Biological Characteristics In Murine Bmdc Under TGF-β1 Stimulation And SMAD6 RNA Interference In Vitro

PARTⅠIdentification on biological features of cultured murine mouse bone marrow-derived dendritic cellsAIM: To establish a method of cultivation and purification of dendritic cells from murine bone marrow in vitro and to observe their morphology.METHODS: Recombinant mouse granulocyte and macrophage colony stimulus factor(GM-CSF) and interleukin-4(IL4) induction was used to induce mouse bone marrow cells to form dendritic cells in vitro.The morphological changes were observed with light inverted microscope.CD11c,MHCII,and CD86 were identified with flow cytometry. The biological function was studied withmixed lymphocyte reaction (MLR).RESULTS: The percentage of the cultured DCs was more than 80% with typical morphology of DCs under light microscope. Immature DC,which had low expression in CD11C,MHCII and CD86,Mature DC,which had high expression in CD11c,MHCII and CD86,could stimulate allogenic mixed lymphocyte proliferation.CONCLUSION: Using of GM-CSF IL-4,LPS in the mouse marrow cell culture can obtain DC witll high quality and high purity.which makes a foundation and pattern for future research of Dc and supply materials on backward position experiment.PartⅡSmad6 gene expression in BMDC transfected by lentiviral shRNAAIM: Use the mouse Smad6 gene RNA interference (RNAi) vector which was successfully constructed ,effectively silence Smad6 gene in BMDC.METHODS: Use the constructed of lentiviral vectors, according to preliminary tests identified of MOI infect BMDC , collect cells after 120h and through the fluorescence microscope, RT-PCR,WB ways check the express status of Smad6 gene in cells. RESULTS : Under fluorescence microscope concluded that the efficiency of viral infection in 75%,PCR and WB confirmed that by the virus way ,the Smad6 gene silencing efficiency about 85% in cells.CONCLUSION: Successful bate the Smad6 gene expression in BMDC of mice.PartⅢBiological characteristics in murine BMDC under TGF-β1 stimulation and Smad6 RNA interference in vitroAIM: to discuss in the case of bate Smad6 and under the action of TGF-β1,if there are changes of biological characteristics in BMDC of mice.METHODS: BMDC were cultivated two groups with cytokine GM-CSF and IL-4,one group of cells infect with lentivirus aim to bata Smad6 gene. 6d, while stimulation with TGF-β1,after another 48h, Morphological characters of BMDC were observed by TEM, surface molecules such as CD11C,CD80,CD86 CD40and MHCⅡwere detected by flowcytometry,Interleukin-6 and interleukin-12 p70 in co-culture medium was quantified by ELISA. RESULTS: The group of not Smad6 gene inhibition, more obvious characteristics of mature DC under TEM, higher expression levels CD11c,CD80,CD86and MHCⅡ.Cell apoptotic index lower,the ability of MLR and secrete IL-6/IL-12p70 are strongger. The group of Smad6 gene inhibition ,mature characteristics of DC form is not clear ,lower expression levels CD11c,CD80,CD86and MHCⅡ. Cell apoptotic index higher,the ability of MLR and secrete IL-6/IL-12p70 are weak.CONCLUSION: In the case of the Smad6 was bated and stimulated with TGF-β1, cells were mostly immature state;but not Smad6 gene inhibition, the cells closer to the mature state.