文章摘要
薛 博,季 敏,管怀进.ATP与P2X7受体在Müller细胞激活导致视网膜神经节细胞株RGC-5凋亡中的作用研究[J].南京医科大学学报,2016,(2):182~188
ATP与P2X7受体在Müller细胞激活导致视网膜神经节细胞株RGC-5凋亡中的作用研究
Involvement of P2X7 receptor and ATP in Müller cell gliosis induced RGC-5 cells apoptosis
投稿时间:2015-07-07  
DOI:10.7655/NYDXBNS20160211
中文关键词: Müller 细胞激活  P2X7受体  ATP  凋亡  RGC-5
英文关键词: glaucoma  Müller cell gliosis  ATP  apoptosis  P2X7 receptor  RGC-5
基金项目:国家自然科学基金资助(81200680)
作者单位
薛 博 南京医科大学附属无锡第二人民医院眼科,江苏 无锡 214000 
季 敏 南通大学附属医院眼科研究所,江苏 南通 226001 
管怀进 南通大学附属医院眼科研究所,江苏 南通 226001 
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中文摘要:
      目的:探讨Müller细胞胶质化激活对RGC-5细胞的影响及ATP与P2X7受体在其中的相关作用?方法:纯化培养Müller细胞?RGC-5细胞株,免疫荧光进行细胞鉴定,并观察加入DHPG后Müller细胞的GFAP表达情况?荧光素酶法检测加入DHPG后Müller 细胞外ATP的含量变化;DHPG刺激后的培养液作为条件培养基加入RGC-5细胞株(RGC-5预先使用MPEP及MPMQ处理2 h),TUNEL法检测RGC-5凋亡情况,预先加入P2X7受体阻断剂亮蓝G(brilliant blue G,BBG)后检测RGC-5的凋亡情况?同时使用Western blot检测抗凋亡蛋白及促凋亡蛋白Bcl-2和Bax的变化情况?免疫荧光及Western blot检测RGC-5上P2X7受体的表达变化?结果:DHPG激活Müller 细胞后可以导致细胞外ATP的增加;激活的Müller 细胞条件培养基(conditioned medium,CM)可以导致RGC-5凋亡的明显增加?而BBG可以抑制RGC-5凋亡的增加,同时Western blot也显示与对照组相比CM显著降低RGC-5细胞上的Bcl-2蛋白水平,但增加了Bax的蛋白表达,而当预先加入BBG后再用CM处理则呈现相反的结果?另外Western blot及免疫荧光显示加入条件培养基后RGC-5细胞的P2X7受体蛋白表达增加?结论:Müller细胞的激活可以导致RGC-5细胞的凋亡,该现象可能与Müller细胞激活引起的ATP释放增加及RGC-5上P2X7受体有关?
英文摘要:
      Objective:To explore the possible involvement of Müller cell gliosis,ATP/ P2X7 receptors and RGC-5 apoptosis in vitro. Methods:We cultured and purificated rat retinal Müller cells,RGC-5. The concentration of extracellular ATP was quantified by bioluminescence assay. After adding the medium of DHPG-activated Müller cells Medium(conditioned medium /CM)to RGC-5 cells,TUNEL assay and Western blot of anti-and pro-apoptotic proteins were used to observe RGCs apoptosis. Immunofluorescence and Western blot were also used to detect the P2X7 expression of RGC-5 with CM. Results:DHPG induced an increasing ATP release from Müller cells. After adding the CM to RGC-5 cells,TUNEL assay showed a significantly increase RGCs apoptosis while P2X7 receptor blocker BBG reduced apoptosis of RGCs. Moreover,CM treatment of RGC-5 cells significantly increased Bax protein level and decreased Bcl-2 protein level,which was also mimicked by BzATP and blocked by BBG,respectively. Conclusion:The current study suggests that DHPG-activated Müller cells could aggravate RGCs apoptosis,while the release of ATP from Müller cells and the activation of P2X7 receptor in the RGC-5 might be involved in this process.
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