Ghrelin通过调节MAPK/ERK通路对脂肪间充质干细胞神经元分化的影响

Effect of Ghrelin on neural differentiation of adipose-derived mesenchymal stem cells by regulating MAPK/ERK pathway

目的:探讨Ghrelin对脂肪间充质干细胞(ADSCs)向神经元分化的影响,并阐明可能的机制。方法:ADSCs随机分为空白组、神经分化诱导剂组、Ghrelin组(给予600μg·L^(-1)Ghrelin)、U0126组(给予40 ng·L^(-1)U0126)、Ghrelin+U0126组(给予600μg·L^(-1)Ghrelin+40 ng·L^(-1)U0126)和Ghrelin受体阻断剂D-赖氨酰3生长激素释放肽6(D-Lys3-GHRP-6)组(给予10-10g·L^(-1)D-Lys3-GHRP-6)。倒置显微镜观察各组细胞病理形态表现,免疫荧光法检测各组细胞中神经丝蛋白(NF)-200和微管蛋白(Tuj-1)阳性表达率,Western blotting法检测各组细胞中丝裂素活化蛋白激酶(MAPK)/细胞外信号调节激酶(ERK)通路蛋白、神经元特异性核蛋白(NeuN)及Tuj-1、神经元特异性烯醇化酶(NSE)、NF、生长激素促分泌受体(GHSR)、胎肝激酶1(Flk1)和CD29蛋白表达水平。结果:细胞培养第10天,空白组ASCs呈长梭形和螺旋状融合;神经分化诱导剂组细胞胞体收缩,长梭形胞体向类圆形转变、突起长度延长及类似神经元样细胞数增多;Ghrelin组细胞胞体突起进一步增多,类圆形细胞数进一步增多;U0126组和D-Lys3-GHRP-6组仅有少量细胞胞体收缩及类圆形转变。免疫荧光法检测,与空白组比较,神经分化诱导剂组细胞中NF-200和Tuj-1阳性表达率升高(P<0.05);与神经分化诱导剂组比较,Ghrelin组细胞中NF-200和Tuj-1阳性表达率升高(P<0.05),U0126组和D-Lys3-GHRP-6组细胞中NF-200及Tuj-1阳性表达率降低(P<0.05);与Ghrelin组比较,U0126组、D-Lys3-GHRP-6组和Ghrelin+U0126组细胞中NF-200和Tuj-1阳性表达率降低(P<0.05)。Western blotting法检测,与空白组比较,神经分化诱导剂组细胞中NSE、NeuN、Tuj-1、NF、GHSR蛋白表达水平和磷酸化MAPK(p-MAPK)/MAPK及磷酸化ERK(p-ERK)/ERK比值升高(P<0.05),Flk1和CD29蛋白表达水平降低(P<0.05);与神经分化诱导剂组比较,Ghrelin组细胞中NSE、NeuN、Tuj-1、NF、GHSR蛋白表达水平和p-MAPK/MAPK及p-ERK/ERK比值升高(P<0.05),Flk1和CD29蛋白表达水平降低(P<0.05),U0126组和D-Lys3-GHRP-6组细胞中NSE、NeuN、Tuj-1、NF、GHSR蛋白表达水平及p-MAPK/MAPK和p-ERK/ERK比值降低(P<0.05),Flk1和CD29蛋白表达水平升高(P<0.05);与Ghrelin组比较,U0126组、D-Lys3-GHRP-6组和Ghrelin+U0126组细胞中NSE、NeuN、Tuj-1、NF和GHSR蛋白表达水平及p-MAPK/MAPK和p-ERK/ERK比值降低(P<0.05),Flk1和CD29蛋白表达水平升高(P<0.05)。结论:Ghrelin可诱导ASCs向神经元分化,其作用机制与激活MAPK/ERK通路有关。

Objective:To discuss the effect of ghrelin on the differentiation of the adipose mesenchymal stem cells(ADSCs)into the neurons,and to clarify its possible mechanism.Methods:The ADSCs were randomly divided into blank group,neural differentiation inducer group,and ghrelin group(given 600μg·L^(-1) Ghrelin),U0126 group(given 40 ng·L^(-1) U0126),Ghrelin+U0126 group(given 600μg·L^(-1) Ghrelin+40 ng·L^(-1) U0126),and Ghrelin receptor blocker D-Lys3-GHRP-6(D-Lys3-GHRP-6)group(given 10-10 g·L^(-1) D-Lys3-GHRP-6).The pathomorphology of the cells in various groups was observed under inverted microscope;the positive expression rates of neurofilament protein(NF)-200 and tubulin(Tuj-1)in the cells in various groups were detected by immunofluorescence method;the expression levels of the mitogen activated protein kinase(MAPK)/extracellular signal-regulated kinase(ERK)pathway proteins,neuron specific nuclear protein(NeuN),Tuj-1,neuron specific enolase(NSE),NF,growth hormone secretagogne receptor(GHSR),fetal liver kinase 1(Flk1),and CD29 proteins in the cells in various groups were detected by Western blotting method.Results:On the 10th day after cell culture,the ADSCs in blank group showed the fusions of long spindle and spiral shapes;the body of the cells in neural differentiation inducer group contracted,and the long spindle like cell body was transformed into the roundlike shape,the protrusion length extended,and the number of neuron-like cells was increased;in Ghrelin group,the number of body protrusions of the cells was increased and the number of round-like like cells was increased;there was a small amount of cell body contraction and circular transformation in U0126 group and D-Lys3-GHRP-6 group.The immunofluorescence assay results showed that compared with blank group,the positive expression rates of NF-200 and Tuj-1 in the cells in neural differentiation inducer group were increased(P<0.05);compared with neural differentiation inducer group,the positive expression rates of NF-200 and Tuj-1 in the cells in Ghrelin group were increased(P<0.05),while the positive expression rates of NF-200 and Tuj-1 in the cells in U0126 group and D-Lys3-GHRP-6 group were decreased(P<0.05);compared with Ghrelin group,the positive expression rates of NF-200 and Tuj-1 in the cells in U0126 group,D-Lys3-GHRP-6 group,and Ghrelin+U0126 group were decreased(P<0.05).The Western blotting results showed that compared with blank group,the expression levels of NSE,NeuN,Tuj-1,NF,and GHSR proteins,and the ratios of phosphorylated MAPK(p-MAPK)/MAPK and phosphorylated ERK(p-ERK)/ERK in the cells in neural differentiation inducer group were increased(P<0.05),while the expression levels of Flk1 and CD29 proteins were decreased(P<0.05);compared with neural differentiation inducer group,the expression levels of NSE,NeuN,Tuj-1,NF,and GHSR proteins,and the ratios of p-MAPK/MAPK and p-ERK/ERK in the cells in Ghrelin group were increased(P<0.05),while the expression levels of Flk1 and CD29 proteins were decreased(P<0.05);the expression levels of NSE,NeuN,Tuj-1,NF,and GHSR proteins,and the ratios of p-MAPK/MAPK and p-ERK/ERK in the cells in U0126 and D-Lys3-GHRP-6 groups were decreased(P<0.05),and the expression levels of Flk1 and CD29 proteins were increased(P<0.05);compared with Ghrelin group,the expression levels of NSE,NeuN,Tuj-1,NF,and GHSR proteins,and the ratios of p-MAPK/MAPK and p-ERK/ERK in the cells in U0126 group,D-Lys3-GHRP-6 group,and Ghrelin+U0126 group were decreased(P<0.05),while the expression levels of Flk1 and CD29 proteins were increased(P<0.05).Conclusion:Ghrelin can induce the differention of the ASCs into the neurons,and it mechanism is related to activating the MAPK/ERK pathway.