The Effect Of HSV-1 Infection On Expression Of Neurotrophic Factors And Related Receptors In Human Glioma Cells

Objective:1. To study the changes of apoptosis in glioma cells caused by HSV-1 infection.2. To study the the effect of HSV-1 infection on expression of neurotrophic factors and its receptors in glioma cells, and in order to explore the mechanism of apoptosis caused by HSV-1 infection in glioma cells.Methods:1. HSV-1 gD expression in human glioma cells (U251) was measured by RT-PCR and Western blot after infection with HSV-16h later; Cell morphological change was investigated under microscope, and effect on apoptosis induced by HSV-1 infected was analysed by flow cytometry.2. After 6h,12h,18h,24h infected with HSV-1, in vitro cultured U251 cells were used to perform RT-PCR to detect the transcription of NGF, BDNF, trkA and P75NTR, Western blot analysis to detect the expression of NGF, BDNF, trkA and P75NTR.Results:1. Glioma cell morphology appeared to be grossly normal in the first 12 hour after HSV-1 infection. By 18h, cytopathic effects, such as cellular swelling, cellular confluence and a honeycomb appearance, were evident in more than 50% of cells. After 24h of infection, these cytopathic effects were observed in almost 100% of cells, with honeycomb-giant cells, and some cell detachment.2. The expression of HSV-1 gD indicated that U251 cell was the permissive cell to HSV-1 infection, which showed that HSV-1 infection cellular model was set up. FACS analysis that rate of apoptosis in different time, which showed it was a gradual increase with the infection time. There were a few apoptosis at 12h, but increased significantly at 24-30h. At 24h and 30h after infection, the rate of apoptosis was 23.1% and 25.1% respectively.3. RT-PCR analysis was used to measure expression of the NGF, BDNF, trkA and P75NTR. After infecting HSV-1, NGF, BDNF and P75NTR gene expression changed similarly. That was, the expression of NGF, BDNF and P75NTR increased first and decreased later with infecting time, thereinto, the expression of NGF and BDNF reached a peak at 6h and the expression of P75NTR reached at 12h. In the case of trkA mRNA relatively lower expression levels were present in infected cells, and decreased steadily. There was a good relationship between the Western blot analysis and the results obtained by RT-PCR. In control and infection groups, NGF was detected with a single 26 kDa band, and NGF protein levels increased to a peak at 12h then decreased with infection time. BDNF and P75NTR levels reached to a peak at 18h then decreased. However, trkA was not detected in all groups.Conclusions:1. U251 cells were the permissive cells to HSV-1 infection.2. HSV-1 infection was able to lead to apoptosis in cultured U251 cells in vitro.3. HSV-1 was able to induce the abnormal expression of neurotrophic factors and receptors in the U251 cells. After infected with HSV-1, the level of transcription and expression of endogenous NGF, BDNF and P75NTR increased and the decreased, while trkA decreased gradually. These indicated that neurotrophic factors and its receptors might be involved in the mechanism of apoptosis in HSV-1 infection.