The Promotion Of VSV Oncolytic Sensitivity By Suppressing Mx1 Gene Expression Through RNAi

Part I: The promotion of VSV oncolytic sensitivity by suppressing Mx1 gene expression through RNAiObject: Studying the oncolytic sensitivities of 5 different tumor cell lines to VSV; Clarifying the relationship between the VSV oncolytic sensitivity and the expression of Mx1 gene, which is an antiviral protein downstream of type I interferon pathway; improving the oncolytic sensitivity of the tumor cells by suppressing Mx1 expression using RNAi technology.Methods: 5 tumor cell lines: RAW264.7, B16, HEPA1-6, MHCC97-H, SMMC7721, were tested for their sensitivity to VSV. After 48 hours attacking, TCID50 assay and MTT assay were performed to determine the VSV oncolytic sensitivity of these 5 tumor cell lines. The baseline expression of Mx1 in three mice tumor cell line was detected by RT-PCR. The Mx1 expression levels in these cell lines were also examined by RT-PCR after exogenous type I IFN induction and VSV infection. Furthermore, Mx1 gene expression was suppressed by RNAi technology, MTT assay and microscopy analysis were used to show the changes in sensitivity to VSV attack after Mx1 gene manipulation.Result: The VSV oncolytic sensitivity of 5 tumor cell lines is B16ï¹¥HEPA1-6ï¹¥MHCC97-Hï¹¥SMMC7721ï¹¥RAW264.7. The basic and induced Mx1 gene expression level showed correlations between Mx1 expression level and sensitivity to VSV. Inhibition of Mx1 gene expression in RAW264.7 cell line (the most resistant tumor cell lines for VSV oncolysis) by RNAi technology promoted VSV oncolytic sensitivity nearly about 35% comparing to control group.At last, in order to the broad applicability of this method and shortly using virus for safety, inhibition of Mx1 gene expression in B16 cell line (the most sensitive tumor cell lines for VSV oncolysis) by RNAi technology promoted VSV oncolytic sensitivity nearly about 30% comparing to control group. Our results suggest that suppression of antiviral gene expression downstream of type I interferon pathway by RNAi technology can improve the sensitivity of the tumor cells to VSV oncolysis, which could be a novel way to promote viral-based oncolytic therapy.Conclusion: Different tumor cell lines showed different VSV oncolytic sensitivities; The Mx1 expression level was correlated with the resistance to VSV oncolysis. Inhibition of Mx1 gene expression could significantly increase the sensitivity of tumor cells to VSV oncolysis.Part II: Early detection of virus infection in pediatric leukemia patients using anti-MxA monoclonal antibody and flow cytometryObject: To detect viral infection in pediatric leukemia patients using anti-human MxA monoclonal antibody and flow cytometry.Methods: Fluorescent label anti-MxA monoclonal antibody and determine its specificity. Collect peripheral blood from pediatric leukemia patients with or without fever. Isolate mononuclear cells and intracellular stain for MxA using the fluorescent labeled anti-MxA monoclonal antibody and fow cytometry.Result: Fluorescent labeled anti-MxA monoclonal antibody showed high specificity detecting MxA expression in A549 cell line induced with type I interferon. Positive detection of MxA expression was correlated with viral infection and it was 7 days earlier than the clinical diagnosis. All the patients without fever and patients with fever, but diagnosed as bacterial infection showed negative in the flow analysis.Conclusion: Anti-human MxA monoclonal antibody and flow cytometry could be used for early detection of virus infection in pediatric leukemia patients with fever. The method can be easily applied in the clinics.