血脂异常ApoE基因缺失小鼠视网膜色素上皮细胞胞浆内黑色素和脂褐素的改变

Changes of melanin and lipofuscin in retinal pigment epithelium intracytoplasm of mice with dyslipidemia and apolipoprotein E-deficiency

目的探讨血脂异常载脂蛋白E(apolipoprotein E,ApoE)基因缺失(ApoE-/-)小鼠视网膜色素上皮(retinal pigment ep-ithelium,RPE)细胞胞浆内黑色素和脂褐素的改变。方法将24只2个月龄ApoE-/-小鼠随机分为高脂组和普食组,每组12只,另用12只2个月龄C57BL小鼠作为普食组,喂养4个月后检测其血浆总胆固醇(total plasma cholesterol,TC)、低密度脂蛋白(low density lipoprotein,LDL)、甘油三酯(plasma triglyceride,TG)含量,Masson三色、银氨液浸染法和醛品红法染色后光镜下观察视网膜光感受器细胞(photoreceptor cell,PRC)、RPE细胞、Bruch膜形态结构,半定量检测视网膜PRC数、RPE细胞数、Bruch膜厚度、RPE细胞胞浆内黑色素和脂褐素含量,采用逐步引入-剔除法多元线性回归分析RPE细胞数与TC、LDL、TG含量的相关性及RPE细胞胞浆内黑色素、脂褐素含量与PRC数、RPE细胞数、Bruch膜厚度的相关性。结果ApoE-/-高脂组、ApoE-/-普食组、C57BL普食组小鼠血浆TC的含量分别为(13.262±0.458)mmol·L-1、(9.722±0.412)mmol·L-1、(5.150±0.308)mmol·L-1,LDL的含量分别为(2.030±0.186)mmol·L-1、(1.047±0.158)mmol·L-1、(0.427±0.080)mmol·L-1,TG的含量分别为(1.233±0.199)mmol·L-1、(0.777±0.161)mmol·L-1、(0.530±0.168)mmol·L-1,PRC细胞核面积分别为(20135.000±1473.272)μm2、(23208.000±716.913)μm2、(26952.000±2449.104)μm2,RPE细胞数分别为8.900±1.148、10.200±0.835、13.800±1.016,Bruch膜厚度分别为(0.982±0.071)μm、(0.870±0.042)μm、(0.594±0.031)μm。ApoE-/-高脂组和普食组小鼠TC、LDL、TG含量较C57BL普食明显增高(P<0.05或0.001)。光镜下观察发现:ApoE-/-高脂组和ApoE-/-普食组小鼠视网膜PRC、RPE细胞排列紊乱、萎缩,RPE细胞内黑色素分布稀疏、面积小,脂褐素分布密集、范围广,Bruch膜粗细不等,胶原分叉,部分小鼠视网膜下可见玻璃膜疣形成;半定量结果显示,ApoE-/-高脂组和ApoE-/-普食组小鼠视网膜PRC细胞核面积、RPE细胞数较C57BL普食组明显减少(P<0.01或0.001),Bruch膜显著增厚(P<0.001),RPE细胞内黑色素含量明显降低(P<0.01或0.001),脂褐素含量明显增高(P<0.001)。多元线性回归分析显示,RPE细胞数与TC、LDL、TG含量呈显著负相关(r=-0.911、-0.888、-0.669,P=0.000、0.00、0.001),黑色素含量与Bruch膜厚度呈明显负相关(r=-0.691,P=0.001),与RPE细胞数、PRC数呈明显正相关(r=0.745、0.408,P=0.000、0.047)。脂褐素含量与Bruch膜厚度呈明显正相关(r=0.830,P=0.000),与RPE细胞数、PRC数呈负相关(r=-0.826、-0.340,P=0.000、0.084)。结论血脂异常ApoE-/-小鼠可出现明显高脂血症,引起视网膜RPE细胞损伤,细胞内黑色素减少、脂褐素增加,导致年龄相关性黄斑变性产生。

Objective To explore the changes of melanin and lipofuscin in retinal pigment epithelium intracytoplasm of mice with dyslipidemia and apolipoprotein Edeficiency （ApoE-/-）. Methods A total of 24 two-month-old ApoE-/- mice were equally and randomly divided into high fat diet group and normal diet group ,and another 12 two-month-old C57BL mice were chosen as normal diet control group. After they were all fed for 4 months,levels of total plasma cholesterol（TC） ,low density lipoprotein（LDL） and plasma trigiyceride（TG） were detected ,morphological characteristics of retinal photoreceptor cell （ PRC ）, retinal pigment epithelium （ RPE ） and Bruch mem- brane were observed by fight microscope after the staining of Masson trichrism staining,fontana staining and aldehydo-magenta staining,and amounts of retinal PRC and RPE, the contents of melanin and lipofuscin in RPE cells and the thickness of Bruch＇ s membrane were examined by semi-quantitative histopathology, the relevance between RPE and TC,LDL,and TG content was analyzed,as well as the relevance between the contents of melanin and lipofuscin in RPE cells and the number of PRC, RPE cells, and the thickness of Bruch＇ s membrane by gradually introduced-excluding method of multiple linear regression. Results The levels of TC were （ 13. 252 ± 0. 458 ） mmol·L-1 , （9. 722 ±0.412）mmol·L-1 and （5. 150 ±0. 308）mmol·L-1 ,levels of LDL were （2. 030 ± 0. 186）mmol·L-1,（1.047 ±0. 158）mmol·L-1 and （0.427 ±0.080）mmol·L-1 ,levels of TG were （1.233±0.199）mol·L-1, （ 0. 777 ± 0.151）mol·L-1 and （0.530±0.158）mml·L-1 ,nucleus areas of PRC were （20 135. 000 ± 1473.272）μm2 , （20 208.000 ±716. 913） μm2 and （26 952.000 ±2449. 104） μm2 and numbers of RPE cells were 8. 900 ± 1. 481,10. 200 ± 0. 835 and 13.800 ± 1. 016, thickness of Bruch＇ s membrane were （ 0. 982 ± 0. 071 ） μm, （0. 870 ± 0. 042 ） μm and （0. 594 ±0.031 ） μm in blood plasma of mice in ApoE-/- high fat diet group,ApoE-/- normal diet group and C57BL normal diet control group, respectively. The contents of TC, LDL and TG in ApoE-/- high fat diet group were significantly higher than those in C57BL normal diet control group （P 〈 0.05 or 0.001 ）. They were observed under light microscope, for ApoE-/- mice in high fat group and normal diet group, their retinal PRC and RPE were disorganized and atrophic, melanin in RPE was sparse and had small area while lipofuscin distributed intensively and ranged widely, Bruch membrane had unequal roughness with fibers forked, and drusen could be found under the retina of some mice. The resultes of semi-quantitative histopathology showed that nucleus areas of PRC and RPE numbers in ApoE-/- high fat group and normal diet group signifi- cantly reduced（P 〈 0.01 or 0. 001 ）, Bruch＇ s membrane markedly thickened （ P 〈 0. 001 ）, melanin in RPE significantly reduced （P 〈0.01 or 0.001 ） and lipofuscin in RPE significantly increased than those in C57BL normal diet groups（P 〈0.001 ）. Results of multiple linear regression analysis showed that the number of RPE cells was obviously negatively correlated with the contents of TC, LDL and TG （ r = - 0. 911, - 0. 888, - 0. 669, P = 0. 000,0.00,0.001, respectively） ; melanin content was obviously negatively correlated with the thickness of Bruch＇ s membrane（ r = -0.691 ,P = 0.001 ） ,while obviously positively correlated with the numl＇er of PRC and RPE cells （ r = 0. 745 , 0. 408 , P = 0. 000,0. 047, respectively）. Lipofuscin content was obviously positively correlated with the thickness of Bruch＇ s membrane （ r = 0. 830, P = 0. 000 ）, while obviously negatively correlated with the numlc er of PRC and RPE cells （ r = - 0. 826, - 0. 340 ,P = 0. 000,0. 084, respectively）. Conclusion Hyperlipidemia obviously appears in dyslipidemia ApoE-/- mice, which leads to that retinal RPE is damaged, melanin of RPE reduces,lipofuscin of RPE increases and age-related macular degeneration generates.