HT22细胞缺血再灌注损伤后钙池操纵钙通道蛋白中STIM的表达及意义

Stromal interaction molecule （STIM1 and STIM2） expressions and its significance in store-operated calcium channel of HT22 cells after hypoxic-ischemic damage

目的 探讨钙池操纵钙通道蛋白组成成分——基质相互作用因子（STIM）在细胞缺血再灌注损伤后的表达变化及意义. 方法 将HT22细胞系随机分为正常对照组及缺血再灌注损伤后1、3、6、12、24h组;正常对照组不做任何处理,缺血再灌注各组细胞通过无糖培养基和氮气箱内培养6h建模.利用荧光免疫组化法检测STIM1和STIM2的空间表达情况;利用实时定量PCR（RT-PCR）、Western blotting法检测STIM1和STIM2的蛋白及mRNA水平表达变化. 结果 （1）荧光免疫组织化学检测发现,缺血再灌注损伤后,特别是损伤后12 h,STIM1染色呈阳性,阳性染色呈颗粒状,分布在胞膜、胞浆和部分胞核上.而STIM2的阳性颗粒则主要表达在细胞核上;（2）RT-PCR检测发现,STIM1在正常对照组和缺血再灌注损伤后各组HT22细胞中高表达,但表达量差异无统计学意义（p＞0.05）.与正常对照组比较,STIM2则在缺血再灌注损伤后1h、6h和24 h有3个表达高峰,表达量差异有统计学意义（P＜0.05）.（3）Western blotting检测结果显示,STIM1持续稳定高表达,各组之间差异无统计学意义（p＜0.05）.STIM2蛋白在缺血再灌注损伤后各组中均明显升高,其中1h、3h、6h及12h组与正常对照组比较差异有统计学意义（p＜0.05）. 结论 缺血性损伤后,STIM1和STIM2均在HT22细胞上表达,但其空间和时间表达变化规律不同,可能通过影响钙库调控的钙内流等机制在脑缺血性损伤的病理生理过程中发挥作用.

Objective To investigate the stromal interaction molecule （STIM1 and STIM2） expressions in store-operated calcium channel （SOCC） of HT22 cells after hypoxic-ischemic damage.Methods HT22 cells were in vitro cultivated; the cells were divided into six groups with 2.5＆#215;105 cells for each group：normal group and 1,3,6,12 and 24 h after ischemia reperfusion injury （IRI） groups,and IRI was induced by cultivation at sugar-free culture medium and nitrogen tank for 6 h.The mRNA and protein expressions of STIM1 and STIM2 were examined by quantitative real time-PCR and Western blotting,respectively.Fluorescent immunohistochemistry was employed to detect the expressions of STIM1 and STIM2.Results Fluorescence immunochemistry showed that antigens of STIM1 distributed in the cytomembrane,cytoplasm and nucleus of HT22 cells,and STIM2 expressed in nucleus.In the mRNA level,STIM 1 stably expressed in cells from normal control group and IRI groups,without significant differences （P〉0.05）; as compared with that normal control group,the STIM2 expression in the IRI groups increased markedly,reaching peak level at 1,6 and 24 h （P〈0.05）.Western blotting indicated that STIM1 was a stable expressed protein,without significant differences between each two groups （P〉0.05）; and STIM2 is a dynamic expressed protein,showing obvious increased expression with that in 1,3,6 and 12 h ofIRI enjoying significantly differences as compared with that in the normal group （P〈0.05）.Conclusion After hypoxic-ischemic neurons damage,both STIM1 and STIM2 express in HT22 cells,but they differ in space and time mode of expression; they maybe play important role in IRI through affecting calcium influx in SOCC.