| Oncolytic viruses?OVs?which are genetically engineered to selectively replicate in and kill cancer cells,represent a research focus of anti-tumor therapy.Nowadays,HSV-1 become one of the heat spot in OVs research because of its high morbidity in the population,low virulence and easy to reconstruct virus.Herpes simplex virus type1?HSV-1?based oncolytic HSV?oHSV?,was the FDA first approved OV,which was treated to patients with advanced melanoma[1].The host response against oHSV is complex,multifaceted,and the induction of cytokine storm by OV is one of severeoutcome.The cancer patients may cause more severe immunopathological damage due to their homeostasis disturbances and weaken immune system once cytokine storm happened.The suppressors of cytokine signalling?SOCS?proteins are pivotal negative regulators of the JAK/STAT pathway which are involved in the control of cytokine networks.The SOCS protein may be closely related in virus infection and organdamage,and SOCS4 protein plays an important role in the regulation of anti-influenza virus infection signal pathway.Ovs,which were modified by HSV-1,are applied to clinical treatment.And its security issues need to be futher explored.Based on the knowledge and technology that we acquired,we had inserted SOCS4 gene into BAC to reconstruct HSV-SOCS4virus,and explore the role of SOCS4 protein in immune pathological injury afterinfected by HSV-1,which could be inducted cytokine storm.ObjectivesIn our study,we had inserted SOCS4 gene into BAC to reconstruct HSV-SOCS4virus.After intranasal infection with HSV-1?F?or HSV-SOCS4 in mice,we use a series of method such as collecting the sample of BALF and serum to detect the levels of cytokine at various time points,testing the quantity and function of immune cells,determinating the virus titer in lung and recording body weights and mortality of mice to compare their differences.In this way,we explore the role of SOCS4 protein in immune pathological injury after the infection of HSV-1,which inducting cytokine storm.Methods1.We had inserted SOCS4 gene into BAC to reconstruct HSV-SOCS4 virus.2.The mice was infected via the intranasal route with HSV-1?F?,HSV-SOCS4 or PBS.After infection,mice were weighed and monitored for morbidity and mortality every day for 12 days.3.After collecting serum and BALF samples,we use the method of double sandwich ELISA to test the levels of cytokines.4.After isolating the immune cells,we use flow cytometric analysis to detect the quantity and function of the cells.5.To evaluate the correlation of cytokines with virus replication/clearance,virus titer from infected mice lung was quantified.6.The lungs of mice were performed histopathology analysis to make sure the extent of the lung issue damage.Results1.After PCR identification of the recombinant,it was suggested that we constructed HSV-SOCS4 recombinant virus successfully.We detected the virus replication capabilities between HSV-1?F?and HSV-SOCS4 via rendering their growth curves.And both of them had no significant difference in replication capacity.2.Serum and Bronchoalveolar lavage fluid samples collection and double sandwich ELISAMCP-1:The maximum level of MCP-1 in BALF of HSV-1?F?mice was detected on day1 and day3,and it is much higher than HSV-SOCS4 mice.IL-1?:An uptrend-downtrend curve of IL-1?production in BALF sample was found of HSV-1?F?infected mice and it fleetly dropped down to on day7.IL-1?values of serum sample make a strong upregulation on day7 in HSV-1?F?mice,which was much higher than HSV-SOCS4 mice.TNF-?:The highest TNF-?level in BALF of HSV-1?F?mice was also observed on day1.The maximum level of TNF-?in serum of HSV-1?F?mice was detected on day7.HSV-1?F?mice were much higher than that of HSV-SOCS4 mice in both samples.IL-6:The IL-6 levels of HSV-1?F?mice were the highest on day7 in both BALF and serum sample.The HSV-SOCS4 mice were significant lower than those of HSV-1?F?mice.IFN-?:There was no signant difference in IFN-?concentration on day3 and day7 after infected by HSV-1?F?,and both of them were much higher than on day1.Concentration of IFN-?in serum on day7 was higher than that in on day1 and day3.And the HSV-SOCS4 mice were significant lower than those of HSV-1?F?mice in 2samples.3.Cell isolation and flow cytometric analysis to comfirm the quantity and function of immune cells:CD11b+cells in BALF from HSV-1?F?mice were predominated over that from HSV-SOCS4 mice and much more cells were stained positive on day1;A large number of CD4+CD62L+and CD8a+CD62L+cells had been detected in spleen on day7,and both of double positive cells from HSV-1?F?mice were much more than from HSV-SOCS4 mice.4.Detection of virus titer in mice lung:The amount of virus was highest on day1 after the infection,it declined greatly on day3,and no virus was detected on day7.5.The pathological changes of lung issue:a severe pathological change was appeared of HSV-1?F?mice lung,especially on day7,and much more slighter pathological change were observed of HSV-SOCS4 or PBS infected mice lung.6.Body weight and mortality of mice after intranasal infection:HSV-1?F?infected mice loss their weight gradually over time,and mice died rapidly and no mouse from HSV-1?F?group survived on day11.HSV-SOCS4 group mice lost weight slightly and the survival rate of HSV-SOCS4 mice maintained at 100%on day12.Conclusions1.we constructed HSV-SOCS4 recombinant virus successfully.And there was no significant difference in replication capacity between HSV-1?F?mice and HSV-SOCS4 mice.2.The mice were infected via the intranasal route with HSV-1?F?or HSV-SOCS4:A significantly higher level of all five cytokines from HSV-1?F?infected mice than that from HSV-SOCS4 mice in both BALF and serum sample;The number of immunity cells was much higher in HSV-1?F?infected mice than in HSV-SOCS4 infected mice,which lead to much more severe pathological damage and contribute to increasemortality.These suggest that SOCS4 protein play an important role of in regulation of antiviral infection signal pathway,which could be alleviated lung pathologicaldamage inducted by cytokine strom after infected HSV-1. |
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