NKCC1抑制剂布美他尼对大鼠脊髓原代少突胶质前体细胞增殖的影响  

Effects of NKCC1 Inhibitor Bumetanide on the Proliferation of Primary Oligodendrocyte Precursor Cells of Rat Spinal Cord

作　　者：付佩彩 叶盛 李志军 骆翔 喻志源[1] Fu Peicai;Ye Sheng;Li Zhijun(Department of Neurology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属同济医院神经内科,武汉430030

出　　处：《华中科技大学学报（医学版）》2021年第4期413-417,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.81771341);湖北省自然科学基金资助项目(No.2020CFB744)。

摘　　要：目的研究钠-钾-氯共转运体1(NKCC1)抑制剂布美他尼(Bumetanide)对脊髓原代少突胶质前体细胞(OPCs)增殖的影响及其机制。方法振摇及差速贴壁法培养大鼠脊髓原代OPCs,使用NKCC1抑制剂布美他尼干预。免疫荧光法鉴定细胞及其NKCC1表达;EdU细胞增殖检测试剂盒检测细胞增殖;流式细胞术检测细胞周期;Western blot检测丝裂原活化蛋白激酶(MAPKs)信号通路蛋白p-p38,p-ERK,p-JNK表达。结果免疫荧光实验显示纯化培养的OPCs 95%以上表达NG2+A2B5,且能分化为少突胶质细胞;OPCs 100%表达NKCC1。EdU结果显示,与对照组相比,布美他尼干预6、12和24 h时,OPCs EdU阳性率增高[6 h:(9.00±0.39)%vs.(20.50±0.47)%,12 h:(10.33±0.33)%vs.(20.66±0.48)%,24 h:(11.30±0.34)%vs.(20.87±0.46)%,n=4,均P<0.01];流式细胞术结果显示,与对照组相比,布美他尼干预6、12和24 h时,S期细胞比例增多[6 h:(10.57±0.48)%vs.(19.75±0.32)%,12 h:(10.38±0.54)%vs.(20.12±0.58)%,24 h:(10.53±0.55)%vs.(20.35±0.43)%,n=4,均P<0.01]。Western blot检测显示,与对照组相比,布美他尼干预6、12和24 h时,p-p38表达上升,p-ERK表达下降(均P<0.01),p-JNK无明显差异。结论 NKCC1抑制剂布美他尼促进脊髓原代OPCs增殖,其机制可能是通过上调p38/MAPKs信号通路,下调ERK/MAPKs信号通路来实现。Objective To investigate the effect and mechanisms of NKCC1 inhibitor bumetanide on the proliferation of primary oligodendrocyte precursor cells(OPCs)of rat spinal cord.Methods The primary OPCs were cultured by shaking process and differential adhesion,and then interfered by bumetanide,an inhibitor of NKCC1.The expression of NKCC1 was detected by immunofluorescence.EdU cell proliferation assay kit was used to detect cell proliferation.Cell cycle was detected by flow cytometry.Western blotting was used to detect the expression of p-p38,p-ERK and p-JNK.Results Immunofluorescence showed that over 95%of the purified OPCs expressed NG2 and A2 B5 and could differentiate into oligodendrocytes.NKCC1 was expressed in 100%of OPCS.EdU results showed that compared with the control group,the EdU positive rate of OPCS increased 6、12 and 24 h after intervention with bumetanide[6 h:(9.00±0.39)%vs.(20.50±0.47)%,12 h:(10.33±0.33)%vs.(20.66±0.48)%,24 h:(11.30±0.34)%vs.(20.87±0.46)%,n=4,all P<0.01].Flow cytometry showed that compared with the control group,the ratio of S-phase cells increased 6、12 and 24 h after bumetanide intervention[6 h:(10.57±0.48)%vs.(19.75±0.32)%,12 h:(10.38±0.54)%vs.(20.12±0.58)%,24 h:(10.53±0.55)%vs.(20.35±0.43)%,n=4,all P<0.01].Western blotting showed that compared with the control group,the expression of p-p38 increased and the expression of p-ERK decreased 6、12 and 24 h after treatment with bumetanide(P<0.01),but there was no significant difference in p-JNK.Conclusion NKCC1 inhibitor bumetanide promotes the proliferation of primary spinal cord OPCs by up-regulating p38/MAPKs signaling pathway and down-regulating ERK/MAPKs signaling pathway.

关 键 词：NKCC1 布美他尼 少突胶质前体细胞 细胞增殖 MAPKS 

分 类 号：R744[医药卫生—神经病学与精神病学]