Ghrelin对脂肪间充质干细胞神经分化的影响

doi:10.13481/j.1671-587X.20220615

摘要/Abstract

摘要：

目的探讨Ghrelin联合LY294002对脂肪间充质干细胞（ADSCs）神经分化的影响，并阐明其作用机制。方法倒置相差显微镜下观察ADSCs形态表现，流式细胞术鉴定ADSCs表面抗体阳性表达率，将第4代ADSCs分为对照组（不进行干预）、LY294002组［给予40 μmol·L^－1磷酸酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）通路特异抑制剂LY294002］、Ghrelin组（给予0.1 μmol·L^－1 Ghrelin）、Ghrelin+LY294002组（给予40 μmol·L^－1 LY294002+0.1 μmol·L^－1 Ghrelin）。倒置相差显微镜下观察各组ADSCs神经分化后的形态表现，免疫荧光染色法检测各组ADSCs中神经元特异性烯醇化酶（NSE）、巢蛋白（Nestin）和胶质纤维酸性蛋白（GFAP）阳性细胞百分率，Western blotting法检测各组ADSCs中 PI3K/Akt通路蛋白表达水平，并计算磷酸化PI3K（p-PI3K）/PI3K和磷酸化Akt（p-Akt）/Akt比值。结果 ADSCs表面抗体CD90和CD13高表达，CD34、CD45和CD106低表达。与对照组比较，LY294002组仅部分细胞由长梭形转变为类圆形胞体，突起较短、数量减少，而Ghrelin组细胞由长梭形转变为类圆形胞体速度增快；与LY294002组比较，Ghrelin+ LY294002组细胞由长梭形转变为类圆形胞体速度增快，且突起数量增加。与对照组比较，LY294002组ADSCs中NSE和Nestin阳性细胞百分率及p-PI3K/PI3K和p-Akt/Akt比值明显降低（P<0.05），Ghrelin组ADSCs中NSE和Nestin阳性细胞百分率及p-PI3K/PI3K和p-Akt/Akt比值升高（P<0.05）；与LY294002组比较，Ghrelin+LY294002组ADSCs中NSE和Nestin阳性细胞百分率及p-PI3K/PI3K和p-Akt/Akt比值升高（P<0.05）；各组ADSCs中GFAP阳性细胞百分率比较差异无统计学意义（P>0.05）。结论 Ghrelin通过激活PI3K/Akt通路促进ADSCs神经分化。

关键词: Ghrelin, LY294002, 脂肪间充质干细胞, 神经分化, 磷酸酰肌醇3激酶/蛋白激酶B信号通路

Abstract:

Objective： To discuss the effect of Ghrelin combined with LY294002 on neural differentiation of the adipose-derived mesenchymal stem cells （ADSCs）， and to clarify its mechanism. Methods The morphology of ADSCs was observed under inverted phase contrast microscope， and the positive expression rates of surface antibodies of the ADSCs in various groups were identificated with flow cytometry； the fourth-generation ADSCs were divided into control group （given non-intervention）， LY294002 group ［given 40 μmol·L^－1 phosphatidylinositol-3-kinase （PI3K）/protein kinase B （Akt） pathway specific inhibitor LY294002］，Ghrelin group （given 0.1 μmol·L^－1 Ghrelin）， and Ghrelin+LY294002 group （given 40 μmol·L^－1LY294002+0.1 μmol·L^－1 Ghrelin）.Inverted phase contrast microscope was used to observe the morphology of the ADSCs in various groups after the neural differentiation， immunofluorescence staining was used to detect the percentages of neuron specific enolase （NSE）， nestin antibody，and glial fibrillary acidic protein （GFAP） positive cells in the ADSCs in various groups， and Western blotting method was used to detect the expression levels of PI3K/Akt pathway proteins in the ADSCs in various groups，and the ratios of phosphorylated PI3K（p-PI3K）/PI3K and phosphorylated Akt（p-Akt）/Akt were calculated. Results The expressions of CD90 and CD13 of ADSCs surface antibodies were high，and the expressions of CD34，CD45，and CD106 were low.Compared with control group，parts of the cells in LY294002 group changed from long spindle shape to round shape and the protuberance was short；compared with LY294002 group，the transformation speed of the cells in Ghrelin+LY294002 group from long spindle shape to round shape was quick，and the number of protuberance was increased.Compared with control group， the percentages of NSE and Nestin positive cells and the ratios of p-PI3K/PI3K and p-Akt/Akt in the ADSCs in LY294002 group were decreased significantly（P<0.05）， the percentages of NSE and Nestin positive cells and the ratios of p-PI3K/PI3K and p-Akt/Akt in the ADSCs in Ghrelin group were increased （P<0.05）； compared with LY294002 group， the the percentage of NSE and Nestin positive cells and the ratios of p-PI3K/PI3K and p-Akt/Akt in the ADSCs in Ghrelin+LY294002 group were increased （P<0.05）； there were no significant differences in the percentage of GFAP positive cells in the ADSCs among various groups （P>0.05）. Conclusion Ghrelin promotes the neural differentiation of the ADSCs by activating the PI3K/Akt pathway.

Key words: Ghrelin, LY294002, Adipose-derived mesenchymal stem cells, Neural differentiation, Phosphatidylinositol-3-kinase/protein kinase B signaling pathway

中图分类号:

杨贺然,李兴江,胡嘉航,李彦伟. Ghrelin对脂肪间充质干细胞神经分化的影响[J]. 吉林大学学报(医学版), 2022, 48(6): 1490-1497.

Heran YANG,Xingjiang LI,Jiahang HU,Yanwei LI. Effect of Ghrelin on neural differentiation of adipose-derived mesenchymal stem cells[J]. Journal of Jilin University(Medicine Edition), 2022, 48(6): 1490-1497.

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Group	NSE	Nestin	GFAP
Control	28.76±2.83	15.59±2.48	7.26±1.03
LY294002	12.51±2.20^*	7.22±1.90^*	7.07±1.51
Ghrelin	49.35±4.63^*	25.37±3.76^*	7.68±1.62
Ghrelin+LY294002	30.30±3.27^△	16.79±2.51^△	7.50±1.74

Group	p-PI3K/PI3K	p-Akt/Akt
Control	1.21±0.18	1.30±0.20
LY294002	0.64±0.16^*	0.72±0.15^*
Ghrelin	1.67±0.20^*	1.79±0.21^*
Ghrelin+LY294002	1.24±0.17^△	1.34±0.11^△

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