PTEN 通过拮抗 PI3K/Akt 信号通路抑制神经干细胞增殖

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24 h 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、48 h 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36 h 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 24 h 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 36 h 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 the cells in the normal group,PTEN expression increased in the model group and PTEN gene transfection group (P <0.05);it was all lower in other groups than in the normal group (P <0.05).The trend of decrease was similar in hypoxia group,Lip2000 group,and PTEN interference group (P >0.05);(4)After PTEN plasmid transfection,there were no apparent changes in the total Akt and bad in the groups,but compared with the normal group,PI3K,p-Akt,and p-bad expressions in model group and PTEN gene transfection group reduced (P <0.05),with the more obvious changes in the PTEN transfection group.PI3K,p-Akt,and p-bad expressions in the hypoxia group, Lip2000 group and PTEN interference group increased (P < 0.05 ). Conclusion Hypoxia contributes to promoting the proliferation of neural stem cells,and the inhibition of PTEN on PI3K/Akt might be the key factor for blocked proliferation of adult neural stem cells.