碱性成纤维细胞生长因子对新生大鼠脑缺血后神经新生的影响

目的 研究碱性成纤维细胞生长因子(bFGF)对新生大鼠脑缺血后神经新生的影响.方法 通过结扎3日龄新生SD大鼠双侧颈总动脉制备脑缺血模型,随机分为治疗组(36只):给予bFGF 10 ng/g侧脑室注射;对照组(36只)给予相同体积的生理盐水.另取36只新生大鼠,仅分离双侧颈总动脉,不结扎,不给予药物,作为假手术组.三组大鼠分别于术后第4、7、14天处死,获取脑组织标本,采用免疫荧光染色、Western印记和实时PCR方法,观察三组大鼠不同时点脑室下区巢蛋白(nestin)、Ⅲ型β-微管蛋白(Tuj1)、胶质纤维酸性蛋白(GFAP)和少突胶质细胞NG2蛋白及其mRNA的表达变化.结果 (1)免疫荧光染色:治疗组BrdU+/nestin+细胞数目术后7 d达高峰[(48.7±5.9)个/视野],高于同时点对照组[(32.2±3.1)个/视野]和假手术组[(17.3±1.6)个/视野],差异有统计学意义(P<0.01);治疗组BrdU+/Tuj1+细胞、BrdU+/GFAP+细胞、BrdU+/NG2+细胞数目术后14 d[分别为(92.6±9.7)、(58.2±6.1)、(57.3±5.4)个/视野]达高峰,高于同时点对照组[分别为(65.8±7.1)、(42.1±4.4)、(37.8±3.2)个/视野]和假手术组[分别为(35.3±3.1)、(33.6±3.4)、(22.4±2.1)个/视野],差异有统计学意义(P<0.01).(2)Western印迹和实时PCR:与免疫荧光染色结果一致,即治疗组nestin蛋白和mRNA表达术后7 d达高峰,高于同时点对照组和假手术组,差异有统计学意义(P<0.01);治疗组Tuj1、GFAP和NG2蛋白和mRNA术后14 d达高峰,高于同时点对照组和假手术组,差异有统计学意义(P<0.01).结论 bFGF可促进新生大鼠脑缺血后脑室下区神经干细胞的增殖以及向功能神经细胞(神经元、星形胶质细胞、少突胶质细胞)的分化,可能对新生大鼠脑缺血后神经细胞的再生具有促进作用.

Effects of basic fibroblast growth factor on neurogenesis in the subventricular zone of 3-day-old rats following brain ischemia

Objective To investigate the effects of exogenous basic fibroblast growth factor (bFGF) on neurogenesis in the subventricular zone (SVZ) of 3-day-old rats following brain ischemia. Methods Seventy-two brain ischemia models of 3-day-old SD rats were established by ligating bilateral common carotid artery. The models were divided into treatment group with 10 ng/g bFGF injection and the control group with saline. Another 36 rats underwent operation without ischemia served as sham-operate group. Proliferating cells were labeled by bromodeoxyuridine (BrdU) through intraperitoneal injection in a cumulative protocol. Rats were killed at 4, 7 and 14 days after ischemic injury. Immunofluoreseence assays, Western blot and real-time PCR were used to observe cellular changes, including the protein expression of nestin, Tuj1, glial fibrillary acidic protein (GFAP) and NG2, and also their mRNA expression in SVZ, respectively. Results (1) Immunofluorescence assay showed that the number of BrdU1/nestin+ cells in SVZ of treatment group was the highest at day 7 after operation [(48. 7±5.9)/field], higher than those in control [(32. 2±3. 1)/field] and sham-operate group [(17.3±1.6)/field] (P<0. 01). The number of BrdU+/Tuj1+ cells, BrdU+/ GFAP+ cells and BrdU+/NG2+ cells in SVZ of treatment group were highest at day 14 after brain ischemia [(92.6±9.7) ,(58.2±6.1) ,(57.3±5.4)/field, respectively], higher than those in control [(65. 8±7. 1), (42. 1 ± 4. 4), (37.8 ± 3. 2)/field, respectively] and sham-operate group [(35.3± 3. 1), (33.6± 3.4), (22.4 ± 2. 1)/field, respectively] (P<0. 01 ). (2) Western blot and real-time PCR analysis found that the expressions of nestin protein and mRNA peaked on day 7, and the expressions of Tuj1, GFAP, and NG2 protein and mRNA peaked on day 14 after operation. The highest expression of nestin, Tuj1, GFAP, and NG2 protein and mRNA were detected in the treatment group. Conclusions bFGF not only increases the proliferation of neural stem cells in SVZ, but also stimulates the differentiation of these cells into neurons, astrocytes, and oligodendroeytes after ischemic injury, bFGF helps repair neonatal ischemic brain injury in rats.