Study On The Effect On Expression Of NGF And Its Receptors In Neurocytoma Cell Lines After HCMV Infection

Background:Human cytomegalovirus(HCMV),a member of the herpesvirus family,is the most common etiologic agent of congenital anomalies of central nervous system(CNS) caused by intrauterine infection in humans.It is estimated that 5-10%of embryos with congenital HCMV infection will have severe neurologic damage at birth,such as microcephaly and perivascular calcification.To date,the mechanism concerning the injuries induced by this kind of viral infection remains uncertain.Nerve growth factor(NGF) is a metabolically active peptide,which was first isolated in nerve tissue.NGF plays a key role in differentiation,development and survival of sympathetic and sensory nerve cells,neural stem cells and cholinergic neurons of the central nervous system(CNS).Apoptosis and cell death may be induced by NGF withdrawal from culture medium of several nerve cells.Objective:1.To study the effect on apoptosis in glioma and neuroblastoma cell line induced by HCMV infected.2.To study the effect on expression of NGF and receptors in glioma and neuroblastoma cell lines after HCMV infection,and explore the pathogenic mechanism in central nervous system after HCMV infection.Materials and Methods:1.Preparation and titration of HCMV AD 169 strain:HCMV AD169 was titered by plaque titration in human embryonic lung fibroblast(HELF) cells and expressed as the number of plaque-forming units(PFU) per milliliter.2.HCMV IE gene level in U251 cells was measured by RT-PCR after infection with HCMV 6h later,cell morphological change was investigated under microscope,and effect on apoptosis induced by HCMV infected was analysed by flow cytometry.3.After 1d,3d,5d,7d infected with HCMV,in vitro cultured U251 cells were used to perform RT-PCR to detect the transcription of NGF,immunofluorescence method and Western blot analysis to detect the expression of NGF. 4.After 48h infected with HCMV,in vitro cultured SH-SY5Y cells were used to perform immunofluorescence method to detect the expression of HCMV IE和pp65.After 2d,4d,6d infected with HCMV,in vitro cultured SH-SY5Y cells were used to perform flow cytometry to detect the effect on apoptosis,and Western blot to detect the expression of NGF and its P75^NTRreceptor.Results:1.Glioma cell morphology appeared to be grossly normal in the first 3 day after HCMV infection.By 5d,cytopathic effects,such as cellular swelling,cellular confluence and a honeycomb appearance,were evident in more than 50%of cells.After 7 d of infection, these cytopathic effects were observed in almost 100%of cells,with honeycomb-giant cells, and some cell detachment.2.The expression of IE gene indicated that glioma(U251 cell) is the permissive cell to HCMV infection,which showed that HCMV infection cellular model was set up.No apparent change was observed at 1 d after infection.A hypodiploid cell peak appeared on the left of Gl peak at 5 d after infection.This peak grew bigger at 7 d after infection.Apoptosis in glioma was induced by HCMV infected,which became enhanced with time.At 5d and 7d after infection,the rate of apoptosis was 4.1%and 42.6%respectively.3.The ratios of NGF and(3-actin transcription in U251 cells were investigated at different time.Compared with control group,there is no significant difference in infection groups at Id,3d and 5d after infection(0.846±0.113,0.721±0.025 and 0.865±0.047,P> 0.05).By 7 days,expression levels were significantly reduced(0.528?.053,P<0.05 ).4.According to immunofluorescence and Western blot data,there is no significant change of NGF expression level during 1 to 5 d after HCMV infection,the level was greatly reduced at 7d after HCMV infection.5.The expression of IE gene indicated that neuroblastoma(SH-SY5Y) is the permissive cell to HCMV infection.No apparent change was observed at 2 d after infection. A hypodiploid cell peak appeared on the left of Gl peak at 4 d after infection.This peak grew bigger at 6 d after infection.At 4d and 6d after infection,the rate of apoptosis was 24.4%and 36.2%respectively.6.According to Western blot data,there is significant change of NGF expression level after HCMV infection,and the expression level of P75^NTR receptor was up-regulated with infection time.Conclusion:1.Both glioma(U251 cell line) and neuroblastoma(SH-SY5Y cell line) is the permissive cell to HCMV infection. 2.HCMV infection can lead to apoptosis in cultured U251 and SH-SY5Y cell line respectively in vitro.3.After infected with HCMV,the level of transcription and expression of endogenous NGF were down-regulated in both glioma(U251 cell line) and neuroblastoma(SH-SY5Y cell line),while those of the NGF receptor P75^NTR were up-regulated only in neuroblastoma (SH-SY5Y).Postgraduate student:Wang haitao(Pathogenic Biology) Directed by Prof.Wang bin...