리순화적대서시망막내피세포표체혈관성혈우병인자(vWF)병재인공기막상형성모세혈관망락결구.AGEs이시간화제량의뢰적방식억제대서시망막내피세포증식능력,재200 mg/L적AGEs조배양지제5천시세포증식능력저우대조조(t=8.9,P＜0.05),7 d화9 d시억제작용경명현(t치분별위15.7화46.1,균P＜0.01).400 mg/L AGEs조재제3천개시출현세포증식감만(t=12.5,P＜0.05),종제5천개시증식속도명현저우대조조(t치분별위22.4、41.5화77.7,균P＜0.01).병차AGEs유도시망막내피세포조망.진일보분석발현AGEs상조료대서시망막내피세포AGEs수체、PKC、ICAM-1화iNOS적mRNA표체(t치분별위91.8、9.22、16화42,균P＜0.01)화단백수평적표체(t치분별위20.2、12.3、7.7화13.9,균P＜0.01).결론 AGEs가능통과상조AGEs수체유도대서시망막미혈관내피세포표형화공능적개변.
    Objective To investigate whether the advanced glycation end products (AGEs) can induce the apoptosis of rat retinal microvascular endothelial cells and the related mechanisms. Methods Twenty SPF male Wistar rats aged 7 to 8 weeks were used. Microvascular endothelial cells was isolated from rat retinal, and identified by immunofluorescence staining and flow cytometry for vWF, and the tube formation on Matrigel. MTT assay was used to analyze the effect of AGEs on the proliferation of rat retinal microvascular endothelial cells. Flow cytometry was used to detect AGEs-induced apoptosis. Reverse transcription PCR and flow cytometry were used to analyze the expression level of RAGE, PKC, ICAM-1 and iNOS with or without AGEs in culture medium. t-test were used for statistical analysis. Results The isolated and purified rat retinal microvascular endothelial cells express vWF, and can form capillary-like network structure in Matrigel. AGEs can inhibit proliferation of rat retinal endothelial cells in dose- and timedependent ways: the proliferation of endothelial cells in 200 mg/L AGEs group was lower than control at 5 -day(t =8.9, P ＜0.05) ,and more significant at 7- and 9-day (t = 15.7 or 46.1, both P ＜0.01 ). The inhibition to proliferation of endothelial cells by 400 mg/L AGEs was significant at 3-day ( t = 12.5, P ＜0.05 ), and became greatly at 5-, 7- and 9-day ( t = 22.4, 41.5 or 77.7, all P ＜ 0.01 ). AGEs also induced apoptosis of retinal endothelial cells. Furthermore, AGEs can up-regulate the expression of RAGE, PKC,iNOS and ICAM-1 at mRNA level(t = 91.8, 9.22, 16 and 42, both P ＜ 0.01 ) and protein level (t = 20.2,12.3, 7.7 and 13.9, all P ＜0.01 ). Conclusion AGEs might induced phenotypic and functional changes of rat retinal microvascular endothelial cells by up-regulating RAGE.
    원문보기 원문다운로드 사이트로이동
저자의 최근 논문