内质网应激在大鼠脑缺血再灌注损伤中的作用研究

Role of endoplasmic reticulum stress in rats after ischemia-reperfusion brain injury

目的观察生长停滞及DNA损伤诱导基因153（GADDl53）和caspase．12在脑缺血再灌注损伤大鼠脑组织中的动态改变，探讨内质网应激在脑缺血再灌注损伤中的作用。方法42只大鼠按随机数字表法分为正常对照组（3只）、假手术组（3只）和脑缺血再灌注损伤组（36只）；脑缺血再灌注损伤组又分为脑缺血2h再灌注6h、12h、24h、72h组，每组各9只。采用线栓法建立大鼠大脑中动脉缺血再灌注损伤模型。采用免疫组化染色、免疫荧光双标染色、Westemblotting检测各组大鼠脑组织中GADDl53和caspase-12的表达。结果免疫组化染色和Westernblotting结果均显示正常对照组和假手术组大鼠脑组织中GADDl53和caspase-12表达为阴性；再灌注6h组GADDl53表达增加，并逐渐增高，持续至72h时较再灌注6h组明显增高，差异均有统计学意义f氏0．05）：再灌注6h组caspase-12表达增加，至24h达高峰，72h时仍维持在较高水平，与再灌注6h组比较差异有统计学意义（P〈0．05）。免疫荧光双标染色结果显示，再灌注6h组可见少量双标阳性细胞，12h、24h时双标阳性细胞数均较再灌注6h组明显增多，差异有统计学意义（氏0．05），至72hcaspase-12单标阳性细胞数减少，GADDl53单标阳性细胞数仍较多，双标阳性细胞数减少。结论GADDl53和caspase-12在脑缺血再灌注损伤大鼠脑组织中的表达随时间呈动态改变，表明内质网应激参与了脑缺血再灌注损伤的病理过程。

Objective To observe the dynamic changes of growth arrest and DNA damage inducible gene 153 （GADD153） and caspase-12 expressions in the brain of rats after ischemia-reperfusion, and explore the role of endoplasmic reticulum stress in ischemia-reperfusion brain injury. Methods Forty-two rats were randomly divided into control group （n=3）, sham-operated group （n=3） and ischemia-reperfusion group （n=36）; the rats of ischemia-reperfusion group were randomly sub-divided into groups of 2 h occlusion and 6, 12, 24, 72 h reperfusion （n=9）. Modified Longa intraluminal thread method was adopted to establish the middle cerebral artery occlusion-reperfusion models. Expression changes of GADD153 and caspase-12 at different time points were detected by immunohistochemistry, double-label immunofluorescence and Western blotting. Results Immunohistochemistry and Western blotting showed that the expressions of GADD153 and caspase-12 in the control group and sham-operated group was negative; the GADD153 expression increased in group of 6 h reperfusion, and that in group of 72 h reperfusion was significantly higher than that in group of 6 h reperfusion （P〈0.05）; the caspase-12 expression increased in group of 6 h reperfusion, enjoyed the highest level in group of 24 h reperfusion which had significant difference as compared with that in group of 6 h reperfusion （P〈0.05）, and still maintained at a high level in group of 72 h repefusion. Double immunofluorescence staining showed that GADD153 and caspase-12 double-positive ceils could be seen in group of 6 h reperfusion; that at groups of 12 and 24 h reperfusion significantly increased as compared with that in group of 6 h reperfusion （/9〈0.05）; the number of caspase-12 single-positive cells reduced, that of GADD153 single-positive cells was still large, and the number of double-positive ceils reduced. Conclusion The expression changes of GADD153 and caspase-12 are time dependent, indicating that endoplasmic reticulum stress involve in the pathological process of ischemia-reperfusion brain injury.