文章摘要
杨永昆,刘雅红,王 群,刘 牧,张永杰.甲状旁腺素相关肽核定位序列与C末端缺失致小鼠少突胶质细胞发育障碍[J].南京医科大学学报,2018,(10):1337~1344
甲状旁腺素相关肽核定位序列与C末端缺失致小鼠少突胶质细胞发育障碍
Nuclear localization sequence and carboxyl terminus of parathyroid hormone related peptide leads to the developmental disabilities of oligodendrocytes in mice
投稿时间:2018-06-12  
DOI:10.7655/NYDXBNS20181002
中文关键词: 甲状旁腺素相关肽  核定位序列  少突胶质细胞  髓鞘
英文关键词: parathyroid hormone related peptide  nuclear localization sequence  oligodendrocytes  myelin sheath
基金项目:国家自然科学基金(81472081,81100942);江苏省自然科学基金(BK2010539)
作者单位
杨永昆 南京医科大学人体解剖学系江苏 南京 211166
泰州市人民医院肿瘤科江苏 泰州 225300 
刘雅红 南京医科大学人体解剖学系江苏 南京 211166 
王 群 南京医科大学人体解剖学系江苏 南京 211166 
刘 牧 南京医科大学基础医学院江苏 南京 211166 
张永杰 南京医科大学人体解剖学系江苏 南京 211166
衰老及相关疾病研究重点实验室江苏 南京 211166 
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中文摘要:
      目的:研究甲状旁腺素相关肽(parathyroid hormone related peptide,PTHrP)核定位序列(nuclear localization sequence,NLS)与C末端缺失对小鼠少突胶质细胞发育及髓鞘生成的影响。方法:采用6日龄PTHrP Knock In(PTHrP KI)小鼠及同窝野生型(wild type,WT)小鼠行5?溴脱氧尿嘧啶核苷(5?bromo?2?deoxyuridine,BrdU)标记,次日取材经免疫荧光染色检测海马区少突胶质前体细胞(oligodendrocyte progenitor cells,OPCs)的增殖能力,通过髓鞘碱性蛋白(myelin basic protein,MBP)免疫组化染色及电镜技术观察中枢神经系统(central nervous system,CNS)的髓鞘发生;体外培养5日龄PTHrP KI小鼠及同窝WT小鼠大脑皮层的O4阳性OPCs,运用免疫荧光染色观察Ki67阳性细胞数,流式分析检测凋亡细胞比例,Western blot检测与增殖凋亡相关蛋白的表达变化。继而行分化培养7 d后,通过2′,3′?环腺苷酸?3′?磷酸二酯酶(2′,3′?cyclic?nucleotide 3′?phosphodiesterase,CNPase)免疫荧光染色检测细胞分化能力。结果:与同窝WT小鼠相比,PTHrP KI小鼠脑内MBP阳性纤维面积减少,轴突髓鞘稀薄,海马区BrdU与少突胶质细胞转录因子2(oligodendrocyte transcription factor 2,Oligo?2)双阳性细胞数减少。体外培养PTHrP KI小鼠OPCs的Ki67阳性细胞百分率降低,AV+/PI?凋亡细胞百分率升高;增殖相关B细胞淋巴瘤滤过性病毒插入位点1(B cell?specific MLV integration site?1,Bmi?1)蛋白、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白表达降低,凋亡相关Caspase?3蛋白、p16蛋白表达升高。分化培养后,PTHrP KI 小鼠来源OPCs的CNPase荧光染色强度降低。结论:PTHrP的NLS与C末端缺失可致小鼠OPCs增殖减少、凋亡增加、分化延缓,进而导致CNS轴突髓鞘形成障碍。
英文摘要:
      Objective:To explore the effects of nuclear localization sequence(NLS) and carboxyl terminus of parathyroid hormone related peptide(PTHrP) on the oligodendrocytes development and myelination in mice. Methods:PTHrP knock in(PTHrP KI)mice and their wild type(WT) littermates were accepted 5?bromo?2?deoxyuridine(BrdU) intraperitoneal injection at postnatal day 6(P6),and were sacrificed at the next day. The myelination in the central nervous system(CNS) was observed by myelin basic protein(MBP)immunohistochemical staining and electron microscope. The proliferation of oligodendrocyte progenitor cells in the hippocampus was detected by BrdU labeling and immunofluorescence staining. O4 positive oligodendrocyte progenitor cells(OPCs)from the cerebral cortex of P5 PTHrP KI mice and their WT littermates were selected and cultured in vitro. Then,the Ki67 positive cells were observed by immunofluorescence staining,the ratio of apoptotic cells was measured by flow cytometer analysis,and the proliferation and apoptosis related proteins were detected by Western blot. The differentiation ability of OPCs was inspected by 2′,3′?cyclic?nucleotide 3′?phosphodiesterase(CNPase)immunofluorescence staining after cultured in differentiation medium for 7 days. Results:Comparison to their WT littermates,PTHrP KI mice showed decreased expression of MBP positive nerve fibers and the thinner myelin sheathes of axons in the brain,decreased BrdU and oligodendrocyte transcription factor 2(Oligo2)double positive cells in hippocampus area. The in vitro cultured OPCs from PTHrP KI mice showed the decreased ratio of Ki67 positive cells,the decreased expression level of proliferation related proteins as B cell?specific MLV integration site?1(Bmi?1)and proliferating cell nuclear antigen(PCNA),the increased ratio of AV+/PI? apoptotic cells,the increased expression level of apoptotic proteins as caspase?3 and p16,and the decreased intensity of CNPase immunofluorescence after 7 days’ differentiation culture. Conclusion:Defect of NLS and C?Terminus of PTHrP leads to the decreased proliferation,increased apoptosis,and delayed differentiation of OPCs,and results in the axonal myelination disability in CNS.
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