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目的探讨脑出血后不同时期血肿周围脑组织中神经营养因子受体同源物2(NRH2)、神经生长因子前体(pro NGF)、sortilin、神经营养因子受体p75(p75NTR)表达及与细胞凋亡的关系。方法收集临床脑出血后实施血肿清除术的周围组织标本,根据脑出血距离标本取出时间,分为6h以内(包括6h)、6h以上至24h(包括24h)、24h以上至72h(包括72h)、72h以上4组,同时取10例手术过程中血肿远隔部位掉落的组织块作为对照组,通过末端脱氧核糖核酸转移酶介导的脱氧尿苷三磷酸缺口末端标记法(TUNEL)测定脑细胞的凋亡指数(AI),采用实时荧光定量PCR、Western blot法分别检测脑组织中NRH2、pro NGF、sortilin、p75NTR mRNA和蛋白表达,Western blot法检测脑组织中Bcl-2、Bax的表达。体外培养大鼠皮层星形胶质细胞,经NRH2 siRNA或scramble siRNA转染后,Western blot法检测pro NGF、sortilin、p75NTR蛋白表达。结果与对照组以及6h以内的脑出血组相比,脑出血后6h以上各组AI升高,以24h以上至72h内组为最高,但6h以内的脑出血组AI与对照组无区别。随着脑出血时间的延长,pro NGF、p75NTR mRNA和蛋白表达水平逐渐升高,24h以上至72h达到高峰,72h后仍处于较高水平。与对照组和6h以内的脑出血组比较,脑出血后6h以上至24h内(包括24h),脑组织NRH2、sortilin的mRNA与蛋白表达水平及Bax表达水平即增加,24h以上至72h内达高峰,72h后仍维持在较高水平。但与对照组比较,6h以内的脑出血组上述指标无显著性差异。与对照组相比,6h以内的脑出血组Bcl-2表达水平无明显变化,脑出血后6h以上的脑出血各组脑组织Bcl-2表达水平降低,以24h以上至72h内处于最低值。NRH2 siRNA组pro NGF、sortilin、p75NTR蛋白表达水平明显低于空白对照组、scramble siRNA组。结论 NRH2在脑出血后血肿周围脑组织中表达增加,通过促进pro NGF、sortilin、p75NTR表达增加Bax/Bcl-2比率,从而诱导脑细胞凋亡。
Objective To investigate the expression of neurotrophic factor receptor 2 (NRH2), pro NGF, sortilin and p75NTR in brain tissue around hematoma after intracerebral hemorrhage The relationship between apoptosis. Methods The samples of peripheral tissues after hematoma removal were collected from patients with intracerebral hemorrhage (ICH). The patients were divided into 6h (including 6h), 6h or more (including 24h), 72h (including 72h) 72h more than 4 groups, while taking 10 cases of surgical hematoma removed from the distant parts of the tissue block as a control group by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) determination of brain cells (AI). The mRNA and protein expressions of NRH2, pro NGF, sortilin and p75NTR in brain tissues were detected by real-time fluorescence quantitative PCR and Western blot respectively. The expressions of Bcl-2 and Bax in brain tissues were detected by Western blot. Rat cortical astrocytes were cultured in vitro. After transfection with NRH2 siRNA or scramble siRNA, the protein expression of pro NGF, sortilin and p75NTR was detected by Western blot. Results Compared with the control group and the intracerebral hemorrhage group within 6h, the AI in all groups above 6h after intracerebral hemorrhage increased to more than 24hrs and reached the highest within 72hs, but there was no difference between the intracerebral hemorrhage groups within 6h and the control group. With the prolongation of ICH, the mRNA and protein expressions of pro NGF and p75NTR gradually increased, reaching the peak at more than 24 hours and reaching the peak at 72 hours and still at a high level after 72 hours. Compared with the control group and the intracerebral hemorrhage group within 6h, the mRNA and protein expression levels of NRH2 and sortilin and the expression of Bax in brain tissue increased from 6h to 24h (including 24h) after cerebral hemorrhage, and reached the peak within 24 h and up to 72h , 72h still remained at a high level. However, compared with the control group, within 6h of cerebral hemorrhage group of the above indicators no significant difference. Compared with the control group, the expression level of Bcl-2 in ICH group within 6h had no significant change, and the Bcl-2 level in ICH group decreased more than 6h after cerebral hemorrhage, and reached the lowest level within 24h to 72h. NRH2 siRNA group pro NGF, sortilin, p75NTR protein expression was significantly lower than the blank control group, scramble siRNA group. Conclusion The expression of NRH2 increases in the perihematomal brain tissue after intracerebral hemorrhage. The expression of proNGF, sortilin and p75NTR increases the ratio of Bax / Bcl-2, thereby inducing apoptosis of brain cells.