高压氧促HIBD新生大鼠内源性神经干细胞增殖过程中Wnt-3蛋白的变化

目的：近年来的研究已经证实高压氧(hyperbaric oxygen, HBO) 能够促进缺氧缺血性脑损伤（Hypoxic ischemic brain damage, HIBD）新生大鼠内源性神经干细胞（Neural stem cells, NSCs）的增殖，并且发现Wnt 信号途径参与神经干细胞的增殖，故本课题将探讨 HBO 对 HIBD 新生大鼠脑内源性 NSCs 增殖分化过程中 Wnt-3 蛋白的变化的影响及其它们之间的相关性。方法：7 日龄 Sprague-Dawley 新生大鼠随机分为正常对照组（CON），缺氧缺血性脑损伤组（HIBD）及高压氧组（HBO）。采用经典的 Rice 法制成 HIBD 模型，HBO 组在 HIBD 后 3 h 内进行 HBO 治疗（压力为 2 个绝对大气压，稳压 1 h，每日 1 次，连续 7 d）。采用 BrdU/nestin， Wnt-3/ nestin 免疫荧光双标法，检测 HIBD 后 6 h，24 h，3 d，7 d，14 d，缺血侧侧脑室室管膜下区（subventricular zone, SVZ）增殖的 NSCs 及 Wnt-3 蛋白的动态变化，激光共聚焦显微镜（LSM 510，Zeiss）分析细胞内 nestin 蛋白与 Wnt-3 蛋白的荧光强度，统计学分析二者相关性。Western blotting 法检测缺血侧大脑半球nestin、Wnt-3 总蛋白的动态变化。结果 ：SVZ 区 BrdU+ nestin+ 细胞于 HBO 治疗后 3 h 开始增加，7 d 达最高水平，显著高于 CON 组与 HIBD组（P<0.01），14 d 时 BrdU+ nestin+ 细胞数开始下降，但仍多于CON 和 HIBD 两组（P<0.05）；NSCs 细胞膜表面 Wnt-3 蛋白于 HBO 治疗后 3 h 开始增加，3 d 时达最高水平，并维持较高水平至 14 d 时开始下降；直线回归分析示细胞内 Wnt-3 蛋白与 nestin 蛋白呈直线相关（r = 0.893, P<0.05）；Western blotting 分析示缺血侧大脑半球Wnt-3 蛋白于 HBO 后 3 d，7 d 一直维持较高水平，显著高于其他各时间点（P<0.05），至 14 d 时表达开始下降；nestin 总蛋白水平于 HBO 治疗后 21 h 开始增加（P<0.05），7 d 达最高水平，后下降。结论：高压氧可以促进 HIBD 新生大鼠内源性 NSCs 的增殖，其机制与 Wnt 信号活化有关。［中国当代儿科杂志，2007，9（3）：241-246］

Changes of Wnt-3 protein during the proliferation of endogenous neural stem cells in neonatal rats with hypoxic-ischemic brain damage after hyperbaric oxygen therapy

OBJECTIVE: Previous studies suggest that hyperbaric oxygen (HBO) treatment promotes the proliferation of neurocytes in neonatal rats following hypoxic-ischemic brain damage (HIBD). The Wnt signaling pathway is associated with neurogenesis. This study examined whether HBO promoted neural stem cells (NSCs) proliferation after HIBD, and whether that the proliferation correlated with Wnt-3 protein expression. METHODS: Seven-day-old Sprague-Dawley rats were randomly divided into three groups: normal control, hypoxia-ischemia (HI), and HI-HBO. HI was induced by the ligation of left common carotid artery, followed by a 2-hr exposure to 8% O2 in the latter two groups. HBO was administered 3 hrs after HI in the HI-HBO group for continuous 7 days (2 atmospheres absolute, once daily). The proliferating NSCs in the subventricular zone (SVZ) was examined by BrdU/nestin immunofluorescence and the expression of Wnt-3 protein in NSCs was examined by nestin/Wnt-3 immunofluorescence at 6 and 24 hrs and at 3, 7 and 14 days of HI. The cellular expressions of nestin and Wnt-3 protein were analyzed by laser scanning confocal microscopy. The linear regression analysis was used to evaluate the correlation between cellular Wnt-3 and nestin protein. The expressions of nestin and Wnt-3 protein in the ischemic cerebral hemisphere were analyzed with Western blotting. RESULTS: The number of BrdU/nestin positive cells in the SVZ increased 3 hrs after HBO therapy, peaked at 7 days and remained at a higher level until 14 days after HBO therapy in the HI-HBO group compared with the normal control and the HIBD groups. The level of Wnt-3 protein in NSCs increased significantly 3 hrs after HBO therapy, peaked at 3 days and remained at high levels until 14 days after HBO therapy in the HI-HBO group compared with the normal control and the HIBD groups. The level of cellular nestin protein was closely correlated with the level of cellular Wnt-3 protein (r = 0.893, P < 0.05). The Western blotting analysis demonstrated increased Wnt-3 and nestin protein expressions in the ischemic cerebral hemispheres. CONCLUSIONS: HBO treatment promotes the proliferation of NSCs in HIBD neonatal rats, which is correlated with the activation of Wnt signaling.