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目的探讨整合素连接激酶(ILK)/磷酸化蛋白激酶B(Akt)信号通路在新生鼠缺氧缺血性脑损伤(HIBD)修复中的作用。方法采用10日龄SD新生大鼠随机分为假手术组(n=3)和缺氧缺血组(HI组,n=23),建立HIBD模型,于HI后0、4、6、12、24、48、72 h处死取脑,免疫荧光法检测ILK表达与分布,Western blot检测ILK、Akt、磷酸化蛋白激酶B(p-Akt)、血管内皮生长因子(VEGF)的表达情况。构建靶向ILK RNA干扰的慢病毒载体,抑制新生鼠脑组织中ILK的表达。右侧侧脑室分别注射含有LV-ILK shRNA慢病毒(n=15)和LV-control慢病毒对照(n=3),建立HIBD模型,于HI后4 h和24 h处死动物取脑,Western blot检测ILK、Akt、p-Akt、VEGF蛋白的表达变化,TUNEL染色检测细胞凋亡。结果 ILK主要表达于皮层和海马区,定位于胞浆和胞膜,在假手术组和HI组均有表达。ILK于HI后表达开始逐渐增加,24 h达高峰,之后表达有所降低。p-Akt于HI后4 h明显增加,后逐渐降低,24 h降至最低,后又增加,在48 h达高峰。VEGF于HI后4 h表达开始增加,12 h达高峰,后维持较高水平。构建的靶向ILK RNA干扰的慢病毒载体在体内应用获得成功。注射慢病毒LV-ILK shRNA组在HI 4 h、24 h时所表达的ILK、p-Akt、VEGF均明显低于同一时间点的LV-control组,同时细胞凋亡明显增加。结论新生大鼠HIBD后,ILK、p-Akt、VEGF蛋白表达均增高,通过抑制ILK的表达,能够降低新生大鼠HIBD后p-Akt和VEGF蛋白表达,增加细胞凋亡。提示HIBD后可能通过ILK/Akt信号通路,增加VEGF表达,进而促进神经细胞存活及血管再生,在新生鼠HI脑损伤修复中发挥作用。
Objective To investigate the role of ILK / Akt pathway in the repair of hypoxic-ischemic brain damage (HIBD) in neonatal rats. Methods 10-day-old SD neonatal rats were randomly divided into sham operation group (n = 3) and hypoxia-ischemia group (HI group, n = 23) The brain was sacrificed at 24, 48 and 72 h, the expression and distribution of ILK were detected by immunofluorescence, and the expressions of ILK, Akt, p-Akt, and VEGF were detected by Western blot. Construction of lentiviral vector targeting ILK RNA interference inhibits ILK expression in brain tissue of neonatal rats. The right lateral ventricle was injected with LV-ILK shRNA lentivirus (n = 15) and LV-control lentivirus (n = 3) respectively to establish HIBD model. Animals were sacrificed at 4 h and 24 h after HI, The expressions of ILK, Akt, p-Akt and VEGF were detected by TUNEL staining. Results ILK mainly expressed in the cortex and hippocampus, localized in the cytoplasm and membrane, and expressed both in sham operation group and HI group. The expression of ILK began to increase gradually after HI, reached the peak at 24 h, and then decreased. p-Akt at 4 h after HI increased significantly, then gradually decreased, 24 h reduced to a minimum, and then increased at 48 h peak. The expression of VEGF began to increase 4 h after HI, peaked at 12 h, and maintained at a high level afterwards. The constructed lentiviral vector targeting ILK RNA interference was successfully used in vivo. The expression of ILK, p-Akt and VEGF in LV-ILK shRNA group at 4 h and 24 h after injection of lentivirus was significantly lower than that of LV-control group at the same time point, meanwhile, the apoptosis rate of LV-ILK shRNA group increased obviously. Conclusions The expression of ILK, p-Akt and VEGF in HIBD of neonatal rats are increased. By inhibiting the expression of ILK, the expression of p-Akt and VEGF in HIBD can be decreased and the apoptosis of neonatal rats can be increased. It is suggested that HIBD could increase the expression of VEGF through ILK / Akt signaling pathway, and then promote neuronal cell survival and angiogenesis, which play a role in the repair of HI brain injury in neonatal rats.