内皮素-B受体在脑缺血大鼠脑内小胶质细胞中的表达

Expression of endothelin-B receptors in the microglia following cerebral ischemia in adult rats

目的研究脑缺血后内皮素-B(ET-B)受体在脑内激活的小胶质细胞中的表达变化,探讨ET-B受体与脑缺血的关系。方法应用微创开颅法建立大鼠大脑中动脉闭塞(MCAO)模型,81只大鼠随机分为2h、6h、12h、1d、2d、3d和1周7个缺血组,以及正常对照组和假手术组(n=9)。用抗凝集素(lectin)和抗ET-B抗体进行免疫荧光双标染色观测脑缺血后激活的小胶质细胞是否表达ET-B受体;实时定量PCR和免疫印迹技术检测脑缺血后ET-B受体mRNA及蛋白的表达变化。结果缺血后1d、2d、3d和1周组中,在缺血坏死与正常组织交界区即半暗区大脑皮质内,可见大量激活的小胶质细胞,且该细胞中ET-B受体表达阳性,以缺血后3d和1周表达最强,假手术组和正常对照组未见小胶质细胞的激活和ET-B受体的表达;脑缺血后ET-B受体mRNA和蛋白表达上调,与对照组比较缺血后2h、6h、12h ET-B mRNA和蛋白表达明显升高(P<0.05),6h达到高峰,伤后1周恢复接近正常水平。结论脑缺血后大鼠脑内大量小胶质细胞聚集和激活,与胚胎时期和生后早期的阿米巴样小胶质细胞相似,能表达ET-B受体,提示脑内内皮素系统可能通过自分泌方式参与脑缺血的病理过程。

Objective To explore the relationship between the endothelin-B （ET-B） receptors and cerebral ischemia by studying the expression of endothelin-B receptors in the activated microglial cells. Methods The rat model of middle cerebral artery occlusion （MCAO） was established by minimal invasive eraniotomy. Eighty one adult male SD rats were randomly divided into ischemia group including 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days and 1 week after ischemia （at each time point, n =9） ; a sham operation group （n =9） and normal control group （n = 9）. ET-B receptor immunoreactivity was observed in activated mieroglial cells following MCAO by double labeling with lectin. The expression of ET-B receptors mRNA and protein were investigated by Western blotting and real-time polymerase chain reactions （RT- PCR）. Results ET-B immunofluoreseence was markedly induced in the activated mieroglial cells in both infarcted and peri-infacted zones by double labeling with lectin at the 1st day, 2nd day, 3rd day and the 1st week, especially at the 3rd day and the 1st week after MCAO. In the control and sham operated rats, microglial cells appeared and relatively unactivated and showed a lack of ET-B immunofluoresecence. ET-B mRNA and protein levels were steadily upregulated from 2 hours to 12 hours （P 〈0. 05） after MCAO as compared with the controls, peaking at the 6th hours. At the 1st week, ET-B mRNA and protein levels were only marginally elevated above the control level. Conclusion A major finding of this study is the massive accumulation of activated mieroglia and the induced expression of ET-B receptors in activated microglial cells following MCAO. It is suggested that ETs via ET-B may act on activated microglia and play an important role in cerebral ischemic injury bearing the receptor.