二乙酰吗啡致大鼠小脑颗粒神经元细胞凋亡过程中C-Jun、Cytc和Caspase-9的作用  

C-jun,Cytc and Caspase-9 in the apoptosis of cerebellar granule neurons induced by diacetylmorphine in rats

作　　者：苏丽萍[1] 路子扬 刘丽 张巍[1] 苏天园 胡夏韵 蒲红伟[5,6] 韩登峰 Su Liping;Lu Ziyang;Liu Li;Zhang Wei;Su Tianyuan;Hu Xiayun;Pu Hongwei;Han Dengfeng(Department of Pathology,First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,Xinjiang Uygur Autonomous Region,China;School of Basic Medicine,Xinjiang Medical University,Urumqi 830054,Xinjiang Uygur Autonomous Region,China;Telemedicine center,First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,Xinjiang Uygur Autonomous Region,China;Second Department of Thoracic Surgery,The First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061,Shaanxi Province,China;Discipline Construction Section,First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,Xinjiang Uygur Autonomous Region,China;Key Laboratory of Forensic Medicine of Hebei Medical University,Shijiazhuang 050000,Hebei Province,China;Department of Neurology,First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,Xinjiang Uygur Autonomous Region,China)

机构地区：[1]新疆医科大学第一附属医院病理科,新疆维吾尔自治区乌鲁木齐市830011 [2]新疆医科大学基础医学院,新疆维吾尔自治区乌鲁木齐市830054 [3]新疆医科大学第一附属医院远程医学中心,新疆维吾尔自治区乌鲁木齐市830011 [4]西安交通大学第一附属医院胸外二科,陕西省西安市710061 [5]新疆医科大学第一附属医院学科建设科,新疆维吾尔自治区乌鲁木齐市830011 [6]河北医科大学法医学重点实验室,河北省石家庄市050000 [7]新疆医科大学第一附属医院神经内科,新疆维吾尔自治区乌鲁木齐市830011

出　　处：《中国组织工程研究》2021年第25期3943-3948,共6页Chinese Journal of Tissue Engineering Research

基　　金：河北省法医学重点实验室开放课题资助项目(KF201603),项目负责人:蒲红伟;国家自然科学基金(81860049,81260464),项目负责人:蒲红伟。

摘　　要：背景:二乙酰吗啡药物可导致神经元损伤,但是目前二乙酰吗啡是否诱导神经元细胞凋亡,C-jun、Cytc和Caspase-9因子是否参与此过程尚未见报道。目的:探讨C-jun、Cytc、Caspase-9是否参与二乙酰吗啡诱导神经元细胞凋亡过程。方法:将SD乳鼠小脑颗粒神经元细胞体外培养7 d,采用不同质量浓度二乙酰吗啡(0,10,40,80,100,120 mg/L)干预神经元细胞24 h,在倒置荧光显微镜下观察神经元细胞形态改变,流式细胞仪检测细胞凋亡率。采用100 mg/L二乙酰吗啡和20μmol/L JNK抑制剂SP600125作用于细胞24 h,通过免疫荧光和蛋白印迹方法检测C-jun、Cytc和Caspase-9蛋白的表达。结果与结论:①在不同质量浓度二乙酰吗啡干预下,随着给药质量浓度的增加,神经元细胞胞体萎缩、亮度降低,部分呈灰黑色,细胞碎裂,神经元网状结构不同程度的消失,细胞改变数量逐渐增加;②随着给药质量浓度的增加,形态改变的细胞数量明显增多(P <0.01),细胞凋亡率也显著增高(P <0.01);③与对照组相比,100 mg/L二乙酰吗啡作用神经元细胞时,二乙酰吗啡组C-jun、Cytc和Caspase-9蛋白呈高表达,差异有显著性意义(P <0.05);与二乙酰吗啡组相比,二乙酰吗啡+SP600125组C-jun、Cytc和Caspase-9蛋白表达明显下调,差异有显著性意义(P <0.05);④结果表明,二乙酰吗啡可诱导小脑颗粒神经元细胞凋亡,C-jun、Cytc和Caspase-9参与了二乙酰吗啡致神经元细胞凋亡的过程。BACKGROUND:Diacetylmorphine drugs can cause neuronal injury,but whether diacetylmorphine induces neuronal apoptosis or whether c-Jun,cytc and caspase-9 factors are involved in this process have not been reported.OBJECTIVE:To investigate whether C-jun,Cytc,and Caspase-9 are involved in apoptosis induced by diacetylmorphine.METHODS:The cerebellar granule neurons of SD suckling mice were cultured in vitro for 7 days.Different concentrations of diacetylmorphine(0,10,40,80,100,120 mg/L)were used to intervene neurons for 24 hours.The morphological changes of neurons were observed by inverted fluorescence microscope,and the apoptosis rate was detected by flow cytometry.The cells were treated with 100 mg/L diacetylmorphine and 20μmol/L JNK inhibitor SP600125 for 24 hours.The expressions of c-jun,cytc and caspase-9 protein were detected by immunofluorescence and western blot assay.RESULTS AND CONCLUSION:(1)With the increase dosages of diacetylmorphine,the neuronal cell body of neurons shrank,the brightness decreased,some of them were gray black,the cells were fragmented,the neuron network structure disappeared in different degrees and the number of cell changes increased gradually.(2)With the increase of the concentration of the drug,the number of morphological changes increased obviously(P<0.01),and the apoptosis rate also increased significantly(P<0.01).(3)Compared with the control group,c-jun,cytc and caspase-9 proteins were highly expressed in the diacetylmorphine group when treated with 100 mg/L diacetylmorphine and the difference was statistically significant(P<0.05).Compared with the diacetylmorphine group,c-jun,cytc,and caspase-9 protein expression was significantly down-regulated in the diacetylmorphine+SP600125 group(P<0.05).(4)The results indicate that diacetylmorphine can induce apoptosis of cerebellar granule neurons.The c-jun,cytc,and caspase-9 are involved in neuronal apoptosis induced by diacetylmorphine.

关 键 词：二乙酰吗啡 小脑 神经元 C-JUN CYTC CASPASE-9 凋亡 

分 类 号：R448[医药卫生—诊断学] R363.21[医药卫生—临床医学]