吉林大学学报(医学版) ›› 2023, Vol. 49 ›› Issue (3): 706-713.doi: 10.13481/j.1671-587X.20230320

• 基础研究 • 上一篇    下一篇

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

杨贺然1,李兴江2,胡嘉航3,李彦伟1()   

  1. 1.牡丹江医学院附属红旗医院检验科,黑龙江 牡丹江 157000
    2.牡丹江医学院解剖教研室,黑龙江 牡丹江 157000
    3.牡丹江医学院附属红旗医院影像科,黑龙江 牡丹江 157000
  • 收稿日期:2022-06-24 出版日期:2023-05-28 发布日期:2023-06-20
  • 通讯作者: 李彦伟 E-mail:609633675@qq.com
  • 作者简介:杨贺然(1988-),女,黑龙江省牡丹江市人,主管技师,医学硕士,主要从事糖尿病微血管病变发病机制方面的研究。
  • 基金资助:
    黑龙江省科技厅自然科学基金联合引导项目(LH2020H074)

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

Heran YANG1,Xingjiang LI2,Jiahang HU3,Yanwei LI1()   

  1. 1.Department of Laboratory,Affiliated Hongqi Hospital,Mudanjiang Medical College,Mudanjiang 157000,China
    2.Department of Anatomy,Mudanjiang Medical College,Mudanjiang 157000,China
    3.Department of Imaging,Affiliated Hongqi Hospital,Mudanjiang Medical College,Mudanjiang 157000,China
  • Received:2022-06-24 Online:2023-05-28 Published:2023-06-20
  • Contact: Yanwei LI E-mail:609633675@qq.com

摘要:

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600 μg·L-1 Ghrelin)、U0126组(给予40 ng·L-1 U0126)、Ghrelin+U0126组(给予600 μg·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10-10 g·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。

关键词: 胃饥饿素, 丝裂素活化蛋白激酶, 细胞外信号调节激酶, 脂肪间充质干细胞, 神经元分化

Abstract:

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 600 μg·L-1 Ghrelin), U0126 group (given 40 ng·L-1 U0126), Ghrelin+U0126 group (given 600 μg·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 round-like 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.

Key words: Ghrelin, Mitogen-activated protein kinase, Extracellular signal-regulated kinase, Adipose-derived mesenchymal stem cells, Neural differentiation

中图分类号: 

  • R394.2