解剖学报 ›› 2021, Vol. 52 ›› Issue (1): 5-13.doi: 10.16098/j.issn.0529-1356.2021.01.001

• 神经生物学 • 上一篇    下一篇

别孕烯醇酮对6-羟基多巴胺损伤的细胞系SH-SY5Y的保护作用

王彤彤1,2,3 叶鑫1,3 边维1,3 陈治池1,3 杜娟娟1,4 傅维达5 陈梦娇5 李俊楠5 孙臣友1,3*   

  1. 1.温州医科大学基础医学院人体解剖学教研室,浙江 温州325035; 2.台州职业技术学院医学与制药工程学院护理教研室,浙江 台州318000; 3.温州医科大学基础医学院神经科学研究所,浙江 温州325035; 4.温州医科大学基础医学院组织学胚胎学教研室,浙江 温州325035; 5.温州医科大学第一临床医学院2015、2016级临床医学、医学影像学本科,浙江 温州325035
  • 收稿日期:2019-06-24 修回日期:2019-08-07 出版日期:2021-02-06 发布日期:2021-02-06
  • 通讯作者: 孙臣友 E-mail:sunchenyou1972@aliyun.com
  • 基金资助:
    别孕烯醇酮促进PD小鼠多巴胺能神经元新生的作用及机制研究;CD133追踪APα诱导的6-OHDA PD小鼠新生多巴胺能神经元起源的应用研究

Allopregnanolone protecting cell line SH-SY5Y against  6-hydroxydopamine induced lesion

WANG Tong-tong1,2,3 YE Xin1,3 BIAN Wei1,3 CHEN Zhi-chi1,3 DU Juan-juan1,4  FU Wei-da5 CHEN Meng-jiao5 LI Jun-nan5 SUN Chen-you1,3*#br#

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  1. 1.Department of Anatomy,School of Basic Medical Sciences of Wenzhou Medical University, Zhejiang Wenzhou 325035, China; 2.Department of Nursing, School of Medicine and Pharmaceutical Engineering of Taizhou Vocational and Technical College, Zhejiang Taizhou 318000, China; 3.Institution of Neuroscience, School of Basic Medical Sciences of Wenzhou Medical University,Zhejiang Wenzhou 325035, China;  4.Department of Histology And Embryology, School of Basic Medical Sciences of Wenzhou Medical University, Zhejiang Wenzhou 325035, China; 5.Bachelor of Clinical Medicine and Medical Imaging, Grade 2015, 2016, the First Clinical Medical College,Wenzhou Medical University,Zhejiang Wenzhou 325035, China

  • Received:2019-06-24 Revised:2019-08-07 Online:2021-02-06 Published:2021-02-06
  • Contact: SUN Chen-you E-mail:sunchenyou1972@aliyun.com

摘要:

目的  明确别孕烯醇酮(APα)对6-羟多巴胺(6-OHDA)损伤的SH-SY5Y细胞系的保护作用,并阐明可能的分子机制。方法  向体外培养的SH-SY5Y细胞系中分别加入6-OHDA、APα、γ-氨基丁酸A受体(GABAAR)拮抗剂荷包牡丹碱(Bic)和电压门控L型Ca2+通道拮抗剂硝苯地平(nifedipine),采用免疫荧光细胞化学染色方法观察不同组别酪氨酸羟化酶(TH)阳性细胞的变化,Western blotting检测胞质钙调蛋白(CaM)、钙离子钙调蛋白依赖性蛋白激酶Ⅱ δ3(CaMKⅡδ3),胞核CaMKⅡδ3、脑源性神经营养因子(BDNF)和细胞周期蛋白依赖性激酶(CDK1)表达的变化,采用蛋白质免疫共沉淀验证CaMKⅡδ3与CDK1/BDNF的相互作用。  结果  APα作用后,6-OHDA损伤的SH-SY5Y细胞TH和5-溴脱氧尿嘧啶核苷(BrdU)阳性细胞数目均明显增加,而TH/BrdU双阳性细胞数目无显著变化;同时Western blotting结果表明,SH-SY5Y细胞胞质和胞核上述蛋白的表达与单纯 6-OHDA 组相比也明显上升,经Bic处理后各蛋白增加更为明显。免疫共沉淀结果表明,CaMKⅡδ3与CDK1/BDNF存在相互作用。  结论  APα对6-OHDA损伤的SH-SY5Y细胞的保护中,GABAAR发挥负性调控作用,通过稳定细胞的内环境达到增加TH阳性神经元数量的目的,其中Ca2+ -CaM-CaMKⅡδ3信号通路和BDNF与CDK1发挥了关键作用。

关键词: 别孕烯醇酮, γ-氨基丁酸A受体, 多巴胺能神经元, 钙/钙调蛋白依赖性蛋白激酶Ⅱ, 脑源性神经营养因子, 免疫印迹法

Abstract:

Objective To clarify the protective effect of allopregnanolone (APα) on cell line SH-SY5Y damaged by 6-hydroxydopamine (6-OHDA) and its possible molecular mechanism.   Methods  6-OHDA, APα, γ-aminobutyric acid A receptor(GABAAR) antagonist, voltage-gated L-type Ca2+ channel antagonist were added to the in vitro cultured cell line SH-SY5Y. Immunofluorescence cell chemical staining was used to observe the changes of tyrosine hydroxylase (TH)-positive cells. The changes of the expression of calmodulin (CaM), Ca2+/calmodulin-dependent protein kinase Ⅱ δ3 (CaMKⅡδ3) in the cytoplasm and CaMKⅡδ3, brain derived neurotrophic factor (BDNF) and cyclin-dependent kinases 1(CDK1) in the nucleus of different groups were detected by Western blotting. The interaction between CaMKⅡδ3 and CDK1/BDNF was verified by co-precipitation.   Results  Having treated with APα, the number of TH and 5-Bromo-2-deoxyuridine (BrdU)-positive cells in 6-OHDA-lesioned SH-SY5Y cells increased significantly, but the number of TH/BrdU-double positive cells did not alter significantly. In the cytoplasmic or nucleus fraction of SH-SY5Y cells, the expression of the aforementioned proteins in the APα+6-OHDA group was higher than that in 6-OHDA+DMSO group by Western blotting, in particular, it increased significantly in APα+Bic+6-OHDA group compared with the APα+6-OHDA group. Immunoprecipitation assay showed that there existed an interaction between CaMKⅡδ3 and CDK1 or BDNF.   Conclusion  In the neuroprotective effect of APα on 6-OHDA-lesioned SH-SY5Y cells, GABAAR plays a negative regulation. As a result, APα increases the number of TH-positive neurons by stabilizing the cellular inner environment, in which the Ca2+ -CaM-CaMKⅡδ3 signaling pathway and BDNF, CDK1 plays a key role.

Key words: Allopregnanolone, γ-Aminobutyric acid A receptor, Dopaminergic neuron, Calmodulin-dependent kinase Ⅱ, Brain-derived neurotrophic factor, Western blotting

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