ATP处理导致PC12细胞通透性变化及NaHS的干预作用  

ATP-induced membrane permeability increase in PC12 cell and NaHS intervention

作　　者：沈慧[1,2] 马洁[1] 王国红[1] 王璐[1] 李新娟[1] 张利彬[1] 魏琳郁 李超堃[1] 赵红岗[1] 李东亮[1] Shen Hui;Ma Jie;Wang Lu;Li Xinjuan;Wang Guohong;Zhang Libin;Li Chaokun;Zhao Honggang;Li Dongliang(Department of Physiology and Neurobiology;The Third Affiliated Hospital, Xinxiang Medical University, Xinxiang 453003, China)

机构地区：[1]新乡医学院生理学与神经生物学教研室,新乡453003 [2]新乡医学院第三附属医院,新乡453003

出　　处：《神经解剖学杂志》2018年第2期184-190,共7页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(81371346;81271376);河南省高等学校重点科研项目计划(15A310009;16A310011;16A310013);河南省高校科技创新团队支持计划(16IRTSTHN020);河南省教育厅科学技术研究重点项目(14A310019)

摘　　要：目的:观察NaHS(H_2S供体)对ATP(三磷酸腺苷)诱导的PC12细胞存活率及膜通透性变化的影响,探讨H_2S对抗ATP致伤作用的可能机制。方法:高分化PC12细胞置于含有高浓度ATP的培养基中3 h制备细胞损伤模型。在ATP处理前30 min,把NaHS或CBX(生胃酮,Pannxin-1通道阻滞剂)加入培养基中,作为预处理。用CCK-8检测细胞存活率,Fura-2/AM荧光染料检测细胞[Ca^(2+)]_i,YO-PRO-1或Lucifer Yellow(荧光黄)荧光染料检测RFU(胞内相对荧光单位),研究NaHS干预后细胞存活率和膜通透性的变化。结果:ATP(3、5mmol/L)处理使PC12细胞存活显著降低,NaHS(200μmol/L)和CBX(10μmol/L)预处理均可显著拮抗ATP(3 mmol/L)诱导的细胞存活率下降。ATP(3、5 mmol/L)可使PC12细胞内[Ca^(2+)]_i明显增加,而NaHS(200μmol/L)和CBX(10μmol/L)预处理可使ATP(3 mmol/L)诱导的胞内[Ca^(2+)]_i的增幅明显降低。ATP(3、5mmol/L)使PC12细胞对YO-PRO-1和Lucifer Yellow通透明显增加,而NaHS(200μmol/L)和CBX(10μmol/L)预处理可显著阻遏ATP诱导的膜通透性增加。结论:Pannxin-1蛋白在ATP诱导PC12细胞膜孔形成中有重要作用,干预膜孔形成可能是NaHS拮抗ATP损伤作用的机制之一。Objective： To explore the possible mechanism of H2S（ hydrogen sulfide） protection against adenosine triphosphate（ ATP） induced PC12 cell injury,the effect of NaHS（ a donor of H2S） on survival or membrane permeability induced by ATP were observed. Methods： PC12 cells were treated with high concentration of ATP for 3 hours to set up the injured neuron-like cell model. NaHS or CBX（ carbenoxolone,Pannxin-1 receptor antagonist） were added into the medium for 30 minutes before the ATP treatment. The viability of PC12 cell was measured by CCK-8 assay. The[Ca^（2＋）]i was detected by Fura-2/AM staining. Fluorescence changes（ RFU） following YO-PRO-1 or Lucifer Yellow uptake were monitored. So the effect of NaHS pretreatment on the cell survival rate and membrane permeability was studied. Results： ATP（ 3,5 mmol/L） made PC12 cell survival significantly reduced,but pretreatment with NaHS（ 200 μmol/L） or CBX（ 10 μmol/L） for 30 min could significantly antagonized cell survival rate drop induced by ATP（ 3 mmol/L）. ATP（ 3,5 mmol/L） made [Ca^（2＋）]i in PC12 cell increased significantly,however,pretreatment with NaHS（ 200 μmol/L） or CBX（ 10 μmol/L） for 30 min could obviously depressed the [Ca^（2＋）]i amplification induced by ATP（ 3 mmol/L）. ATP（ 3,5 mmol/L） made YO-PRO-1 and Lucifer Yellow transparent increased significantly in PC12 cell,but pretreatment with NaHS（ 200 μmol/L） or CBX（ 10 μmol/L） for 30 min could inhibited membrane permeability increase induced by ATP obviously. Conclusion： Pannexin-1 protein plays an important role in PC12 cell membrane pore formation induced by ATP at high concentration and the intervention of membrane pore formation may be one of the mechanisms of NaHS against ATP-induced injury.

关 键 词：ATP 硫化氢 Pannxin-1 PC12细胞 CBX 

分 类 号：R346[医药卫生—基础医学]