P13K／AKT信号通路在一氧化氮对肠神经干细胞增殖分化中的作用

Role of PI3K/AKT signaling pathway in nitric oxide on the proliferation and differentiation of enteric neural stem cells

目的通过抑制P13K／AKT信号通路，探讨该信号通路在一氧化氮对体外培养的大鼠肠神经干细胞增殖、分化的调控情况。方法从孕15d胚鼠肠道提取肠神经干细胞，传代后的肠神经干细胞分为LY294002（25μmol/L）组、LY294002（50μmol／L）组及空白对照组，培养48h后，形态学观察分析各组的神经球大小情况，流式细胞仪检测各组肠神经干细胞nestin、BrdU标记的增殖细胞、子代细胞神经元Tuj-1染色阳性细胞百分比；传代后的肠神经干细胞分为50〉tool／1DETA／NO＋20ng／mlEGF组、100μmol／LL-NAME＋20ng／mlEGF组及空白对照组（20ng／mlEGF），培养1hA，利用WesternBlot法检测及gel—pro图像软件分析各组P-AKT磷酸化水平。结果形态学观察发现LY294002（50μmol／L）组神经球较小、部分细胞有退化现象、LY294002（25ffmol／L）组次之、空白对照组神经球较大；流式检测发现LY294002（25μmol／L）组与空白对照组相比，肠神经干细胞nestin百分比显著降低[（18．42±1．22）％比（27．44±1．08）％，（P〈0．05）]，BrdU标记的增殖细胞百分比显著降低[（2．20±0．30）％比（12．27±0．59）％，（P〈0．05）]，神经元Tuj-1百分比显著增加[（17．23±0．78）％比（13．98±0．53）％，（P〈0．05）]；而LY294002（50μmol／L）组与空白对照组相比，肠神经干细胞nestin百分比[（8．77±0．90）％比（27．44±1．08）]、BrdU标记的增殖细胞百分比[（0．62±0．03）％比（12．27±0．59）]及神经元百分比[（9．88±i．22）％比（13．98±0．53）％]差异均有统计学意义（P〈0．05）。WesternBlot法检测发现100μmol／LL-NAME＋20ng／mlEGF组P-AKT磷酸化的程度显著增强[（15．71±1．09）％比（39．42±1．02）％，P〈0．053，50μmol／1 DETA／NO＋20ng／mlEGF组显著降低[（58．44±1．73）％比（39．42±1．02）％，P〈0．05]。结论一氧化氮能抑制肠神经干细胞增殖、促进其向神经元分化，其作用机制与抑制P13K／AKT信号通路有关。

Objective To explore the role of phosphoinositide-3-kinase （PBK）/AKT signaling pathway in nitric oxide on the in vitro proliferation and differentiation of enteric neural stern cells （ENSCs）. Methods ENSCs were isolated from gut of 15-day-old rat embryos and cultured into 3 groups of LY294002 25 μmol/L （PI3-K inhibitor） intervention, LY294002 50 μmol/L intervention and control. After 48h culture, neurospheric sizes in each group were observed. The percentages of positive cells for nestin, 5-bromo-2-deoxyuridine （BrdU） and Tuj-1 were measured by flow eytometry. And ENSCs were also cultured into 3 groups of DETA/NO 50μmol/L ± 20 ng/ml EGF intervention, L-NAME100μmol/L± 20 ng/ml EGF intervention and control （20 ng/ml EGF）. After lh culture, quantitative Western blot and gel-pro imaging system were used to detect the levels of phosphorylated- AKT level. Results Neurospheric sizes were the smallest, some cells had a phenomenon of degradation in LY294002（50/%mol/L） group and the largest in control and LY294002 （25 /μmol/L） groups. Flow cytometry showed that positive rates for nestin decreased significantly [（ 18. 42 ± 1.22） %//0 vs （27. 44 ± 1.08） %, P〈0. 05], BrdU decreased significantly [2. 20 ± 0. 30） % vs （12. 27 ±0. 59）, P〈0. 05] and Tuj-1 increased significantly [17. 23 ± 0. 78） % vs （13. 98 -± 0. 53） %, P〈0. 05] in LY294002 （25 μmol/L） group. There were all significant decreases （P〈0. 05） for nestin, BrdU and Tui-1 in LY294002 （50μmol/L） group （8. 77 ± 0. 90） % vs （27. 44 ± 1.08） % （0. 62 ± 0. 03） % vs （12. 27 ± 0. 59）% （9. 88 ± 1.22）% vs （13. 98 ± 0. 53）%. Western blot quantitative analysis showed that the expression level of P-AKT increased significantly （P〈0. 05） in L-NAME （100μmol/L） ± 20 ng/ml EGF （ 15.71 ± 1.09 ）% vs （ 39. 42 ± 1.02 ） and decreased significantly in DETA/NO （50μmol/L）＋20ng/mlEGF[（15.71±1. 09）% vs （39.42±1.02）, P〈0.05]. Conclusions Nitric oxide decreases the proliferation of ENSCs and promotes its differentiation into neurons through an inhibition of PI3K/AKT pathway.