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目的观察博莱霉素(BLM)造模大鼠肺微血管内皮细胞(EC)紧密连接及Cx43表达的变化特征,探讨其在肺纤维化发生中的作用和意义。方法健康SD大鼠随机分为对照组和实验组每组20只,采用ELISA法测定对照组和实验组第3、7、14、28天血浆血管性假血友病因子(vWf)水平。对采血后的大鼠,分别在第3、7、14、28天行硝酸镧灌注,制作肺组织病理标本,用透射电镜观察血管EC紧密连接及镧颗粒分布;采用组织块培养法行肺微血管EC体外培养,用激光共聚焦显微镜观察Cx43表达。结果各实验组大鼠血清中vWf水平均高于对照组,以第3天最高,差异有统计学意义(P<0.01)。对照组EC结构完整,EC间连接紧密,未见镧盐颗粒通过EC间的紧密连接。实验组均可见EC连接间隙增宽,并有高密度的镧盐颗粒在细胞连接间隙中线状沉淀。实验组Cx43阳性反应的范围及强度均明显减弱,第3、7、14、28天Cx43蛋白的荧光反应强度及表达阳性率(25%、38%、45%、71%),均明显低于对照组,差异有统计学意义(P<0.05)。结论EC紧密连接呈持续开放状态,导致炎性细胞渗出及与之相应的细胞因子分泌持续增加,启动成纤维细胞增殖过度可能是BLM诱发肺间质结构发生异常改变的重要病理基础。
Objective To observe the changes of tight junctions and expression of Cx43 in pulmonary microvascular endothelial cells (ECs) of bleomycin (BLM) -induced model rats, and to explore their roles and significance in the pathogenesis of pulmonary fibrosis. Methods Healthy Sprague-Dawley rats were randomly divided into control group and experimental group with 20 rats in each group. Plasma vWF levels in control group and experimental group at 3, 7, 14, and 28 days were measured by ELISA. After the blood was collected from the rats, respectively, on the 3rd, 7th, 14th and 28th days, respectively. Lanthanum nitrate was injected into the lung to make histopathological specimens. Transmission electron microscopy was used to observe the vascular EC tight junctions and lanthanum particles distribution. EC cultured in vitro, the Cx43 expression was observed by laser confocal microscopy. Results The serum levels of vWf in rats in each experimental group were higher than those in the control group, with the highest on the third day (P <0.01). ECs in the control group were intact with tight junctions between ECs. No tight junctions between lanthanide particles and ECs were observed. The experimental group showed widening of the EC connection gap, and high-density lanthanide particles precipitated linearly in the cell connection gap. The extent and intensity of Cx43 positive reaction in the experimental group were significantly weakened, and the fluorescence intensity and the positive rate of Cx43 protein expression (25%, 38%, 45%, 71%) on the 3rd, 7th, 14th and 28th days were significantly lower than Control group, the difference was statistically significant (P <0.05). Conclusions EC tight junctions are continuously open, resulting in the exudation of inflammatory cells and the corresponding increase of cytokine secretion. Proliferating hyperplasia of fibroblasts may be an important pathological basis for the abnormal changes of pulmonary interstitial structure induced by BLM.