非受体酪氨酸激酶对糖氧剥夺诱导的少突胶质细胞凋亡的影响及其机制  

EFFECT AND MECHANISM OF THE NON-RECEPTOR TYROSINE KINASE C-ABL IN OLIGODENDROCYTE APOPTOSIS INDUCED BY GLUCOSE-OXYGEN DEPRIVATION

作　　者：张竣 张莹 王树坤 侯琳[1] 袁增强 ZHANG Jun;ZHANG Ying;WANG Shukun;HOU Lin;YUAN Zengqiang(School of Basic Medicine,Qingdao University,Qingdao 266071,China)

机构地区：[1]青岛大学基础医学院,山东青岛266071 [2]军事医学研究院军事认知与脑科学研究所

出　　处：《精准医学杂志》2021年第3期207-211,共5页Journal of Precision Medicine

基　　金：国家自然科学基金重点项目(81930029);国家重点研发计划青年项目(SQ2017ZY050253)。

摘　　要：目的探讨非受体酪氨酸激酶(c-Abl)对糖氧剥夺(OGD)诱导的少突胶质细胞(OLs)凋亡的影响及其机制。方法体外培养少突胶质细胞系(oli-neu),分别经OGD处理第0、3、6、9小时时采用CELL TITER-GLO(CTG)法检测oli-neu的活力,使用Anexin V-FITC/PI试剂盒检测oli-neu的凋亡率,采用蛋白质免疫印迹实验(Western blot)检测oli-neu中酪氨酸245位点磷酸化的c-Abl(Y245-c-Abl)和酪氨酸182位点磷酸化的p38(p-p38)蛋白相对表达水平。从C 57 BL/6 J小鼠脑组织中分离并培养原代OLs,分别检测经OGD处理第0、3、6、9小时时OLs中Y245-c-Abl、p-p38蛋白相对表达水平;检测oli-neu经100 nmol/L ABL001预处理后再经OGD处理第6小时时的细胞活力、细胞凋亡率,以及oli-neu内Y245-c-Abl、p-p38的相对表达水平和活化的半胱氨酸蛋白酶-3(cleaved-caspased-3)和前体半胱氨酸蛋白酶-3(pro-caspased-3)蛋白比值;检测经100 nmol/L ABL001预处理后再经OGD处理第6小时时OLs中Y245-c-Abl、p-p38蛋白的相对表达水平。结果oli-neu经OGD处理第0、3、6、9小时后细胞活力逐渐下降、细胞凋亡率逐渐升高(F=249.10、68.18,P<0.05);Western blot结果显示,oli-neu和OLs经OGD处理第0、3、6、9小时时,各时间点细胞内Y245-c-Abl、p-p38蛋白的相对表达水平差异均有显著性(F=4.68~15.93,P<0.05);oli-neu经ABL001预处理再经OGD处理6 h后细胞活力明显升高、细胞凋亡率明显降低,oli-neu中Y245-c-Abl和p-p38蛋白的相对表达水平及cleaved-caspase-3/pro-caspase-3比值明显降低(t=2.80~4.23,P<0.05)。OLs经ABL001预处理再经OGD处理6 h后,OLs中Y245-c-Abl和p-p38蛋白的相对表达水平明显下降(t=3.06、3.79,P<0.05)。结论c-Abl参与了OGD诱导的oli-neu和OLs凋亡过程,而c-Abl抑制剂可以减轻细胞的凋亡,c-Abl促进细胞凋亡的机制可能是通过c-Abl-p38通路介导的。Objective To investigate the effect and mechanism of the non-receptor tyrosine kinase c-Abl in oligodendrocyte(OL) apoptosis induced by glucose-oxygen deprivation(OGD). Methods The oligodendrocyte cell line oli-neu was cultured in vitro;at 0, 3, 6, and 9 hours of OGD treatment, CELL TITER-GLO(CTG) was used to measure the viability of oli-neu cells, Annexin V-FITC/PI apoptosis kit was used to measure the apoptosis rate of oli-neu cells, and Western blot was used to measure relative protein expression levels of phosphorylated c-Abl at tyrosine 245(Y245-c-Abl) and phospho-p38 MAPK(p-p38) at tyrosine 182. Primary OLs were isolated from the brain tissue of C57BL/6J mice and cultured, and the relative protein expression le-vels of Y245-c-Abl and p-p38 in OLs were measured at 0, 3, 6, and 9 hours of OGD treatment;the viability and apoptosis rate of oli-neu cells were measured after pretreatment with 100 nmol/L ABL001 followed by 6 hours of OGD treatment, as well as the re-lative expression levels of Y245-c-Abl and p-p38 and cleaved-caspase-3/pro-caspase-3 ratio in oli-neu cells;the relative protein expression levels of Y245-c-Abl and p-p38 in OLs were measured after pretreatment with 100 nmol/L ABL001 followed by 6 hours of OGD treatment. Results After 0, 3, 6, and 9 hours of OGD treatment, there was a gradual reduction in the viability of oli-neu cells and a gradual increase in the apoptosis rate of oli-neu cells(F=249.10, 68.18,P<0.05). Western blot showed that at 0, 3, 6, and 9 hours of OGD treatment, there were significant differences in the relative protein expression levels of Y245-c-Abl and p-p38 between oli-neu cells and OLs(F=4.68-15.93,P<0.05). After pretreatment with ABL001 followed by 6 hours of OGD treatment, there was a significant increase in the viability of oli-neu cells and a significant reduction in the apoptosis rate of oli-neu cells, as well as significant reductions in the relative protein expression levels of Y245-c-Abl and p-p38 and cleaved-caspase-3/pro-caspase-3 ratio in oli-neu cells(t=2.80-4.

关 键 词：少突神经胶质 糖氧剥夺 细胞低氧 原癌基因蛋白质c-abl P38丝裂原活化蛋白激酶类 细胞活力 细胞凋亡 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学] R742[医药卫生—基础医学]