| Objective: This study was focused on the effect of transformed growth factor-β1 on the cytoskeleton organization of human CD14+ monocyte-derived DCs and underlying molecular mechanism. It is significant for further understanding the immune escape mechanism of cancer as well as improving the clinical efficiency of DCs-based cancer immune therapy. Methods: CD14+ monocytes were enriched from fresh harvested whole blood(within anticoagulant heparin) by immunomagnetic beads. Immature dendritic cells(im DCs) were induced by GM-CSF and IL-4 for 5 days as well as mature dendritic cells(m DCs) were induced by TNF-α(tumor necrosis factor-α),GM-CSF(granulocyte-macrophage colony stimulating factor) and IL-4(interleukin-4) for 3 days in vitro. Collected im DCs and m DCs were co-cultured with hepatoma cells respectively, and the cells were randomized into the im DCs control groups(I), im DCs and Bel7402 cell co-culture groups(II), m DCs control groups(III). m DCs and Bel7402 cell co-culture groups(IV). After 48 h of treatment, total RNA of the cells was collected,and c DNA microarrary was used to uncover the function related genes of DCs. In addition, m DCs were treated with 5 ng/ml of TGF-β1 for 15, 30, 60, 120, 360, 720,1440 minutes, and then the influence of F-actin and the expression and location of p-Smad2, Smad2, cofilin1, profilin1 were tested.The changes of m DCs’ F-actin, motilities and antigen stimulatory capabilities were decected and compared after TGF-β/Smad signaling pathway blocked. Results:There were 1531 significantly regulated genes(fold changes≥ 2 compared with that of control group) involved in II/I of which 598 up-regulated and 933 down-regulated, 663 significantly regulated genes(fold changes≥ 2 compared with that of control group) involved in IV/III of which 290 up-regulated and 373 down-regulated.The function of these genes involve signal transduction, immune response, cell adhesion, etc. The results of western blot showed that the expression levels of Smad2/3 was up-regulated while p-Smad2/3 and some actin-binding proteins, including cofilin1, profilin1 were down-regulated at 120 minute. Immunofluorescence experiment showed that after treatment with TGF-β1, Smad2/3 in m DCs mainly distributed in the cytoplasm and most of p-Smad2/3 localized in nucleus, the organizations of F-actin were markedly remodeled, furtherrmore, the protrusions of cells became much shorter and sparser. This phenomenon could be reverted by SB431542 treatment.Transwell and mixed lymphatic reaction results suggest that the impaired motilities and antigen stimulatory capabilities of m DCs by TGF-β1 could be converted by blocking the TGF-β1-Smad2/3 signaling pathway with SB431542. Conclusion: The effects of hepatocellular carcinoma micro-environment on DCs involve in multi-regulation and expression of genes, these genes control and influence the functions, differentiation and maturation of DCs.TGF-β1 is a king of inhibitory cytokines in tumor microenvironment.Our data found that TGF-β1 change the cytoskeleton organization,motilities and antigen stimulatory capabilities of m DCs via smad signaling pathway.It’s significant for understanding the biological behaviors and immune escape mechanism of DCs as well as improving the efficiency of DCs’ clinical therapy. |
