| OBJECTIVE:Influenza , a kind of transmissible disease caused by influenza virus, does a lot of harm to people's heath and the social economy. DRP is a TCM compound preparation comprised of scutellaria, cape jasmine fruit,pig gall, HERBA ERIGERONTIS.It has the effect of heat-clearing and detoxicating, promoting blood circulation to remove meridian obstruction .In this thesis,we investigated the effect of Dureping injection(DRP) on the function of FM1 infected mice spleen T cell in vitro and on the function of FM1 infected mice lung T cell in vivo,in order to understand the target of DRP antiviral effect,and provide theoretical evidences for application in clinical treatment and further study of DRP.METHODS:The experiment includes two sections:in vitro and in vivo.The first section is to study the effect of Dureping injection(DRP) on the function of mice spleen T cell during FM1 infection in vitro. We take the spleen T cells of BALB/c mice(SPF grade) as our major object,and set groups,SHL group,Sham group,and FM1 infected Model group. The experiment include :①the effect of DRP on the multiplication,and IFN-γ,IL-10 production of normal spleen T cells;②the effect of FM1 on Con A induced spleen T cells and the effect of DRP intervention;③the effect of FM1 infected Mф(Ana-1) on T cells multiplication and IFN-γproduction and the effect of DRP intervention;④the effect of DRP on the function of spleen T cells killing FM1 infected Mф(Ana-1).The second section is to study the effect of Dureping injection (DRP) on the function of FM1 infected mice lung T cell in vivo. We take the ICR mice(SPF grade) as our object,and set 7 groups: DRP1, DRP2, DRP3,SHL,Ribavirin,Sham,and Model.On the first day we infected the ICR mice with FM1, the mice were treated with medicine, one time per day.The mice were sacrificed on the 7rd after inoculation,we weight the mice's body and lung .Then the mice's lung tissue was prepared, IFN-γmRNA,IL-10mRNA in the lung tissue was measured with RT-PCR, CD40L protein in the lung tissue was measured with Western-blot.RESULTS:Part 1 The experiments in vitro.1 The effect of DRP on the multiplication and IFN-γ,IL-10 production of normal spleen T cellsDRP 17μg/ml and DRP 8.5μg/ml groups showed inhibitory action on the multiplication of normal spleen T cells and IL-10 production.,at the same time ,DRP 17μg/ml group showed promotion actin on the level of IFN-γproduction .2 the effect of FM1 on Con A induced spleen T cells and the effect of DRP intervention⑴After the spleen T cells were infected with FM1 and treated with medicine for 12 hours ,we changed the culture fluid and continue culturing to 36 hours , compared with sham group ,the level of model group spleen T cell decreased , DRP 17μg/ml group showed up-regulation on the level of spleen T cell; After the spleen T cells were infected with FM1 and treated with medicine for 24 hours,we changed the culture fluid and continue culturing to 36 hours, compared with sham group ,the level of model group spleen T cell decreased , DRP 17μg/ml,8.5μg/ml and 4μg/ml groups showed up-regulation on the level of spleen T cell; After the spleen T cells were infected with FM1 and treated with medicine for 36 hours, compared with sham group ,the level of model group spleen T cell decreased ,DRP groups did not show any significant function .⑵After the spleen T cells were infected with FM1 and treated with medicine for 12 hours , the level of IFN-γ,IL-10 pruduction in model group significantly higher than sham group ,the level of IFN-γproduction in DRP 8μg/ml and DRP 2μg/ml groups was significantly lower than model group,and the level of IL-10 production in DRP 17μg/ml,2μg/ml groups was significantly higher than sham group and lower than model group,but the level of IL-10 production in DRP 8μg/ml groups was significantly lower than sham group and model group; the level of IFN-γproduction in DRP17μg/ml,8μg/ml and DRP 2μg/ml groups was significantly higher than model group,After the spleen T cells were infected with FM1 and treated with medicine for 24 hours, the level of IFN-γ,IL-10 pruduction in sham group increased and significantly higher than in model group which decreasd; and the level of IL-10 production in DRP 17μg/ml,8μg/ml groups was significantly lower than model group;After the spleen T cells were infected with FM1 and treated with medicine for 36 hours,the he level of IFN-γ,IL-10 pruduction in all groups decreased and have no significant difference.3 the effect of FM1 infected Mф(Ana-1) on T cells multiplication and IFN-γproduction and the effect of DRP interventionAna-1 (treated with medicine beforehead) was infected with FM1 , 8 hours latter,the cell cultural supernatant were added into Con A induced T cells,the multiplication of T cell in model group was the same as sham group ,but the level of IFN-γpruduction was insignificantly highter than sham group ; DRP 8μg/ml and DRP 2μg/ml groups showed up-regulation on the multiplication of spleen T cells, DRP 17μg/ml showed up-regulation on the level of IFN-γpruduction in spleen T cells;Ana-1 (treated with medicine beforehead) was infected with FM1 ,12 hours latter,the cell cultural supernatant were added into Con A induced T cells,the multiplication of T cell in model group was significantly lower than in sham group ,and the the level of IFN-γproduction in model group was insignificantly lower than in sham group; DRP 17ug/ml , DRP 8μg/ml and DRP 2μg/ml groups showed up-regulation on the multiplication of spleen T cells ,DRP 17μg/ml showed up-regulation on the level of IFN-γpruduction in spleen T cells; Ana-1 (treated with medicine beforehead) was infected with FM1 ,24 hours latter,the cell cultural supernatant were added into Con A induced T cells, the multiplication of T cell in model group was significantly lower than in sham group ,and the the level of IFN-γproduction in model group was insignificantly lower than in sham group; DRP groups did not showed any function on the multiplication of spleen T cells, DRP 17ug/ml and DRP 8μg/ml groups showed up-regulation on the level of IFN-γpruduction in spleen T cells;4 the effect of DRP on the function of T cells killing FM1 infected Mф(Ana-1)After T cells were added to FM1 infected Ana-1 for 12 hours, When E/T=100 :1, T cells killing rate in model group was significantly lower than sham goup ,DRP 17μg/ml showed up-regulation on the T cells killing rate; When E/T=50 :1, T cells killing rate in model group was also significantly lower than sham goup ,DRP 17μg/ml and DRP 8.5μg/ml groups showed up-regulation on the T cells killing rate.After T cells were added to FM1 infected Ana-1 for 24 hours, T cells killing rate in model group have no significantly difference with sham group .When E/T=100 :1 ,DRP 17μg/ml and DRP 8.5μg/ml showed up-regulation on the T cells killing rate; When E/T=50 :1, T cells killing rate in model group was also significantly lower than sham goup ,DRP 17μg/ml ,DRP 8.5μg/ml and DRP 2μg/ml groups showed up-regulation on the T cells killing rate.Part 2 The experiments in vivo1 the effect of DRP on the lung index of FM1 infected miceAfter FM1 infected mice was treated with DRP for 7 days, the lung index of mice in model group was significantly higher than sham group ;compared with model group, the lung index of mice in Ribarivin,SHL, DRP1,DRP2 and DRP3 groups was significantly decreased,and the function of DRP2 was the best ;we can see the body weight of mice in DRP2 and DRP3 groups have no significant diffirence with sham group.2 the effect of DRP on the transcription of IL-4,INF-γmRNA in FM1 infected mice lungAfter FM1 infected mice was treated with DRP for 7 days,the transcription of IL-4,INF-γmRNA in model group was significantly higher than sham group ;compared with model group, DRP showed down-regulation on the transcription of IL-4 in FM1 infected mice lung ,and up-regulation on the transcription of INF-γin FM1 infected mice lung.3 The effect of DRP on the expression of CD40L protein in FM1 infected mice lungAfter FM1 infected mice was treated with DRP for 7 days, the expression of CD40L protein in model group was significantly higher than sham group ;compared with model group, the expression of CD40L protein in Ribarivin,SHL, DRP1,DRP2 and DRP3 was significantly decreased,but the expression of CD40L protein in DRP groups was significant highter than sham group and Ribarivin group.CONCLUSION DRP in vitro could inhibit Th2 cell factor IL-10 production,and enhance Th1 cell factor IFN-γproduction,protect spleen T cells from death caused by FM1, inhibit FM1 infected T cells produce Th2 cell factor IL-10,and enhance Th1 cell factor IFN-γ; the cell cultural supernatant of FM1 infected Ana-1 (treated with DRP beforehead) could enhance the multiplication and IFN-γproduction of T cells; DRP could enhance the function of CTL killing FM1 infected Mф(Ana-1).DRP in vivo could down-regulation the lung index of FM1 infected mice,and have no significant inhibition on the mice weight; DRP could inhibit the transcription IL-4mRNA , and enhance the transcription of INF-γin FM1 infected mice lung; DRP could inhibit the expression of CD40L protein in FM1 infected mice lung. |
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