PTEN通过拮抗PI3K/Akt信号通路抑制神经干细胞增殖
PTEN inhibits the proliferation of neural stem cells via the antagonism of PI3K/Akt signaling pathway

摘要：目的 探讨PTEN抑制神经干细胞增殖的作用，以阐明是否可以通过抑制PTEN蛋白表达来促进神经干细胞增殖。方法 取新生24h昆明小鼠海马组织，分离培养原代神经干细胞，并采用免疫荧光检测鉴定；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)、PTEN转染组(低氧+缺血模型组+Ad5-PTEN转染组)、PTEN干扰组(低氧+缺血模型组+Lip2000+PTENsiRNA干扰组)，检测0、6、24、36、48h不同时间点各组神经干细胞的增殖情况，同时检测PTEN转染后PTEN蛋白在各组神经干细胞的表达情况，以及对PI3K-Akt信号通路上标志性蛋白表达的影响。结果 免疫荧光检测Nestin以鉴定神经干细胞球，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。MTT测定发现低氧组、Lip2000组培养36h时神经干细胞出现明显增殖，与模型组、PTEN转染组及PTEN干扰组相比，差异有统计学意义(P<0.05)，其中PTEN干扰组的细胞增殖又较模型组、PTEN转染组明显，差异亦有统计学意义(P<0.05)；与正常组细胞比较，模型组与PTEN转染组的PTEN表达升高(P<0.05)，且均较正常组降低(P<0.05)，其中低氧组、Lip2000组与PTEN干扰组降低的趋势相当(P>0.05)。PTEN质粒转染后各组总的Akt和bad未出现明显变化，但是与正常组比较，模型组与PTEN转染组的PI3K、p-Akt、p-bad表达降低(P<0.05)，其中PTEN转染组的变化较为明显。低氧组、Lip2000组与PTEN干扰组的PI3K、p-Akt、p-bad表达升高(P<0.05)。结论 低氧有助于促进神经干细胞增殖，PTEN对PI3K/Akt的抑制作用可能是成体神经干细胞增殖受阻的关键因素。
ABSTRACT: Objective To explore the role of PTEN in the suppression of neural stem cells so as to clarify whether neural stem cell proliferation can be promoted by regulating the PI3K-Akt/PTEN expression level. Methods We removed the hippocampus from neonatal 24h Kunming mice, isolated and cultured the generation of neural stem cells, which were then identified by immunofluorescence test. We randomly grouped the cultured neural stem cells into normal group, ischemia model group, hypoxia group (hypoxia+ischemia model group), Lip2000 group (hypoxia+ischemia model group+Lip2000 null transfection group, PTEN transfection group (hypoxia+ischemia model group+Ad5-PTEN transfection group), and PTEN interference group (hypoxia+ischemia model group+Lip2000+PTENsiRNA interference group). We detected the proliferation of neural stem cells in the groups at different time points, and PTEN protein expression of neural stem cells in each group after PTEN transfection, and the effect on iconic protein on Akt-PI3K signaling pathway. Results (1) Nestin identification of the neural stem cells was tested by immunofluorescence. We observed green bright and typical cell spheroids under fluorescence microscope; the clear structure could be seen within spheroids. (2) We determined the proliferation of neural stem cells at different time points by MTT. After 36h of culture, the neural stem cells had obvious proliferation in the hypoxia group and Lip2000 group compared with the model group, the PTEN transfection group and PTEN interference group (P<0.05), including the cell proliferation of PTEN interference group compared to that of the model group, and PTEN transfection group obviously, the differences were also significant (P<0.05); (3) Compared with