糖基化终产物对视网膜Mükker细胞表达碱性成纤维细胞生长因子的影响(1)
[摘要] 目的 探讨糖基化终产物(AGEs)对体外培养兔视网膜Müller细胞表达碱性成纤维细胞生长因子(bFGF)的影响。方法 体外分离并培养兔视网膜Müller细胞。37℃条件下制备牛血清白蛋白AGEs(AGEs-BSA)及其对照物。应用AGEs-BSA及其对照物作用于Müller细胞，采用免疫细胞化学(ICC)方法半定量检测AGEs对视网膜Müller细胞表达bFGF的影响。结果 与对照组相比，AGEs作用下体外培养的兔视网膜Müller细胞bFGF的表达增高(P且具有一定的时间和浓度依赖性。结论 AGEs可以上调Müller细胞表达bFGF，推测AGEs可能通过增加bFGF的表达从而加速糖尿病视网膜病变(DR)新生血管的形成。
[关键词] 糖基化终产物；碱性成纤维细胞生长因子；视网膜Müller细胞；免疫细胞化学
[中图分类号] R774.1;R-33 [文献标识码] A
, http://www.100md.com [文章编号] 1671-7562(2008)02-0107-04
Effect of advanced glycosylation end products on expression of basic
fibroblast growth factorin cultured rabbit retinal Müller cells
AI Jing1,LIU Yao1,LUAN Jie1,CHEN Ping-sheng2
(1.Department of Ophthalmology,Zhongda Hospital,Southeast University,Nanjing 210009,China;2.Department
of Pathology,School of Basic Medical Science,Southeast University,Nanjing 210009,China)
, 百拇医药
Abstract:Objective To investigate the effect of advanced glycosylation end products(AGEs)on expression of basic fibroblast growth factor(bFGF)in rabbit retinal Müller cells in vitro.Methods Rabbit retinal Müller cells were cultured according to the descriptions in literatures.AGEs-BSA and its control were made in 37℃ in vitro.Müller cells were divided into AGEs-BSA group,AGEs-BSA control group and blank control group.AGEs-BSA group and AGEs-BSA control group were respectively treated with 5 different concentration series of AGEs-BSA and AGEs-BSA control for1,3,6 and 9 days,while blank control group was incubated without any intervention.Then bFGF expression in Müller cells was half-quantitatively detected by immunocytochemistry(ICC).Results Comparing with blank control and AGEs-BSA control group,AGEs-BSA evoked a time and concentration-dependent increase of bFGF expression by retinal Müller cells.Conclusion AGEs might promote the neovascularization of diabetic retinopathy through up regulating the expression of bFGF in Müller cells.
, http://www.100md.com
Key words:advanced glycosylation end products;basic fibroblast growth factor;retinal Müller cells;immunocytochemistry
(Modern Medical Journal,2008,36:107-110)
糖尿病视网膜病变(diabetic retinopathy,DR)是糖尿病最常见、最严重的并发症之一。糖尿病患者葡萄糖代谢发生异常，产生大量糖基化终产物(advanced glycosylation end products,AGEs)。传统观点认为AGEs集中作用于视网膜血管系统，导致血-视网膜屏障功能的损害，进而产生不同程度的DR。视网膜Müller细胞在维持视网膜结构和功能上具有重要作用，但长期以来关于AGEs对该细胞作用的研究较少，而碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)在Müller细胞参与的视网膜多种病理过程中具有重要作用。因此，本实验拟通过观察AGEs对视网膜Müller细胞表达bFGF的影响，进一步探讨DR的发生机制。
, 百拇医药
1 材料和方法
1.1 细胞培养
参照刘瑶等[1]报道的方法并稍加改进进行Müller细胞的分离培养并鉴定。简述如下：取健康新西兰大白兔，重约2.0~2.5kg，雌雄不限(由东南大学医学院动物实验中心提供)。经耳缘静脉注入空气处死，无菌条件下摘除眼球，快速解剖分离视网膜，制成微小组织块进行贴壁培养。细胞培养液为含20%胎牛血清的DMEM培养液。培养环境为恒温恒湿培养箱(37℃，体积分数为5%CO2)。
1.2 牛血清白蛋白糖基化终产物(AGEs-BSA)及AGEs-BSA对照物的制备与检测
AGEs-BSA的制备参照我校心血管研究所制备AGEs-BSA的方法进行[2]。AGEs-BSA对照物的制备，除不加无水葡萄糖外，其他与制备AGEs-BSA相同。孵育完成后，将AGEs-BSA及其对照物用荧光分光光度计(Varian Cary Eclipse,美国)测定其荧光值(pH 7.4，0.2μmol•L-1 PBS用于调零，激发波长360nm，峰峡5nm，发射波长450nm)。AGEs-BSA的荧光强度较AGEs-BSA对照物约高12倍，证实AGEs-BSA制备成功。最后将制备物用0.2μm针头滤器过滤灭菌封口后置4℃冰箱中待用。
1.3 AGEs-BSA作用观察, 百拇医药(艾 静 刘 瑶 栾 洁 陈平圣)
[关键词] 糖基化终产物；碱性成纤维细胞生长因子；视网膜Müller细胞；免疫细胞化学
[中图分类号] R774.1;R-33 [文献标识码] A
, http://www.100md.com [文章编号] 1671-7562(2008)02-0107-04
Effect of advanced glycosylation end products on expression of basic
fibroblast growth factorin cultured rabbit retinal Müller cells
AI Jing1,LIU Yao1,LUAN Jie1,CHEN Ping-sheng2
(1.Department of Ophthalmology,Zhongda Hospital,Southeast University,Nanjing 210009,China;2.Department
of Pathology,School of Basic Medical Science,Southeast University,Nanjing 210009,China)
, 百拇医药
Abstract:Objective To investigate the effect of advanced glycosylation end products(AGEs)on expression of basic fibroblast growth factor(bFGF)in rabbit retinal Müller cells in vitro.Methods Rabbit retinal Müller cells were cultured according to the descriptions in literatures.AGEs-BSA and its control were made in 37℃ in vitro.Müller cells were divided into AGEs-BSA group,AGEs-BSA control group and blank control group.AGEs-BSA group and AGEs-BSA control group were respectively treated with 5 different concentration series of AGEs-BSA and AGEs-BSA control for1,3,6 and 9 days,while blank control group was incubated without any intervention.Then bFGF expression in Müller cells was half-quantitatively detected by immunocytochemistry(ICC).Results Comparing with blank control and AGEs-BSA control group,AGEs-BSA evoked a time and concentration-dependent increase of bFGF expression by retinal Müller cells.Conclusion AGEs might promote the neovascularization of diabetic retinopathy through up regulating the expression of bFGF in Müller cells.
, http://www.100md.com
Key words:advanced glycosylation end products;basic fibroblast growth factor;retinal Müller cells;immunocytochemistry
(Modern Medical Journal,2008,36:107-110)
糖尿病视网膜病变(diabetic retinopathy,DR)是糖尿病最常见、最严重的并发症之一。糖尿病患者葡萄糖代谢发生异常，产生大量糖基化终产物(advanced glycosylation end products,AGEs)。传统观点认为AGEs集中作用于视网膜血管系统，导致血-视网膜屏障功能的损害，进而产生不同程度的DR。视网膜Müller细胞在维持视网膜结构和功能上具有重要作用，但长期以来关于AGEs对该细胞作用的研究较少，而碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)在Müller细胞参与的视网膜多种病理过程中具有重要作用。因此，本实验拟通过观察AGEs对视网膜Müller细胞表达bFGF的影响，进一步探讨DR的发生机制。
, 百拇医药
1 材料和方法
1.1 细胞培养
参照刘瑶等[1]报道的方法并稍加改进进行Müller细胞的分离培养并鉴定。简述如下：取健康新西兰大白兔，重约2.0~2.5kg，雌雄不限(由东南大学医学院动物实验中心提供)。经耳缘静脉注入空气处死，无菌条件下摘除眼球，快速解剖分离视网膜，制成微小组织块进行贴壁培养。细胞培养液为含20%胎牛血清的DMEM培养液。培养环境为恒温恒湿培养箱(37℃，体积分数为5%CO2)。
1.2 牛血清白蛋白糖基化终产物(AGEs-BSA)及AGEs-BSA对照物的制备与检测
AGEs-BSA的制备参照我校心血管研究所制备AGEs-BSA的方法进行[2]。AGEs-BSA对照物的制备，除不加无水葡萄糖外，其他与制备AGEs-BSA相同。孵育完成后，将AGEs-BSA及其对照物用荧光分光光度计(Varian Cary Eclipse,美国)测定其荧光值(pH 7.4，0.2μmol•L-1 PBS用于调零，激发波长360nm，峰峡5nm，发射波长450nm)。AGEs-BSA的荧光强度较AGEs-BSA对照物约高12倍，证实AGEs-BSA制备成功。最后将制备物用0.2μm针头滤器过滤灭菌封口后置4℃冰箱中待用。
1.3 AGEs-BSA作用观察, 百拇医药(艾 静 刘 瑶 栾 洁 陈平圣)