Research On The Effect Of Sinomenine Induce DC2.4 Maturation And On The Mechanism Of Sinomenine Induce Matured DC2.4 Apoptosis

Objectives1.Compare the differences between DC2.4 and BMDC, choose the better one to complete the subsequent experiment.2.Explore the mechanism of SIN to treat rheumatoid diseases, and whether it is related to theDC2.4.3.Discuss the influence of SIN on the apoptosis of DC2.4, and reveal the the mechanism of SIN on the treatment of autoimmune diseases.Methods1.Use the morphology methods and the phenotypes CD11c,CD80 and CD86 of cytomembrane to identify the difference between DC2.4 with BMDC,and assess the maturity of DC2.4.2. The experiment was divided into four groups:LPS control group.LPS with different concentrations of SEN group((10ug/ml?20ug/ml and 40ug/ml).Applied ELISA to explore the amount-effect and a time-effect relationship of cytokines INF-??TNF-??IL-6?IL-10 and IL-12 on different times(24h,48h and 72h);use the flow cytometry to detect the expression of the co-stimulating factor CD80 and CD86 on DC2.4 cell membrane. Through one-way mixed lymphocyte experiment,each group DC2.4 play as stimulate cells and the T cell separated from BALB/c mice spleen through nylon as reaction cells.The result is dentermined by MTT method which is represent the response inhibition of T cell proliferation with the condition of stimulate cells vs reaction cells as 1:10?1:40?1:80 and 1:100.With the experiment,we can observe whether SIN have a effect on the maturation of DC2.43. After LPS co-culture 24h,the experiment is divided into four groups:control group,different concentrations of SIN group(10ug/ml?20ug/ml and 40ug/ml). Analyze the proliferation effect of SIN on matured DC2.4, detect inhibition effect of SIN on DC2.4. With the double dye of AnnexinV-FITC and PI,apply the flow cytometry to matured the apoptosis percentage of DC2.4,apply the relative quantitative qPCR to detect the influence of SIN on Fas,Bcl-2 and Bax.apply the western blot to detect the caspase3, cleaved caspase9 and cleaved PARP.Results1. Morphology:DC2.4 is adherent state with polygonal, star or fusiform.After LPS co-cultured, the change of morphology of DC2.4 was not obvious. BMDC is first adherent then change to suspension,with irregular shape.After LPS co-cultured,BMDC become bigger with colony increased significantly. The shape of BMDC is irregular,which is star or fusiform with burr around cell.There is a onthe CD80 and CD86 of DC2.4 positive percentage of LPS stimulating group was significantly increaseand than the control group. The CD11c level was no statistical significance between the two groups.There are differences between DC2.4 and BMDC in the form and function. The result refers to DC2.4 is a unmatured DC.2. There was no significantly difference in the difference times,the INF-y, TNF-??IL-6 and IL-12 level of medium and high concentration group was lower than the LPS group (P<0.05), while a significantly increase in IL-10(P<0.05). So it refer to SIN can inhibit DC2.4 mature and secretion.It shows that the co-stimulating factor CD86 is significantly reduce with SIN intervention on medium and high concentration by flow cytometry. While the expression of CD80 was lower in matured or immatured DC2.4,so use the average fluorescent intensity to analyze.The result shows that CD80 decreased significantly with high concentration of SIN and SIN with high concentration inhibit DC2.4 mature. But there is not significantly difference on the group of low and medium concentration of SIN.In the experiment of mixed lymphocyte culture,it shows that the group of DC2.4 with SIN intervention lower the stimulating effect on T cell proliferation than control.But the result shows most obvious effect on T cell when the stimulus cells:reaction cells is 1:40.There was a statistically difference in different concentrations..With the concentration of SIN increased (>20ug/ml),the proliferation rate of DC2.4 was decreased.(P<0.05).Apoptosis detected by flow cytometry shows that the rate of apoptosis in medium and high concentration are significant higher than the control group.It primary caused by early apoptosis.Through relative quantitative qPCR,it reveal that SIN in medium and high concentration can promote Fas and Bax transcription and inhibit Bcl-2 transcription on matured DC2.4.Caspase-3?cleaved caspase-9 and cleaved PARP are detected by western blot.The result shows cleaved caspase-3 obvious concentration-dependent increased in the group with SIN intervention comparing with the control group.Cleaved PARP also obvious increased in the group with SIN than the control group.Cleaved caspase-9 increase in the group with SIN than the control group.But there is no obvious difference between various concentration of SIN in cleaved caspase-9 than in cleaved PARP and cleaved caspase-3.Conclusions1. Although there are some differences between DC2.4 with BMDC,the from and function are same.it refers that the DC2.4 is a unmatured DC and it can change into matured DC by LPS intervention.2.SFN can inhibit the active of T cells through preventing the maturation of DC2.4.3.SIN can promote the apoptosis of matured DC2.4 primary by ways of Fas to active caspases8,then active caspase-3 and PARP.At the same time,mitochondria death pathway takes part in the process of apoptosis.Through the change of Bcl-2/Bax,mitochondrial membrane permeability transition pore open,which induce the outflow of cytochrome C.It actives caspaseS and PARP and cause apoptosis.