实验性自身免疫性脑脊髓炎大鼠紧密连接蛋白表达的变化研究

Effect of GFAP and MMP9 expressions on tight junction proteins in experimental autoimmune encephalomyelitis rats

目的观察紧密连接蛋白在实验性自身免疫性脑脊髓炎（EAE）大鼠小脑中的表达变化，并探讨其相关病理机制。方法将24只SD大鼠按随机数字表法分为正常对照组、EAE组（采用足垫皮内注射自身免疫性抗原构建大鼠EAE模型），免疫后第12天起持续记录大鼠的行为学评分，免疫后第23天取材行HE和坚牢蓝（LFB）染色观察大鼠小脑组织病理学变化，行免疫荧光染色评估小脑组织中紧密连接蛋白Occludin、ZO-1、Claudin-5的表达变化，行逆转录PCR和Western blotting检测小脑组织中星形胶质细胞表面标志物胶质纤维酸性蛋白（GFAP）和基质金属蛋白9（MMP9）mRNA和蛋白的表达变化。结果与正常对照组相比，免疫后第15~23天的EAE组大鼠行为学评分及组织病理学评分明显增加，差异均有统计学意义（P〈0．05）；EAE组大鼠小脑白质区域呈现大量的炎性细胞浸润和髓鞘缺失症状；与正常对照组相比，EAE组紧密连接蛋白Occludin、ZO-1及Claudin-5的表达明显降低，GFAP和MMP9的mRNA和蛋白的表达明显上调，差异均有统计学意义（P〈0．05）。结论EAE发病过程中星形胶质细胞的活化以及MMP9表达的增加可能导致紧密连接蛋白Occludin、ZO-1、Claudin．5的缺失，破坏血脑屏障，加剧炎性细胞浸润和脱髓鞘。

Objective To observe the expressions of tight junctional proteins in rats with experimental autoimmune encephalomyelitis （EAE）, and to explore their relevant pathological mechanism. Methods Twenty-four Sprague Dawley rats were selected and randomly divided into a control group and a EAE group, and rats in the EAE group were subcutaneously injected at the footpad with autoimmune antigens to induce EAE models. The clinical behavioral scale scores of the rats 12 d after immunization were observed and recorded daily. The rats were sacrificed 23 d after immunization, and the histopathological changes of the brain tissues were observed by hematoxylin-eosin （HE） staining and fast blue （LFB） staining; the expressions of tight junction proteins Occludin, ZO-1 and Claudin-5 in the brain tissues were evaluated by immunofluorescence staining; the mRNA and protein levels of glial fibrillary acidic protein （GFAP, surface marker of astrocytes） and matrix metalloproteinase-9 （MMP9） in brain tissues were detected by reverse transiption-PCR （RT-PCR） and Western blotting, respectively. Results As compared with those in the control group, the clinical behavioral scale scores from 15 to 23 d of immunization and histopathological scale scores in the EAE group were significantly increased （P〈 0.05）. In EAE group, a lot of inflammatory cell infiltration and myelin loss in the cerebellar white matter were noted. As compared with those in the control group, the expressions of Occludin, ZO-1, and Claudin-5 in EAE group were significantly decreased （P〈0.05）, and the mRNA and protein expressions of GFAP and MMP9 were significantly up-regulated （P〈0.05）. Conclusion The activation of astrocytes and increased expression of MMP9 may reduce expressions of Occludin, ZO-1 and Claudin-5, disrupt blood-brain barrier, promote inflammatory cell infiltration, and increase myelin loss during EAE.