| Part oneEffect of cigarette smoke exposure on lung dendritic cells and thecorrelation research with Th1/Th2 balance in asthmatic ratsObjective To study the effect of cigarette smoke exposure on the number and distribution oflung DCs and expression of IFN-γ,IL-4,10,12 in asthmatic rats,and to explore immunologicalmechanism between smoke and asthma.Methods 50 male Wistar rats were randomly divided into five groups:a control group,aasthma group,a cigarette smoke exposure combined asthma group,a asthma group treated bybudesonide,a cigarette smoke exposure combined asthma group treated by budesonide.Established the rats model of every group.The changes in the number of leukocyte and differentkinds of leukocyte in bronchoalveolar lavage fluid(BALF) have been studied.The number anddistribution of OX-62+ cells(DCs) was determined by immunohistochemistry and OX-62protein was determined by Western blot.The concentration of IFN-γand IL-4,10,12 inperipheral blood and BALF were measured by enzyme-linked immunosorbent assay (ELISA).Results1. Compared with the control group,the number of leukocytes,the percentage of neutrophils,andlymphocytes of BALF in the asthma group and the cigarette smoke exposure combined asthmagroup remarkably increased (P<0.01,respectively).Compared with the asthma group,the numberof leukocytes and the percentage of neutrophils of BALF remarkably increased and thepercentage of eosnophils decreased in the cigarette smoke exposure combined asthmagroup(P<0.01,respectively).Compared with the asthma group treated by budesonide,the numberof leukocytes and the percentage of neutrophils of BALF remarkably increased meanwhile thepercentage of eosnophils decreased in the cigarette smoke exposure combined asthma grouptreated by budesonide(P<0.01 P<0.05 and P<0.01).2. Compared with the control group,the number of DCs in the asthma group and the cigarettesmoke exposure combined asthma group remarkably increased(P<0.01).Compared with theasthma group,the number of DCs remarkably increased in the cigarette smoke exposurecombined asthma group(P<0.01).Compared with the asthma group treated by budesonide,thenumber of DCs increased remarkably in the cigarette smoke exposure combined asthma grouptreated by budesonide(P<0.01).3. Compared with the control group,the expression of OX-62 protein increased remarkably inthe asthma group and the cigarette smoke exposure combined asthmagroup(P<0.01,respectively).Compared with the asthma group,the expression of OX-62 protein increased remarkably in the cigarette smoke exposure combined asthmagroup(P<0.01).Compared with the asthma group treated by budesonide,the expression ofOX-62 protein increased remarkably in the cigarette smoke exposure combined asthma grouptreated by budesonide(P<0.01).4. Compared with the control group,IFN-γof peripheral blood and BALF decreased remarkablyin the asthma group and the cigarette smoke exposure combined asthmagroup(P<0.01).Compared with the asthma group,IFN-γof peripheral blood and BALFdecreased remarkably in the cigarette smoke exposure combined asthmagroup(P<0.01).Compared with the asthma group treated by budesonide,IFN-γof peripheralblood and BALF decreased remarkably in the cigarette smoke exposure combined asthmagroup treated by budesonide(P<0.01).Compared with the control group,IL-4 of peripheral bloodand BALF increased remarkably in the asthma group and the cigarette smoke exposurecombined asthma group(P<0.01).Compared with the asthma group,IL-4 of peripheral blood andBALF increased remarkably in the cigarette smoke exposure combined asthmagroup(P<0.01).Compared with the asthma group treated by budesonide,IL-4 of peripheral bloodand BALF increased remarkably in the cigarette smoke exposure combined asthma grouptreated by budesonide(P<0.01).5. Compared with the control group,IL-10 and IL-12 of peripheral blood and BALF decreasedremarkably in the asthma group and the cigarette smoke exposure combined asthma group(P<0.01).Compared with the asthma group,IL-10 and IL-12 of peripheral blood and BALFdecreased remarkably in the cigarette smoke exposure combined asthma group(P<0.01).Compared with the asthma group treated by budesonide,IL-10 of peripheral blood andBALF remarkably increased(P<0.01) and IL-12 remarkably decreased(P<0.01) in the cigarettesmoke exposure combined asthma group treated by budesonide.6. The correlation analysis: the number of lung DCs was positively correlated with the numberof leukocytes,neutrophils,lymphocytes and was negatively correlated with IL-10 and IL-12 ofperipheral blood and BALF.IL-10 of peripheral blood was positively correlated with IFN-γ/IL-4ratio of peripheral blood.IL-12 of peripheral blood and BALF was positively correlated withIFN-γ/IL-4 ratio of peripheral blood and BALF.Conclusion Cigarette smoke exposure can up-regulate the number of dendritic cells,theexpression of IL-4 and down-regulate the expression of IFN-γ,IL-10 and IL-12,which may playan important role in aggravating airway inflammation and Th1/Th2 balance in asthmatic rats. Part twoEffect of cigarette smoke exposure on function in marrow-deriveddendritic cells activating lymphocytes and the correlationwith Th1/Th2 balance in asthmatic ratsObjective To study the effect of cigarette smoke exposure on the expression ofCD80,CD86,IL-10,IL-12 and relationship with both the activity of lymphocytes and Th1/Th2balance by marrow-derived DCs from asthmatics rat,and to explore immunological mechanismbetween smoke and asthma.Methods 50 male Wistar rats were randomly divided into five groups as the partone.Established the rats model of every group.Then the bone marrow-derived DCs of eachgroup were cultured for five days and then added LPS for 48 hours in every group.Inaddition,rat spleen lymphocytes and bone marrow derived DCs were cultured together formixed lymphocyte responses(MLR).Then collected cells and cell supernatants.The expressionof IFN-γ,IL-4,10,12 were determined by ELISA,the expression of CD80 and CD86 weredetermined by flow cytometric analysis,and the proliferations of lymphocytes were examinedwith MTT colorimetric assay.Results1. Compared with the control group,the expression of CD80 decreased and CD86 increasedremarkably in the cigarette smoke exposure combined asthma group(P<0.01,respectively).Compared with the asthma group,the expression of CD80 and CD86 decreased remarkably in thecigarette smoke exposure combined asthma group (P<0.01).Compared with the asthma grouptreated by budesonide,the expression of CD80 and CD86 decreased remarkably in the cigarettesmoke exposure combined asthma group treated by budesonide (P<0.01).2. Compared with the control group,the expression of IL-10 increased remarkably in thecigarette smoke exposure combined asthma group(P<0.01). Compared with the asthma group,the expression of IL-10 decreased remarkably in the cigarette smoke exposure combinedasthma group(P<0.01).Compared with the asthma group treated by budesonide,the expressionof IL-10 increased remarkably in the cigarette smoke exposure combined asthma group treatedby budesonide(P<0.01);Compared with the control group,the expression of IL-12 decreasedremarkably in both the cigarette smoke exposure combined asthma group and the asthmagroup(P<0.01,respectively).Compared with the asthma group, the expression of IL-12decreased in cigarette smoke exposure combined asthma group remarkably(P<0.01). Comparedwith the asthma group treated by budesonide,the expression of IL-12 decreased remarkably inthe cigarette smoke exposure combined asthma group treated by budesonide(P<0.01).3. results of MLR (1) Compared with the control group and the asthma group, the stimulating activity of DCs onallogeneic lymphocytes decreased remarkably in the cigarette smoke exposure combinedasthma group(P<0.01,respectively).Compared with the asthma group treated by budesonide,thestimulating activity of DCs on allogeneic lymphocytes decreased remarkably in the cigarettesmoke exposure combined asthma group treated by budesonide(P<0.01) .(2) Compared with the control group, the expression of IFN-γdecreased and the expression ofIL-4 increased remarkably in both the cigarette smoke exposure combined asthma group andthe asthma group(P<0.01,respectively).Compared with the asthma group,the expression ofIFN-γdecreased and the expression of IL-4 increased remarkably in the cigarette smokeexposure combined asthma group(P<0.01,respectively).Compared with the asthma grouptreated by budesonide,the expression of IFN-γdecreased remarkably and the expression of IL-4increased remarkably in the cigarette smoke exposure combined asthma group treated bybudesonide(P<0.01,respectively).4. The expression of CD80 was positively correlated with the activity of lymphocytes and theexpression of IL-12(P<0.01,respectively).The expression of CD86 was positively correlatedwith the activity of lymphocytes and the expression of IL-10(P<0.01,respectively).Theexpression of IL-10 was positively correlated with the activity of lymphocyte s and theexpression of IL-12 was positively correlated with IFN-γ/IL-4 ratio(P<0.01,respectively).Theexpression of IL-10 was negatively correlated with IL-12(P<0.05).Conclusions Cigarette smoke exposure may inhibit expression of CD80,CD86,IL-10,IL-12 bymarrow-derived DCs in asthmatics rat.what's more,these changes decrease the ability of DCsactivating lymphocytes,enhance Th1/Th2 imbalance and reduce the effectiveness ofglucocorticoid,which may play an important role in aggravating airway inflammation inasthma. Part threeEffect of nicotine on the role of dendritic cells in asthmaChapter oneEffect of nicotine on function in marrow-derived dendritic cellsactivating lymphocytes and the correlation researchwith Th1/Th2 balance in asthmatic ratsObjective To study the effect of nicotine on the expression of IL-10,12,CD80,CD86 andrelationship with both the activity of lymphocytes and Th1/Th2 balance by marrow-derivedDCs from asthmatics rat,and to explore immunological mechanism about nicotine aggravatingasthma.Methods Established asthma rats model and the bone marrow-derived DCs werecultured,then DCs were randomly divided into the control group and the nicotine-treated group.①concentration-dependent effect of nicotine on the expression of IL-10,IL-12,CD80,CD86:DCswere incubated with 50,100,200,400μg/ml nicotine for 72 hours.②time-dependent effect ofnicotine on the expression of IL-10 and IL-12:The cells were exposured to 400μg/ml nicotinefor 0,4,8,12,24,72 hours respectively.The cells of the control groups were exposured to thesame volume of PBS instead of nicotine at each time points. In addition,rat spleen lymphocytesand bone marrow derived DCs were cultured together for MLR.Then collected cells and cellsupernatants.The expression of IFN-γ,IL-4,IL-10 and 12 were determined by ELISA,theexpression of CD80 and CD86 were determined by flow cytometric analysis,and theproliferations of lymphocytes were examined with MTT colorimetric assay.Results1. Compared with the control group,the expression of CD80 and IL-12 protein of 400μg/mlnicotine-treated group decreased remarkably(P<0.01,respectively) and the expression of IL-10protein of both 200μg/ml and 400μg/ml nicotine-treated group decreasedremarkably(P<0.01,respectively).2. Compared with the control group, the stimulating activity of DCs on allogeneic lymphocytesof 100,200,and 400μg/ml nicotine-treated group decreased remarkably(P<0.01,respectively).Compared with the control group,the expression of IFN-γand IL-4 ofboth 200μg/ml and 400μg/ml nicotine-treated group decreasedremarkably(P<0.01,respectively).3. After stimulation with 400μg/ml nicotine for 0, 4, 8, 12, 24, 72 hours, the expression of IL-10decreased over time and reached the lowest point at 72 hours,which were remarkably lowerthan the control groups at 12,24,72hours(P<0.01,respectively).But compared with the control group,the expression of IL-12 increased remarkably at 12 hours(P<0.01,respectively),and theexpression of IL-12 decreased remarkably at 72 hours(P<0.01,respectively).4. After stimulation with 400μg/ml Nicotine for 0, 4, 8, 12, 24, 72 hours, the stimulatingactivity of DCs on allogeneic lymphocytes and the expression of IL-4 protein decreased overtime and reached the lowest point at 72 hours,which were remarkably lower than control groupsat 12,24,72 hours(P<0.01,respectively).But compared with control group,the expression ofIFN-γincreased remarkably at 12 hours(P<0.01,respectively),and the expression of IFN-γdecreased remarkably at 72 hours(P<0.01,respectively).Conclusion A certain concentration of nicotine can inhibit the asthma rats DCs to decreaseexpression of CD80,CD86,IL-10 and IL-12 in a period of time,which may increase the imbalanceof Th1/Th2, and aggravate airway inflammation in asthma. ChapteChapter twoRoles of myeloid differentiation factor 88 on bone marrow-deriveddendritic cells in asthmatic rat and interference effects by nicotineObjective To study the effect of nicotine on the expression of myeloid differentiation factor88 (MyD88),nuclear factor-κB(NF-κB),and relationship with both the activity of lymphocytesand Th1/Th2 balance by marrow-derived DCs from asthmatics rat,and to exploreimmunological mechanism about nicotine aggravating asthma.Methods Established the asthma rats model and the bone marrow-derived DCs were cultured.DCs were randomly divided into four groups as follows:the control group,the nicotine group,theasthma group and the nicotine combined asthma group.The rat bone marrow derived DCs werecultured,then added a certain volume of PBS for 72 hours in both the control group and theasthma group while the bone marrow-derived DCs were cultured and then adding liquidnicotine(400μg/ml)for 72 hours in both the nicotine group and the nicotine combined asthmagroup,Then collected cells and cell supernatants.In addition,rat spleen lymphocytes and bonemarrow derived DCs were cultured together for MLR.The expression of IFN-γ,IL-4,10,12 weredetermined by ELISA.The expression of MyD88 were determined by Western blot.Theproliferations of lymphocytes were examined with MTT colorimetric assay. The expression ofNF-κB were determined by immunocytochemistry.Results1. Compared with the control group, the expression of IL-10 increased remarkably and IL-12decreased remarkably in the asthma group and the nicotine combined asthmagroup(P<0.01,respectively).Compared with the asthma group, the expression of IL-10 andIL-12 decreased remarkably in the nicotine combined asthma group(P<0.01 andP<0.05,respectively).2. Compared with the control group,the expression of MyD88 and NF-κB decreased remarkablyin the nicotine group,the asthma group and the nicotine combined asthma group(P<0.05 andP<0.01,respectively).Compared with the asthma group and nicotine group,the expression ofMyD88 and NF-κB decreased remarkably in the nicotine combined asthma group(P<0.01 andP<0.05,respectively).3. results of MLR(1) Compared with the control group,the stimulating activity of DCs on allogeneic lymphocytesincreased remarkably in asthma group and decreased in the nicotine group and the nicotinecombined asthma group(P<0.01 and P<0.05,respectively). Compared with the asthma group,thestimulating activity of DCs on allogeneic lymphocytes decreased in the nicotine combinedasthma group(P<0.01,respectively). (2) Compared with the control group, the expression of IFN-γdecreased remarkably in theasthma group and the nicotine combined asthma group(P<0.01,respectively).Compared with thecontrol group, the expression of IL-4 increased remarkably in the asthma group and decreasedremarkably in the nicotine group and the nicotine combined asthmagroup(P<0.01,respectively).Compared with the asthma group, the expression of IL-4 and IFN-γdecreased remarkably in the nicotine combined asthma group(P<0.01,respectively).4. The expression of MyD88 was positively correlated with the expression of NF-κB and IL-12,and the expression of NF-κB was positively correlated with the expression ofIL-12(P<0.01,respectively).The expression of IL-10 was positively correlated with the activityof lymphocytes and negatively correlated with IFN-γ/IL-4 ratio(P<0.01,respectively).Theexpression of IL-12 was positively correlated with IFN-γ/IL-4 ratio(P<0.01,respectively).Conclusion Nicotine can inhibit the expression of IL-10,IL-12,NF-κB and MyD88 bymarrow-derived DCs from asthmatics rat,which exacerbate the imbalance of Th1/Th2,and itseffects on IL-12 may be caused by MyD88-dependent and NF-κB signaling pathways.
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