缺氧对脑脊液-视神经屏障中脑膜上皮细胞内质网和线粒体膜电位的影响

目的　探讨脑膜上皮细胞（ｍｅｎｉｎｇｏｔｈｅｌｉａｌｃｅｌｌｓ，ＭＥＣｓ）参与视神经疾病的发病机理，研究缺氧对ＭＥＣｓ功能变化的影响。方法　将培养的ＭＥＣｓ分别在体积分数２１％Ｏ２（对照组）和体积分数１％ Ｏ２（缺氧组）环境下孵育１ｄ和２ｄ，采用酶联免疫吸附实验测定和比较ＭＥＣｓ的总抗氧化能力（ｔｏｔａｌａｎｔｉ-ｏｘｉｄａｔｉｖｅｃａｐａｃｉｔｙ，Ｔ-ＡＯＣ）；采用ＭｉｔｏＴｒａｃｋｅｒＲｅｄ检测和比较ＭＥＣｓ在正常氧和缺氧环境下作用２ｄ的线粒体膜电位的变化；通过测定钙联蛋白的荧光染色，以评价ＭＥＣｓ暴露于氧化应激环境后对内质网功能的影响。结果　与对照组相比，缺氧组的氧化应激能力降低，结果显示Ｔ-ＡＯＣ下降。对照组和缺氧组细胞分别培养１ｄ后，细胞中Ｔ-ＡＯＣ分别为（０．０１２４＋０．００１１）Ｕ?Ｌ－１和（０．０１１９＋０．００９０）Ｕ?Ｌ－１，差异无统计学意义（ｔ＝０．９１５、Ｐ＝０３８２）。分别培养２ｄ后，缺氧组细胞中Ｔ-ＡＯＣ（０．０１０７＋０．００８３）Ｕ?Ｌ－１低于对照组（０．０１２９＋０．００８７）Ｕ?Ｌ－１，差异有统计学意义（ｔ＝４．６１６、Ｐ＝０．００１）。不同氧环境作用于细胞２ｄ后，采用共焦显微镜荧光探针方法分析作用于对照组和缺氧组的ＭＥＣｓ线粒体膜电位的变化，结果发现缺氧组细胞荧光强度与对照组相比明显减低；同时通过荧光显微镜观察发现，缺氧组ＭＥＣｓ钙联蛋白绿色荧光强度较对照组明显增强。结论　缺氧通过影响ＭＥＣｓ内质网及线粒体功能而影响其氧化应激能力。

Effects of hypoxia on endoplasmic reticulum and mitochondrial membrane potential in human meningothelial cells in cerebral fluid-optic nerve barrier

Objective To investigate the possible role of meaningothelial cells ( MEC) involving in the pathogenesis of optic nerve diseases , and explore the effects of hypoxia on functional changes in MEC. Methods The total anti-oxidative capacity ( TAOC) of MEC were investigated and compared by enzyme linked immunosorbent assay ( ELISA) after cells exposed t0 21% 0, ( normoxia group) and l% 02 ( hypoxia group) condition for I day and 2 days , respectively. Mitochondrial membrane potential was detected and compared by staining with MitoTracker Red after MEC were incubated in normoxia and hypoxia environment for 2 days, and fluorescence staining of calnexin was performed to asses the impact of oxidative stress on the function of endoplasnuc reticulum of MEC. Results Compared with the control group, the oxidative stress was decreased in cells treated with hypoxia. An inhibitory effect of hypoxia on T-AOC of MEC exhibited in our study. T-AOC of MEC in the normoxia and hypoxia group for I day was ( 0. 012 4 +0. 001 I ) U . L -l and ( 0. 011 9 + 0. 009 0 ) U . L ’I , respectively , there was no statistical difference( t = 0. 915 ,P = 0. 382) . The production of T-AOC of MEC decreased after cells exposure to hypoxia(0. 010 7 +0. 008 3)U . L-l for 2 days compared with the normoxia group (0. 012 9 + 0. 008 7) U . L-l for the same length of exposure time . there was significant difference ( t = 4. 616 .P = 0. 001 ) . After incubation with different oxygen environment for 2 days , the mitochondrial membrane potential decreased in hypoxia group compared with the control group by the analysis of fluorescent probe under the confocal microscope ;meanwhile fluorescense stairung revealed that intensity of the green fluorescence of calnexin of MEC in the normoxia group was weaker than that in the hypoxia group by immunofluorescense microscope. Conclusion Hypoxia may affect the oxidative stress of MEC by disturbing the endoplasmic reticulum and mitochondria.