Nogo-66蛋白对视网膜小胶质细胞OX40L表达的影响

Proteinum Nogo-66 on expression of retinal microglia OX40L

目的研究免疫共刺激分子OX40配体(OX40 ligand,OX40L)在小胶质细胞上的表达,探讨Nogo-66蛋白刺激小鼠视网膜小胶质细胞株N9表达细胞膜黏附分子OX40L的可能性。方法根据不同浓度(250μg·L-1、500μg·L-1、1000μg·L-1、2000μg·L-1)的Nogo-66蛋白细胞培养液将实验组分为1、2、3、4组。应用免疫细胞化学方法,鉴定小胶质细胞在不同浓度的Nogo-66蛋白细胞培养溶液(实验组)中的表达情况,并与无刺激因子的细胞培养液(对照组)中小胶质细胞OX40L表达对比,Image-Pro Plus Version6.0图像分析软件处理,比较激活后小胶质细胞OX40L表达差异。结果Nogo-66蛋白质作为刺激因子处理后的小胶质细胞表现激活状态下的典型形态,细胞突起减少,体积明显增大,相同倍率拍照后图像分析可知,对照组细胞相对平均体积为(93.0020±17.0101)pixel,实验1、2、3、4组相对平均体积分别为(134.2258±23.5828)pixel、(242.7430±72·9439)pixel、(228.1760±37.1582)pixel、(204.7210±43.4950)pixel,与对照组相比,差异均有显著统计学意义(P均<0.001),实验1组与2、3、4组相比差异亦有显著统计学意义(P均<0.01)。对照组OX40L表达的细胞体积比为0.12%,实验1、2、3、4组分别为34.30%、58.91%、60.25%、59.71%,与对照组相比,差异均有显著统计学意义(P均<0·01)。结论Nogo-66蛋白能够刺激小胶质细胞处于激活状态,表达OX40L,从而可能进一步影响中枢神经系统疾病治疗的临床前研究。

Objective To study the expresssion of costimulatory molecules OX40 ligand（OX40L） on microglia and discuss the activation of Proteinum Nogo-66 on probability of retinal microglia cells N9 expressing OX40L.Methods The experimental group was subdivided into four groups with the different concentration levels of protein Nogo-66 culture fluid（250 μg·L-1,500 μg·L-1,1 000 μg·L-1,2 000 μg·L-1）.The expression of OX40L in different concentration levels of protein Nogo-66 culture fluid was observed with immunocyto chemical method,and compared with that in culture fluid without protein Nogo-66.The differentiation of microglia OX40L expression was compared with that after activation using Image-Pro Plus Version 6.0 Analyse Managing Software.Results Retinal microglia OX40L expressed at more significant level after stimulated by protein Nogo-66 with cell prominence reduced and the bulk of cell increased.With image analysis after taking photographs at the same multiplying factor,the relative cell average volume of control group was（93.002 0±17.010 1）pixel,and that of experimental group one,two,three,four were（134.225 8±23.582 8）pixel,（242.743 0±72.943 9）pixel,（228.176 0±37.158 2）pixel,（204.721 0±43.495 0）pixel,respectively.There was significant difference between control group and each experimental group（P〈0.001）.There was statistical difference between experimental group one and experimental two,three and four（P〈0.01）.The volume ratio of OX40L in control group was 0.12%,and that experimental groups were 34.30%,58.91%,60.25%,59.71%,respectively.There was significant difference between control group and each experimental group（P〈0.01）.Conclusion The retinal microglia could be activated by protein Nogo-66 and express higher level of OX40L.It may contribute to the preclinical analysis of central nervous system disease.