| Objective:Critically ill patients with sepsis as the main cause of death with high morbidity and mortality,the pathogenesis of complex features,the lack of specific treatment,and costly too much.It is a heavy burden to the families and society,thus revealing sepsis the pathogenesis,finding rational treatment is an urgent problem.It has been confirmed that immune suppression and out of control inflammatory response is one of the major pathophysiological mechanisms of sepsis.As an immune inhibitor,TGF-β1 can suppress the proliferation and differentiation of B and T lymphocytes, reduce natural killer cell activity,regulate monocyte macrophage function and antagonize the inflammatory cytokines such as IL-1/IL-2.It involved in the pathogenesis of sepsis,but its out of control in the role of inflammation remain unclear.IL-10 inhibits monocyte-macrophage activation and antigen presenting function,prevents the activation of Th-1 cytokine synthesis of antigen presenting cells,which suppress the immune inflammatory response. TNF-a also known as bad quality,is an important pro-inflammatory cytokines.In this study,suppression of endogenous expression of TGF-β1 by RNA interference,we observe the changes of this immune cell to secrete pro-inflammatory cytokines(TNF-α) and anti-inflammatory factor(IL-10). So as to provide new ideas to clarify the mechanisms of TGF-β1 regulate immune and inflammatory response,and find new ways to overcome the sepsis.Methods:1.transient transfection of human leukemic monocytic cell line THP-1,using siRNA to silence cells TGF-β1 gene.Cultured cells were divided into four groups:①50nMsiRNA group;②30nMsiRNA group; ③negative control group(NC group);④control group(routine culture). After 24 hours transfection,collecte of cells in each group,making slide,observed the cell transfection results under a fluorescence microscope.With the kit to extract total RNA,RT-PCR to detect the level of TGF-β1 gene silence effects. After 48 hours transfection,collecte of cells in each group,with a total protein extraction kit,BCA protein quantification,WB to detect cell TGF-β1 protein inhibition;2.ELISA assay of cell culture medium in the TNF-αand IL-10 concentration;3.the results of data analysis software application spssl6.0 statistics,P<0.05 as significantly different.Results:①observed by fluorescence microscope,50nMsiRNA group cells with green fluorescence more than 30nMsiRNA group;②RT-PCR results showed the 30nMsiRNA group,NC group,control group has a bright band at 288bp,50nMsiRNA group has brightness weak bands here,contrast internal reference (β-actin) IOD,calculated the relative expression,and control compared,50nMsiRNA TGF-β1mRNA group decreased significantly, P<0.05,significantly different,and 30nMsiRNA group and control group was not statistically significant.③WB results indicated 50nMsiRNA group protein bands darker than the other three groups,compared to internal reference(β-actin)IOD,compared with control group,50nMsiRNA group of TGF-β1 protein expression was decreased,P<0.05,There were significant differences.④50nMsiRNA cells TNF-αhigher than the control group, P<0.05,statistically significant.⑤IL-10 were not significantly different in four groups (P>0.05).Conclusions:①Application of RNA interference technology,transiently transfected human leukemic monocytic cell line THP-1,can be successful in the inhibition of gene and protein level of endogenous production of TGF-β1;②TGF-β1 can inhibit the proinflammatory cytokine TNF-αproduction;③TGF-β1 had no effect on the antifungal inflammatory cytokines IL-10 production. |
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