人巨细胞病毒诱导分化中的神经干细胞分泌神经生长因子前体  

Human cytomegalovirus infection induces nerve growth factor precursor expression in neural stem cells during differentiation into astrocytes

作　　者：李玲[1] 王斌[2] 白志强[2] 李鹏[2] 王海涛[2] 刘海燕[2] 姜光域[2] 宋旭霞[2] 钱冬萌[2] 闫志勇[2] 丁守怡[2] 

机构地区：[1]青岛大学医学院组胚教研室,青岛医学博士研究生266071 [2]青岛大学医学院微生物教研室,青岛266071

出　　处：《医学研究生学报》2010年第9期904-909,共6页Journal of Medical Postgraduates

基　　金：国家自然科学基金(30770105);青岛市科技计划项目(08-1-3-30-jch);泰山学者建设工程专项经费资助

摘　　要：目的研究人巨细胞病毒(human cytomegalovirus,HCMV)感染对人神经干细胞(neural stem cells,NSC)向星形胶质细胞分化过程中神经生长因子(nerve growth factor,NGF)及其受体p75NTR、trkA表达的影响,为阐明HCMV感染致NSC损伤的分子机制提供理论依据。方法体外培养人海马NSC,感染组以感染复数(multiplicity of infectin,MOI)为5的HCMVAD169株感染NSC,对照组只加入与病毒悬液等体积的培养液,在感染病毒的同时,诱导感染组和对照组NSC向星形胶质细胞分化,分别在感染后0、1、3、5、7、9 d收获细胞,用RT-PCR、免疫荧光和Western blot方法检测细胞内NGF及其受体p75NTR、trkA的转录水平和蛋白表达水平。结果对照组NSC不表达NGF及其受体P75NTR和trkA。感染组细胞在第3天开始出现NGFmRNA的表达,第5天出现P75NTRmRNA的表达,且其表达强度随时间增强;第5天出现相对分子质量为40000和100000NGF前体蛋白(ProNGF)和P75NTR蛋白,随时间延长,表达强度升高。免疫荧光检测显示,NGF前体蛋白位于细胞的细胞质;整个感染过程中未检测到trkA的表达。结论 HCMV感染可诱导向星形胶质细胞分化的海马NSC分泌NGF前体蛋白及其受体p75NTR。Objective The objective of present study is to investigate the effect of HCMV infection on the expression of neural growth factor,NGF receptor（p75NTR） and trkA in human hippocampal neural stem cells（NSC） during its differentiation into astrocytes,so as to provide theoretical evidence for elucidating the molecular mechanism of NSC damage caused by HCMV infection.Methods NSCs were isolated and prepared using NSCs proliferating medium including hEGF and hFGF.To drive NSC differentiate toward astrocyte phenotype,growth factors were withdrawn and 10% FBS was added into the culture medium（DMEM/F12）.NSCs and astrocytes were identified by detecting Nestin and GFAP using immunofluorescence method respectively.At the beginning of differentiation,NSCs were infected with HCMV AD169（MOI=5） and collected at 0,1,3,5,7,9 d.p.i（days of postinfection）.Unfected NSCs were collected at the corresponding point of differentiation and used as control group.NGF,p75NTR,trkA mRNA and protein expression products were analyzed via RT-PCR,immunofluorescence and western blotting.Results NGF,p75NTR and trkA were not detected in undifferentiated NSCs or control NSCs with normal differentiation.The mRNAs of NGF and p75NTR were detected at 3 d.p.i.and 5 d.p.i respectively,and the expression of them occurred in a time-dependent manner.The specific 40 KD and 100 KD bands were detected in Western blot,but the mature form of NGF（26 KD） band was not detected.The 40 KD and 100 KD bands should be the precurosors of NGF（proNGF）.ProNGFs were detected at 5 d.p.i.and also with time dependent manner.Furthermore,p75NTR was expressed at 5.d.p.i.and proNGF was expressed in the cytoplasm via immunofluorescence.TrkA was undetectable in this experiment.Conclusion HCMV infection induces proNGF and p75NTR expression during the differentiation of human hippocampal NSCs into astrocytes.

关 键 词：人巨细胞病毒 神经干细胞 神经生长因子 神经生长因子前体蛋白 P75NTR 细胞凋亡 

分 类 号：R373.11[医药卫生—病原生物学]