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目的观察创伤性脑损伤(traumatic brain injury,TBI)后大鼠损伤皮质区miRNA-9的表达变化规律,探讨其对脑微血管内皮细胞的保护作用。方法选取成年雄性SD大鼠60只制作控制性皮质撞击损伤模型(controlled cortical impact,CCI),分别应用荧光定量PCR和Western blot检测伤后6 h及1、3、7、14、28 d各时间点伤灶周围组织中miRNA-9及CD31的表达情况。培养原代大鼠脑微血管内皮细胞,将内皮细胞分为正常组、损伤模型组[用依托泊苷(etoposide,ETO)损伤]、miRNA-9过表达损伤模型组、空转染损伤模型组,应用CCK-8检测各组细胞活力;应用Western blot检测各组B细胞淋巴瘤/白血病-2蛋白(B-cell lymphoma-2,Bcl-2)、B细胞淋巴瘤/白血病-2相关X蛋白(Bcl-2 associated X,Bax)、活化半胱氨酸天冬氨酸特异性蛋白酶-3蛋白(cleaved cysteinyl aspartate specific proteinase 3,cl-caspase-3)表达水平。结果 (1)脑创伤后伤灶周围区域miRNA-9表达明显增加,于伤后第14天达高峰(P<0.01);内皮细胞标志物CD31蛋白表达水平从伤后第3~28天持续高于正常组(P<0.05);(2)内皮细胞建模转染后,qPCR结果提示损伤模型组较正常组miRNA-9表达显著降低(P<0.01),但miRNA-9过表达损伤模型组miRNA-9表达显著高于损伤模型组(P<0.01);CCK-8结果同样显示miRNA-9过表达损伤模型组细胞活力明显高于损伤模型组(P<0.01);(3)相比于损伤模型组,miRNA-9过表达损伤模型组内皮细胞Bcl-2蛋白表达增加(P<0.01),Bcl-2/Bax值增加(P<0.05),但Bax蛋白、cl-caspase-3蛋白表达降低(P<0.05)。结论创伤性脑损伤后伤灶周围区域miRNA-9表达增多且过表达miRNA-9可提高依托泊苷诱导损伤的内皮细胞活力,提示脑创伤后miRNA-9表达增加有助于脑血管重塑的发生。
Objective To observe the changes of miRNA-9 expression in injured cortex after traumatic brain injury (TBI) in rats and explore its protective effect on cerebral microvascular endothelial cells. Methods Sixty adult male Sprague-Dawley rats were selected to make a controlled cortical impact (CCI) model. Fluorescent quantitative PCR and Western blot were used to detect the expression of CCI at 6 hours and 1,3,7,14,28 days The expression of miRNA-9 and CD31 in the tissues around the point wounds. The primary rat brain microvascular endothelial cells were cultured, and the endothelial cells were divided into normal group, injury model group [etoposide (ETO) injury], miRNA-9 overexpression injury model group, Cell viability was measured by CCK-8. Western blot was used to detect the expression of B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma / leukemia- Bcl-2 associated X, Bax) and the expression of cleaved cysteinyl aspartate specific proteinase 3 (cl-caspase-3). Results (1) The expression of miRNA-9 in the peri-injured area increased significantly (P <0.01) on the 14th day after injury, and the expression of CD31 protein increased from the 3rd to 28th day (2) After transfection with qPCR, the expression of miRNA-9 in the injury model group was significantly lower than that in the normal group (P <0.01), but the expression of miRNA-9 in the injury model group The results of CCK-8 also showed that the cell viability of miRNA-9 overexpression group was significantly higher than that of the injury model group (P <0.01); (3) Compared with the model group, The expression of Bcl-2 and Bcl-2 / Bax increased (P <0.05), but the expression of Bax protein and cl-caspase-3 protein in the injury model group increased Decreased (P <0.05). Conclusion The increased expression of miRNA-9 in the peri-injured area after traumatic brain injury and the overexpression of miRNA-9 can increase the activity of etoposide-induced endothelial cell injury, suggesting that increased expression of miRNA-9 may contribute to cerebral vascular remodeling occur.