脑死亡状态下肾脏差异蛋白质的筛选与功能初步鉴定

Screening and functional identification of differential proteins in kidney under brain-death state

目的用蛋白质组学法筛选并鉴定脑死亡状态下肾脏差异蛋白质的表达。方法建立稳定的兔脑死亡模型，同时设立假手术对照组。收集脑死亡后2、4、6、8h的肾脏组织，采用双向凝胶电泳技术和基质辅助激光解析／电离飞行时间质谱（MALDI—TOF—MS）鉴定脑死亡后肾脏差异蛋白质，并用免疫组织化学法进一步验证。结果脑死亡组与假手术组可检测到的蛋白质点分别为（916±25）、（904±19）个。与假手术组比较，脑死亡组共有24个蛋白质上调，21个蛋白质下调。其中10个差异较大的蛋白质经肽质量指纹图谱分析初步鉴定为：抑制素、PRP38预rnRNA处理因子38、N-乙酰氨基半乳糖转移酶、膜粘连蛋白5、钙调磷酸酶B1、超氧化物歧化酶、V—type质子ATP酶亚基Cl、细胞色素b-cl复合体亚单位1、NADH脱氢酶亚基10、过氧化物酶3。进一步通过免疫组织化学法检测抑制素的表达，结果发现随着脑死亡时间的延长，抑制素在肾脏中的表达逐渐增加。结论抑制素可以作为判断脑死亡后肾脏损伤的检测指标，为揭示脑死亡状态下肾脏损伤机制提供新的实验证据。

Objective To screen and identify the differentially expressed proteins in brain-death kidney. Methods Stable brain death model was established and kidney tissues at 2, 4, 6 and 8 h after brain death were collected. Differentially expressed proteins were identified by two-dimensional gel electrophoresis and matrix-assisted laser-desorptionionization time-of-flight mass spectrometry （MALDI-TOF-MS）, which were further confirmed by immunohistochemistry. Results Protein spots detected in brain death group and sham group were 916± 25 and 904 ± 19, respectively. As compared with sham group, 24 protein spots were upregulated, and 21 downregulated in brain death group. Ten differentially expressed protein spots were identified as follows： Prohibitin, PRP38 pre-mRNA processing factor 38 （yeast） domain containing A, beta-l,3-N-acetylgalactosaminyltransferase 1, annexin A5, calcineurin subunit B type 1, superoxide dismutase [ Mn], mitochondrial,V-type proton ATPase subunit G1, eytochrome b-el complex subunit 1, mitochondrial precursor, NADH dehydrogenase [ ubiquinone] 1 beta subcomplex subunit 10, peroxiredox-in-3. The expression of prohibitin was further verified by immunohistochemistry, and it was found that with the extension of the brain-dead time, the expression of prohibitin in the kidney was increased gradually. Conclusion Prohibitin could be used as an indicator to judge the extent of kidney damage, which could provide new experimental evidence for the study on the kidney injury mechanism under the brain-death state.