PTCSC3对谷氨酸诱导SH-SY5Y神经细胞凋亡的影响  

Effects of PTCSC3 on apoptosis of SH-SY5Y neurons induced by glutamate

作　　者：苗峥 胡雨 米山 焦明丽 韩明明 王轶博 胡勇 黄锋[1] 詹合琴[1] MIAO Zheng;HU Yu;MI Shan;JIAO Mingli;HAN Mingming;WANG Yibo;HU Yong;HUANG Feng;ZHAN Heqin(School of Pharmacy,Xinxiang Medical University,Xinxiang 453003,China)

机构地区：[1]新乡医学院药学院,河南新乡453003

出　　处：《沈阳药科大学学报》2024年第9期1222-1235,共14页Journal of Shenyang Pharmaceutical University

基　　金：河南省自然科学基金项目(162300410219)。

摘　　要：目的探讨乳头状甲状腺癌易感性候选基因3(papillary thyroid carcinoma susceptibility candidate 3,PTCSC3)对谷氨酸(glutamicacid,Glu)诱导人神经母细胞瘤细胞(SH-SY5Y)凋亡的影响及其机制。方法体外培养SH-SY5Y细胞,谷氨酸损伤SH-SY5Y细胞24 h,转染技术对PTCSC3进行抑制及过表达,采用Hoechst 33258染色法、流式细胞技术、AO/EB染色及四甲基偶氮唑盐比色(methylthiazolyl tetrazolium,MTT)法检测抑制或过表达PTCSC3后对各组细胞凋亡、生存的影响;实时荧光定量PCR(quantitative real-time PCR,qPCR)技术检测各组细胞中PTCSC3、CCAAT/增强子结合蛋白β(CAAT/enhancer binding protein beta,C/EBP-β)及Cbp/p300结合转化激活因子4(Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 4,CITED4)的基因表达情况;Western blot检测各组细胞中C/EBP-β、脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)、蛋白激酶B(protein kinase B,Akt)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)及CITED4、细胞周期蛋白D1(Cyclin D1)、原癌基因n-Myc的蛋白表达水平。结果2000μmol·L^(-1)Glu处理24 h对PTCSC3细胞有明显的损伤作用;与模型组比较,PTCSC3抑制剂可有效改善Glu诱导SH-SY5Y细胞凋亡率的增加和细胞存活率的下降;明显降低C/EBP-β基因及蛋白、Bax蛋白的表达,促进BDNF、p-Akt、Bcl-2蛋白的表达;增加CITED4、Cyclin D1和n-Myc的表达水平。反之,过表达PTCSC3则出现与上述结果相反的结果。结论PTCSC3可能通过调控C/EBP-β,进而调控下游信号通路BDNF/Akt和CITED4/Cyclin D1信号通路,改善Glu诱导SH-SY5Y神经细胞的凋亡和生存状态。Objective To investigate the effects and mechanism of papillary thyroid carcinoma susceptibility candidate 3(PTCSC3)on apoptosis of human neuroblastoma cells(SH-SY5Y)induced by glutamate(Glu).Methods SH-SY5Y cells were cultured in vitro and SH-SY5Y cells were injured by glutamate for 24 h.PTCSC3 was inhibited and overexpressed by transfection technique.Hoechst 33258 staining,flow cytometry,AO/EB staining and methylthiazolyl tetrazolium,MTT colorimetric assay were used to detect the effects of inhibition or overexpression of PTCSC3 on apoptosis and survival of cells in each group.Real-time fluorescence quantitative PCR(qPCR)was used to detect the gene expressions of PTCSC3,CCAAT/enhancer binding proteinβ(C/EBP-β)and Cbp/p300 binding transforming activator 4(Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 4)in cells of each group.Western blot was used to detect the protein expressions of C/EBP-β,brain-derived neurotrophic factor(BDNF),protein kinase B(Akt),B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax)and CITED4,Cyclin D1 and proto-oncogene n-Myc.Results 2000μmo·L^(-1)Glu could significantly damage PTCSC3 cells for 24 h.Compared with the model group,PTCSC3 inhibitor could effectively improve the increase of apoptosis rate and decrease of cell survival rate of SH-SY5Y cells induced by Glu,significantly reduce the expressions of BDNF-βgene and protein and Bax protein,promote the expressions of BDNF,p-Akt and Bcl-2 protein,and increase the expression levels of CITED4,Cyclin D1 and n-Myc.On the contrary,the results of overexpression of PTCSC3 were contrary to the above results.Conclusion PTCSC3 may improve the apoptosis and survival status of SH-SY5Y neurons induced by Glu by regulating C/EBP-β,and then regulating downstream signal pathways BDNF/Akt and CITED4/Cyclin D1.

关 键 词：乳头状甲状腺癌易感性候选基因3 人神经母细胞瘤细胞 细胞凋亡 作用 分子机制 

分 类 号：R96[医药卫生—药理学]