PI3K-AKT通路对脑缺血损伤神经干细胞的增殖作用

关键词：PI3K-AKT信号通路  神经干细胞  腺病毒AKT载体  AKT干扰

Keywords:PI3K - AKT pathway  neural stem cells  adenovirus AKT carrier  AKT interference

摘要：

      摘要 目的：利用低氧干预分离的小鼠神经干细胞，通过构建腺病毒载体将腺病毒5-丝氨酸-苏氨酸蛋白激酶3（Ad5-AKT3）基因和丝氨酸-苏氨酸蛋白激酶3小干扰RNA（AKT3 siRNA）干扰转染至缺血神经干细胞，探索磷酸肌醇三磷酸激酶-丝氨酸-苏氨酸蛋白激酶（PI3K-AKT）通路对缺血性神经干细胞的增殖作用。 方法：取新生24h昆明小鼠海马，分离培养原代神经干细胞，并采用巢蛋白（Nestin）免疫荧光检测和5-溴脱氧尿嘧啶核苷（Brdu）渗入实验对其进行鉴定；将所培养的传代神经干细胞随机分组：正常组、缺血模型组、低氧组（低氧+缺血模型组）、Lip2000组（低氧+缺血模型组+Lip2000空转染组）、AKT转染组（低氧+缺血模型组+Ad5-AKT3转染组）、AKT干扰组（低氧+缺血模型组+Lip2000+AKT3 siRNA干扰组）。建立脑缺血损伤的体外模型，低氧干预，构建腺病毒Ad5-AKT3载体和AKT3 siRNA后，各组行MTT检测；RT-qPCR检测AKT3 mRNA表达情况；Werstern Blot检测PI3K、AKT3、PCNA。 结果：通过免疫荧光检测Nestin鉴定神经干细胞球，在荧光显微镜下观察到绿色明亮且典型的细胞球，球内可见清晰结构；渗入Brdu免疫荧光检测可见整个着色细胞球。MTT OD值和RT-qPCR检测AKT3 mRNA均显示，与正常组相比，模型组和AKT干扰组下降（P＜0.05），其余各组均升高（P＜0.05），其中低氧组和Lip2000组之间无明显差异（P＞0.05）。与模型组相比，除AKT干扰组下降外（P＜0.05），其余各组均升高（P＜0.05）。Western Blot检测PI3K、AKT3、PCNA蛋白表达与正常组相比，模型组和AKT干扰组表达量减少（P＜0.05），低氧组、Lip2000组和AKT转染组均增加（P＜0.05），其中AKT转染组差异最明显（P＜0.01）。 结论：PI3K-AKT信号通路在低氧干预下有效促进神经干细胞的增殖过程。

Abstract:

      Abstract Objective: Using low oxygen intervention in the separation of neural stem cells in mice, by constructing adenovirus vector Ad5 - AKT3 gene and AKT3 siRNA interference transfection to ischemia neural stem cells, to determine the role of PI3K - AKT pathway effect on in ischemic neural stem cell proliferation. Method: Twenty-four hours Kunming mice’s hippocampus was isolated. The original generation of neural stem cells were cultured, identifying by the Nestin immunofluorescence test and Brdu infiltration experiments. Passage neural stem cell randomly assigned to normal group, model group, ischemia and hypoxia group (low oxygen + ischemia model group), Lip2000 group (low oxygen + ischemia model + Lip2000 empty transfection group), AKT transfection group (low oxygen + Ad5-AKT3 transfection group + ischemia model group), AKT interference group (low oxygen + Lip2000 + ischemia model group + Akt3 siRNA interference group). After establishment of in vitro model of cerebral ischemia injury, low oxygen intervention, constructing adenovirus Ad5 - AKT3 carrier and AKT3 siRNA,each group was determined by MTT detection, RT - qPCR; and Western Blot detection of PI3K, AKT3 and PCNA. Result: neural stem cells are identified by immunofluorescence test of Nestin, Typical cell balls with green bright and its clear structure were observed under fluorescence microscope. Infiltration of Brdu immune fluorescence detected the entire staining cells. Determined by MTT OD value and RT-qPCR detection and comparing with normal group, AKT3 mRNA in model group and AKT interference group decreased (P＜0.05), the rest of the groups increased (P＜0.05). There is no significant difference (P＞0.05) between the low oxygen group and Lip2000 group Comparing with model group, except falling in AKT interference group (P＜0.05), the other groups increased (P＜0.05). Comparing with normal group, PI3K, AKT3, PCNA protein expression, decreased in model group and AKT interference group(P＜0.05), increased in low oxygen group, Lip2000 group and AKT transfection group(P＜0.05)., including Among those, AKT transfection group increased most obviously (P＜0.01). Conclusion: Low oxygen promotes neural stem cells proliferation effectively through PI3K - Akt signaling pathway.