| Objective:1.To investigate the effect of human cytomegalovirus(HCMV)infection at multiplicities of infection(MOI)of 0.2,1 or 5 on the proliferation,differentiation and apoptosis of U251 glioma stem cells(GSCS)in vitro.2.To observe the effect of HCMV infection on U251 GSCS tumorigenesis and angiogenesis in the brain of nude mice,after U251 GSCS were infected with HCMV AD169 strain at MOI of 1.Methods:1.Human embryonic lung fibroblasts were infected with HCMV AD169 strain.The supernatant was collected and the virus titer was determined by plaque assay.2.The GSCS were isolated from the U251 glioma cell line by monoclonal culture method.The proportion of CD133 positive cells in U251 GSCS was detected by flow cytometry.3.U251 GSCS were infected with HCMV AD169 strain at MOI of 0.2,1,5.The proliferation of U251 GSCS were detected by MTT at 12,24,36,and 48 h.4.After HCMV infected 48 h,the expressions of IE-1 m RNA,Bcl-2 m RNA and Bax m RNA were detected by RT-PCR and the expressions of IE-1,Bcl-2,Bax and CD133 proteins were detected by western blot.5.U251 GSCSwere infected with HCMV AD169 strain at MOI of 1,then inoculated into the brains of ten nude mice,set as experimental group;in control group,U251 GSCS were un-infected with HCMV.Observate the growth and appearance change of nude mice.6.Killed moribund nude mice,remove brain tissue and isolate tumor tissue for HE staining.The expression of IE-1 and CD34 protein was detected by immunohistochemistry.7.Use weidner vascular counting method to calculate the tumor microvessel density of experimental group and group.Results:1.U251 glioma cell suspension growth in the serum free medium.Flow cytometry results showed that the percentr of CD133 positive cells in U251 GSCS was 98.00%.2.The MTT results showed that the proliferation of U251 GSCS infected with HCMV at MOI of 1 and 5 was significantly increased compared with the control group(P <0.05).there was no significant difference in cell proliferation between U251 GSCS infected with HCMV at MOI of 0.2 and control group at 12,24,36 and 48h(P> 0.05).3.RT-PCR results showed that the expression of IE-1 m RNA and Bcl-2 m RNA was up-regulated and the expression of Bax m RNA was down-regulated with the infection of HCMV(MOI=1,5)when compared with the control group(P <0.05),there was no significant difference in the expression of IE-1 m RNA,Bcl-2 m RNA and Bax m RNAbetween U251 GSCS infected with HCMV at MOI of 0.2 and control group(P>0.05).4.Western blot results showed that the expressions of IE-1,CD133 and Bcl-2 proteins were up-regulated and the expression of Bax protein was down-regulated after infected with HCMV at MOI of 1,5(P <0.05).5.After transplanting the HCMV(MOI=1)-positive U251 GSCS three weeks later,most of nude mice occur skin shrinkage and neurological symptoms.6.HE staining results showed tumor cells were dysplastic.Immunohistochemical results showed that IE-1 positive expression in HCMV(MOI=1)infected transplanted tumor compared with control group.7.Weidner vascular count showed that the microvessel density of HCMV(MOI=1)infected U251 GSCS transplanted tumor was significantly higher than that of the control group(P < 0.05).Conclusions:1.A certain titer of HCMV infected with U251 GSCS can promote its proliferation and maintain its dry characteristics.tumor.2.After HCMV infection with U251 GSCS,IE-1 positive-express in the cells and U251 GSCS apoptosis was inhibited via to down regulating the expression of Bax protein and up regulating the expression of Bcl-2 protein.3.HCMV infection can promote the growth of U251 GSCS transplanted tumor by promoting the differentiation of CD133 positive U251 GSCS into CD34 vascular endothelial cells,then increasing its malignancy. |
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