中脑星形胶质细胞源性神经营养因子在视网膜Müller细胞中诱导激活p44/42丝裂原活化蛋白激酶信号通路的实验研究  

MANF induced p44/42MAPK phosphorylation in retinal Müller cells

作　　者：裴雪婷 卢建民[2] Yiwen Li Rong Wen Pei Xueting;Lu Jianmin;Li Yiwen;Wen Rong(Beijing Tongren Eye Center,Beijing Tongren Hospital,Capital Medical University,Beijing Ophthalmology&Visual Science Key Laboratory^Beijing 100730,China;Department of Ophthalmology,First Affiliated Hospital,Dalian Medical University,Dalian 116011,China;Bascom Palmer Eye Institute University of Miami,Miami,FL 33136,USA)

机构地区：[1]首都医科大学附属北京同仁医院,北京同仁眼科中心,北京市眼科学与视觉科学重点实验室,100730 [2]大连医科大学第一附属医院眼科,116011 [3]美国迈阿密大学巴斯康•帕默眼科研究所,33136

出　　处：《中华眼科医学杂志（电子版）》2019年第6期328-334,共7页Chinese Journal of Ophthalmologic Medicine(Electronic Edition)

基　　金：国家自然科学基金项目(81300737);美国国立卫生研究院资助项目(R01EY015289,R01EY018586);美国佛罗里达州视觉希望计划(08KN-09,2KF02)

摘　　要：目的中脑星形胶质细胞源性神经营养因子(MANF)可能对视网膜光感受器细胞和视网膜神经节细胞具有保护性作用。但是其作用机制还不清楚。本研究探索MANF在视网膜上的作用通路,为其在眼科的应用奠定基础。方法选用Sprague-Dawley雄性大鼠25只,体重0.2~0.25 kg。采用数字表法,随机将大鼠分为未处理组和处理组。其中,未处理组5只,处理组左眼注射重组人MANF10μg (3μl),右眼注射磷酸盐缓冲液(PBS)3μl,分别于注射后0.5 h、1 h、2 h及4 h四个时间点取材视网膜,每个时间点5只大鼠。新生大鼠视网膜分离培养原代Müller细胞,100ng/ml MANF处理细胞后,在不同时间点收集细胞Western blot检测磷酸化p44/42丝裂原活化蛋白激酶(MAPK)的表达水平,冰冻切片免疫组化检测MANF诱导活化的磷酸化p44/42MAPK在视网膜上的定位。以Image J图像分析软件分析图像,Western blot条带密度比值和免疫荧光强度为计量资料以(x±s)表示;各时间点与未处理组组间,采用独立样本t检验的方法进行比较。结果 SD大鼠玻璃体腔注射MANF后,磷酸化p44/42MAPK的表达在0.5 h就开始明显升高,1 h达到高峰,较对照玻璃体腔注射PBS组和未处理组差异均具有统计学意义(t=21.18,4.41;P<0.05),然后逐渐下降,4 h降至未处理组水平。冰冻切片免疫组化检查结果显示磷酸化p44/42MAPK阳性的细胞位于视网膜内核层的Müller细胞,其荧光强度显著高于阴性未处理组,差异有统计学意义(t=11.41,P<0.05)。原代培养的Müller细胞经MANF处理后,磷酸化p44/42MAPK在5 min时较未处理组就有显著升高,差异有统计学意义(t=4.48,6.43;P<0.05),15 min达到高峰,差异有统计学意义(t=19.57,9.18;P<0.05),到30 min降至正常水平。结论 p44/42MAPK信号通路参与了MANF的神经保护作用机制。MANF直接作用于视网膜Müller细胞,可能通过与Müller细胞的相互作用起到细胞保护性作用。Objective Mesencephalic astrocyte-derived neurotrophic factor(MANF)is a potent neurotrophic factor for photoreceptors and retinal ganglion cells.But the mechanism underlying its neuroprotective effects is not well understood.Our present work investigate potential signaling mechanism and pathway underlying these action effects.Methods 25 Sprague-Dawley male rats(weight 0.2 to 0.25 kg)were selected.They were randomly divided into untreated group and 4 treated groups with different treating time.There were 5 rats in each group.In treated group,the left eyes of SD rats were injected intravitreally with 10μg MANF(3μl),and the right eyes were injected with 3μl PBS(phosphate buffered saline)as negative controls.Retinas were collected at 0.5 h,1 h,2 h,4 h after injection.Müller cells were isolated from rat retina and cultured in DMEM+10%fetal bovine serum.Cells were treated with MANF(100 ng/ml)and collected at given time points after addition of MANF.The levels of phospho-p44/42MAPK wereassessed by Western blot analysis.Immunohistochemical analysis using frozen section was employed to localize the MANF-induced phospho-p44/42MAPK.Images were analyzed using Image J analysis system.Relative density ratio of Western blot bands and immune fluorescence intensity were represented as means±standard errors(x±s).Independent sample t test was used to compare MANF treated and control groups.Results A dramatic significant increase in phospho-p44/42MAPK was detected 30 min after MANF injection into the vitreous,and increase to the peak at 1 hour(t=21.18,4.41;P<0.05),then declined to normal level at 4 hours.Immunostaining for phospho-p44/42MAPK detected a group of positive cells in the inner nuclear layer,with characteristics of Müller cells.The immune fluorescence intensity was significantly stronger than control group(t=11.41,P<0.05).The MANF-induced phospho-p44/42MAPK was confirmed in cultured Müller cells.Addition of MANF(100 ng/ml)to culture medium induced a significant increase in phospho-p44/42MAPK as soon as 5 min(t=4.4

关 键 词：中脑星形胶质细胞源性神经营养因子 P44/42丝裂原活化蛋白激酶信号通路 视网膜 Miiller细胞 

分 类 号：R774[医药卫生—眼科]