普罗布考对高糖培养人视网膜Müller细胞特化蛋白1/细胞骨架相关蛋白/核因子E2相关因子2/半胱氨酸连接酶催化亚基表达的影响  

Effects of probucol on high glucose-induced specificity protein 1/Keapl/Nrf2/glutamate-cysteine ligase catalytic in the cultured human miiller cells

作　　者：李陈香 艾诗禧 陈忠平[3] 周旭霞 Li Chenxiang;Ai Shibei;Chen Zhongping;Zhou Xuxia(Aier School of Ophthalmology, Central South University, Changsha 410015, China;The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518000, China;Changsha Aier Eye Hospital, Changsha 410015, China)

机构地区：[1]中南大学爱尔眼科学院,长沙410015 [2]中山大学附属第七医院眼科,深圳518067 [3]长沙爱尔眼科医院眼底病科,长沙410015

出　　处：《中华眼底病杂志》2019年第2期187-191,共5页Chinese Journal of Ocular Fundus Diseases

基　　金：湖南省白然科学基金(2018JJ2001);湖南省卫计委科研计划课题项目(C2015-69);爱尔眼科医院集团科技基金(AF1601D5).

摘　　要：目的观察普罗布考对高糖环境下人视网膜Müller细胞中特化蛋白1(SP1)/细胞骨架相关蛋白(Keap1)/核因子E2相关因子2(Nrf2)/半胱氨酸连接酶催化亚基(GCLC)表达的影响;初步探讨普罗布考的抗氧化作用。方法体外培养的人Müller细胞分为正常糖组(5.5 mmol/L)、高糖组(25.0 mmol/L)、正常糖+普罗布考组、高糖+普罗布考组;后两组中加入100 μmol/L普罗布考。免疫荧光染色法鉴定Müller细胞;免疫荧光染色法和实时RP-PCR(qRT-PCR)检测各组Müller细胞中SP1、Keap1、Nrf2、GCLC蛋白、mRNA的表达。两组间数据比较采用独立样本t检验。结果体外培养的人Müller细胞谷氨酰胺合成酶染色阳性率>95%。免疫荧光染色结果显示,Müller细胞中SP1、Keap1、Nrf2、GCLC蛋白均呈阳性表达。qRT-PCR结果显示,与正常糖组比较,高糖组Müller细胞中SP1(t=28.30,P<0.000)、Keap1(t=5.369,P=0.006)、Nrf2(t=10.59,P=0.001)mRNA表达显著上调,GCLC显著下调(t=4.633,P=0.010),差异均有统计学意义。与高糖组比较,高糖+普罗布考组Müller细胞中SP1(t=12.60,P=0.000)、Keap1(t=4.076,P=0.015)mRNA表达显著下调,Nrf2(t=12.90,P=0.000)、GCLC(t=15.96,P<0.000)mRNA表达显著上调,差异均有统计学意义。结论普罗布考通过抑制高糖诱导Müller细胞中SP1、Keap1的表达,上调Müller细胞中Nrf2、GCLC的表达,发挥抗氧化作用。Objective To observe the expression ofprobucol on high glucose-induced specificity protein 1(SP1), kelchlike ECH associated protein 1 (Keapl), NF-E2-related factor 2 (Nrf2) and glutamatecysteine ligase catalytic (GCLC) in the cultured human muller cells and preliminary study the antioxidation of the probucol on muller cells. Methods Primary cultured human muller cells were randomly divided into four groups: normoglycaemia group (5.5 mmol/L glucose), normoglycaemia with probucol group (5.5 mmol/L glucose+100 pmol/L probucol), hyperglycemia group (25.0 mmol/L glucose), hyperglycemia with probucol group (25.0 mmol/L glucose + 100 gmol/L probucol). Immunofluorescence staining was used to assess distribution of SPI, Keapl, Nrf2, GCLC in human Muller cells. SP1, Keap 1, Nrf2 and GCLC messenger RNA (mRNA) expression was evaluated by quantitative real-time RT-PCR (qRT-PCR). Independent sample t test was used to compare the data between the two groups. Results All muller cells expressed glutamine synthetase (>95%), which confirmed the cultured cells in vitro were the purification of generations of muller cells. The expressions of SP1, Keap 1, Nrf2, and GCLC protein were positive in human miiller cells. qRT-PCR indicated that SP1 (t=28.30, PV0.000), Keapl (t=5.369, P=0.006), and Nrf2 (t=10.59, P=0.001) mRNA in the hyperglycemia group increased obviously compared with the normoglycaemia group;GCLC (t=4.633, P=0.010) mRNA in the hyperglycemia group decreased significantly compared with the normoglycaemia group. However, SP1 (t=12.60、P=0.000) and Keapl (t=4.076、P=0.0l5) in the hyperglycemia with probucol group decreased significantly compared with the hyperglycemia group;Nrf2 (t=12.90, P=0.000) and GCLC (t=15.96, P<0.000) mRNA in the hyperglycemia with probucol group increased obviously compared with with the hyperglycemia group. Conclusion Probucol plays an antioxidant role by inhibiting the expression of SP1, Keapl and upregulating the expression of Nrf2, GCLC in muller cells induced by high glucose.

关 键 词：普罗布考/治疗应用 Spl转录因子 NF-E2相关因子2 谷氨酸-半胱氨酸连接酶 MÜLLER细胞 细胞骨架相关蛋白 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]