激活素A在鸡胚背根神经节发育过程中的作用
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摘要
目的:探讨激活素A(Activin A)与鸡胚背根神经节(DRG)发育的关系,进一步分析Activin A在DRG发育过程中的作用机制。方法:采用8 d的鸡胚DRG原代培养法,观察Activin A对DRG神经突起生长的影响,选择不同日龄(8、10、12、14、16、18 d)的鸡胚DRG,通过RT-PCR、免疫组织化学染色等方法,分析Activin A、激活素ⅡA型受体(ActRⅡA)以及激活素ⅡB型受体(ActRⅡB)、信号传导蛋白Smads等在不同日龄鸡胚DRG中的表达情况,酶联免疫吸附法检测神经递质γ-氨基丁酸等分泌水平。结果:Activin A组及神经生长因子(NGF)组鸡胚神经节长度分别为(85.70±4.36)μm和(114.34±4.09)μm,均高于对照组(2.42±0.42)μm(P<0.05),RT-PCR检测结果显示,随着时间的推移,Activin A及ActRⅡA、ActRⅡB mRNA表达量逐渐下降,而激活素结合蛋白FS mRNA表达量逐渐增加,激活素信号传导蛋白Smad3 mRNA表达量逐渐降低。Activin A及神经生长因子(NGF)组γ-氨基丁酸(GABA)水平分别为(644.09±20.20) mmol/L和(627.32±12.533) mmol/L,均高于对照组的(501.00±0.71)mmol/L(P<0.05)。结论:Activin A在DRG发育早期发挥重要作用,其作用与调控神经递质GABA的释放有关。
Objective:To analyze the relationship between activin A and DRG development, and then the neurotransmitters released by neurons of DRG were detected to further analyze the mechanism of activin A in the development of DRG. Methods: DRG were dissected from 8 d chicken embryos under a dissecting microscope and cultured in vitro. Then the role of activin A in stimulating neurite outgrowth were analyzed. Reverse transcription PCR(RT-PCR) and immunohistochemical staining were used to examine the expressions of activin A and its receptors, Smads in DRG of chicken of different embryonic days(8, 10, 12, 14, 16, 18 d). Enzyme linked immunosorbent assay(ELISA) was used to detect the neurotransmitters of GABA levels in the supernatant of cultured DRG cells in vitro. Results: Activin A promoted the neurite outgrowth of embryonic DRG of chicken. RT-PCR was used to examine the expressions of activin A and its receptors of embryonic DRG of chicken of different embryonic days(8, 10, 12, 14, 16, 18 d) respectively. The results showed that the expressions of activin A, ActRⅡA and ActRⅡB m RNA decreased gradually, but FS m RNA, an activin-binding protein, m RNA increased day by day. The expression of Smad3 m RNA, a downstream signaling molecule of activin, also decreased with the development of embryonic DRG. ELISA results showed that activin A and NGF could enhance the levels of GABA in the supernatant of cultured embryonic DRG cells significantly(P <0.05). Conclusion: Activin A may play an important role mainly in the early development of embryonic DRG, which is associated with inhibiting proliferation of glial cells and regulating release of neurotransmitter GABA.
Objective:To analyze the relationship between activin A and DRG development, and then the neurotransmitters released by neurons of DRG were detected to further analyze the mechanism of activin A in the development of DRG. Methods: DRG were dissected from 8 d chicken embryos under a dissecting microscope and cultured in vitro. Then the role of activin A in stimulating neurite outgrowth were analyzed. Reverse transcription PCR(RT-PCR) and immunohistochemical staining were used to examine the expressions of activin A and its receptors, Smads in DRG of chicken of different embryonic days(8, 10, 12, 14, 16, 18 d). Enzyme linked immunosorbent assay(ELISA) was used to detect the neurotransmitters of GABA levels in the supernatant of cultured DRG cells in vitro. Results: Activin A promoted the neurite outgrowth of embryonic DRG of chicken. RT-PCR was used to examine the expressions of activin A and its receptors of embryonic DRG of chicken of different embryonic days(8, 10, 12, 14, 16, 18 d) respectively. The results showed that the expressions of activin A, ActRⅡA and ActRⅡB m RNA decreased gradually, but FS m RNA, an activin-binding protein, m RNA increased day by day. The expression of Smad3 m RNA, a downstream signaling molecule of activin, also decreased with the development of embryonic DRG. ELISA results showed that activin A and NGF could enhance the levels of GABA in the supernatant of cultured embryonic DRG cells significantly(P <0.05). Conclusion: Activin A may play an important role mainly in the early development of embryonic DRG, which is associated with inhibiting proliferation of glial cells and regulating release of neurotransmitter GABA.
引文
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[12]Morel M,Shynkar V,Galas JC,et al. Amplification and temporal filtering during gradient sensing by nerve growth cones probed with a microfluidic assay[J]. Biophysical journal,2012,103(8):1648-1656.
[2]Mu Y,Gudey SK,Landstr?m M. Non-Smad signaling pathways[J]. Cell and tissue research,2012,347(1):11-20.
[3]方琳,王轶楠,刘海岩,等.激活素A促进鸡胚背根神经节神经突起生长的作用机制[J].中国兽医学,2012(7):1020-1022,1032.
[4]侯亮,张扬,孙轶峰,等.激活素-卵泡抑素系统在病理性瘢痕中的表达[J].中国实验诊断学,2017,21(12):2093-2096.
[5]Su X,Huang L,Xiao D,et al. Research Progress on the Role and Mechanism of Action of Activin A in Brain Injury[J]. Journal of neuroimmunology,2018(12):697.
[6]范钰奇,王明华,谢东雪,等.激活素A通过下丘脑室旁核调节动脉压的作用及其机制[J].吉林大学学报(医学版),2016,42(4):699-703,844.
[7]Fang L,Wang YN,Cui XL,et al. The role and mechanism of action of activin A in neurite outgrowth of chicken embryonic dorsal root ganglia[J]. Journal of cell science,2012,125(6):1500-1507.
[8]方琳,王轶楠,葛敬岩,等.激活素A与NGF协同刺激鸡胚背根神经节神经突起生长[J].中国免疫学杂志,2015,31(9):1206-1209.
[9]成洪聚,相龙全,亚白柳,等.背根神经节神经元GABA A型受体对痛觉调制的研究进展[J].吉林大学学报(医学版),2018,44(6):1322-1325.
[10]Wayman GA,Bose DD,Yang D,et al. PCB-95 modulates the calcium-dependent signaling pathway responsible for activity-dependent dendritic growth[J]. Environmental health perspectives,2012,120(7):1003.
[11]Domínguez S,de Sevilla DF,Bu?o W. Postsynaptic activity reverses the sign of the acetylcholine-induced long-term plasticity of GABAA inhibition[J]. Proceedings of the National Academy of Sciences,2014,111(26):E2741-E2750.
[12]Morel M,Shynkar V,Galas JC,et al. Amplification and temporal filtering during gradient sensing by nerve growth cones probed with a microfluidic assay[J]. Biophysical journal,2012,103(8):1648-1656.