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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 To study the effect of human cytomegalovirus (HCMV) infection on the expression of nerve growth factor (NGF) and its receptor p75NTR, trkA during the differentiation of neural stem cells (NSCs) into astrocytes Expression, to clarify the molecular mechanism of NSC damage caused by HCMV infection provide a theoretical basis. Methods The NSC of human hippocampus was cultured in vitro. The infected group was infected with HCMVAD169 strain with a multiplicity of infectin (MOI) of 5, and the control group was incubated with equal volumes of virus suspension. At the same time of infecting the virus, And control group, NSCs differentiated into astrocytes, and the cells were harvested at 0, 1, 3, 5, 7 and 9 days after infection respectively. The expression of NGF and its receptor was detected by RT-PCR, immunofluorescence and Western blot p75NTR, trkA transcriptional level and protein expression level. Results In control group, NSC did not express NGF and its receptors P75NTR and trkA. The expression of NGF mRNA began to appear on the 3rd day in the infected cells, and the expression of P75NTR mRNA appeared on the 5th day, and its expression intensity increased with time. On the 5th day, the relative molecular mass was 40,000 and 100000NGF precursor protein (ProNGF) and P75NTR protein, With time, expression intensity increased. Immunofluorescence showed that the NGF precursor protein was localized in the cytoplasm of the cells; no trkA expression was detected during the whole infection. Conclusion HCMV infection induces secretion of NGF precursor protein and its receptor p75NTR into astrocytes differentiated hippocampal NSCs.