C57BL/6J小鼠衰老耳蜗血管纹微血管周细胞对内皮细胞通透性影响的研究

Effect of microvascular pericytes of cochlear stria vascularis on endothelial cell permeability in C57BL/6J mice

目的研究小鼠衰老耳蜗血迷路屏障通透性的变化, 并通过建立内皮细胞(endothelial cells, EC)系和周细胞(pericytes, PC)系非接触式共培养模型, 进一步探讨耳蜗血管纹微血管PC对EC通透性的影响。方法 C57BL/6J小鼠分为2组:3月龄为年轻组, 12月龄为衰老组;细胞实验分为4组:EC组、EC+PC共培养组、D-半乳糖(D-gal)+EC组和D-gal+EC+PC共培养组。听性脑干反应(auditory brain response, ABR)检测2组小鼠听觉功能;伊文思蓝(evans blue)染色检测2组小鼠耳蜗血迷路屏障的通透性;透射电镜观察2组小鼠血迷路屏障EC、PC和紧密连接结构;免疫荧光检测2组小鼠耳蜗血管纹上紧密连接蛋白表达水平;Transwell小室检测EC通透性;Western blot及免疫荧光技术检测EC上紧密连接蛋白的表达水平。以SPSS 20.0软件进行统计学分析。结果与年轻组相比, 衰老组小鼠ABR阈值明显升高且Ⅰ波潜伏期延长(t=10.25, P<0.01;t=5.61, P<0.05)、耳蜗血迷路屏障通透性升高且血管纹上紧密连接蛋白的表达降低(P值均<0.05);耳蜗超微结构显示衰老小鼠耳蜗血管纹微血管管腔变形、基底膜增厚、内皮间紧密连接间隙增大;原代培养的EC和PC阳性率达95%以上;并确定15 g/L D-gal诱导的细胞为衰老细胞;与EC组相比, D-gal+EC组EC的紧密连接蛋白表达降低(t=7.42, P<0.01;t=13.19, P<0.05), 通透性增加(t=11.17, P<0.01);在共培养组中, EC+PC共培养组和D-gal+EC+PC共培养组EC间紧密连接蛋白表达均增加, 通透性均降低。结论衰老小鼠耳蜗血迷路屏障的通透性升高, 紧密连接蛋白水平降低;衰老状态下, 耳蜗血管纹微血管PC可能通过调节紧密连接蛋白的表达进而影响EC通透性。

Objective To study the changes in the permeability of the blood labyrinth barrier of the aging cochlea in mice,and to establish a non-contact co-culture model of endothelial cells(EC)and pericytes(PC)to furtherly investigate the cochlear stria vascularis microvascular pericytes impact on the permeability of endothelial cells.Methods C57BL/6J mice were divided into two groups,three months old as young group,12 months old as senile group.Cell experiment was divided into four groups,EC group,EC+PC co-culture group,D-gal+EC group and D-gal+EC+PC co-culture group.Auditory brainstem response(auditory brain response,ABR)was used to detect the auditory function of the two groups of mice.Evans blue staining was applied to detect the permeability of the cochlear blood labyrinth barrier of the two groups of mice.Transmission electron microscopy was used to observe the ultrastructure of blood labyrinth barrier endothelial cells,pericytes and tight junctions in the two groups of mice.Immunohistochemistry was used to detect the expression levels of tight junction proteins in the stria vascularis of the cochlea of the two groups of mice.Transwell chamber was used to detect the permeability of endothelial cells.Western blot and immunofluorescence technology were used to detect the expression level of tight junction protein on endothelial cells.SPSS 20.0 software was used to analyze the data.Results Compared with the young group,the ABR threshold of the aging group was significantly increased,the latency of wave I was prolonged(t=10.25,P<0.01;t=5.61,P<0.05),the permeability of the cochlear blood labyrinth barrier was increased and the expression of tight junction protein on the vascular stria was decreased(P<0.05).The cochlear ultrastructure showed that the cochlear vascular stria microvascular lumen was deformed,the basement membrane thickened and the tight junction gap between endothelium enlarged.The positive rate of ECs and PCs in primary culture was more than 95%.The cells induced by 15 g/L D-gal were determined to be senescent cells.Compared with EC group,the expression of tight junction protein in endothelial cells of D-gal+EC group decreased(t=7.42,P<0.01;t=13.19,P<0.05)and the permeability increased(t=11.17,P<0.01).In the co-culture group,the expression of tight junction protein between endothelial cells in EC+PC co-culture group and D-gal+EC+PC co-culture group increased and the permeability decreased.Conclusions In aging mice,the permeability of cochlear blood labyrinth barrier will increase and the level of tight junction protein will decrease;in aging state,cochlear vascular stria microvascular pericytes may affect endothelial cell permeability by regulating the expression of tight junction protein.