Expression of dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin on dendritic cells generated from human peripheral blood monocytes

AIM： To generate dendritic cells （DCs） from human peripheral blood and to detect the expression of dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin （DC-SIGN; CD209） for the further study of DC-SIGN in hepatitis C virus （HCV） transmission. METHODS： Peripheral blood monocytes were isolated from blood of healthy individuals by Ficoll--Hypaque sedimentation and cultured in complete medium containing rhGM-CSF and rhIL-4. Cells were cultured for seven days, with cytokine addition every two days to obtain immature DCs. Characteristics of the cultured cells were observed under light and scanning microscope, and the expression of DC-SIGN was detected by immunofluorescence staining. RESULTS： After seven-day culture, a large number of cells with typical characteristics of DCs appeared. Their characteristics were observed under light and scanning electron microscope. These cells had a variety of cell shapes such as those of bipolar elongate cells, elaborate stellate cells and DCs. DC-SIGN was detected by immunofluorescence staining and its expression level on cultivated dendritic cells was high. CONCLUSION： DCs with a high expression of DC-SIGN can be generated from human peripheral blood monocytes in complete medium containing rhGM-CSF and rhIL-4.

Changes of NLRP1-ASC-Caspase-1 signaling pathways in peripheral blood monocytes from patients with atrial fibrillation

Objective:To detect the expression of(Peripheral blood mononuclear cells,PBMCs)NLRP3 signal pathway in peripheral blood monocytes of patients with chronic heart failure,and to explore the expression of NLRP3 signal pathway and its induced inflammatory response in PBMCs of patients with different types of chronic heart failure.Methods:patients with chronic heart failure(NYHAⅡ~Ⅳ),Ⅱ(nude 20),Ⅲ(nude 20)andⅣ(nude 20)admitted to our hospital from 2019 to 2020 were selected,and 20 normal subjects were selected as the control group.The peripheral venous blood of all subjects was collected,and the plasma and monocytes were extracted respectively.The monocytes were identified by magnetic beads sorting.The mRNA and protein expression levels of NLRP3,ASC and Caspase-1 in PBMCs were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and Western blot,and the level of interleukin-1β(IL-1β)in plasma was detected by enzyme-linked immunosorbent assay((ELISA)).Results:compared with the normal control group,the expression of NLRP3,ASC,Caspase-1 protein and mRNA in PBMCs of patients with gradeⅡ,ⅢandⅣincreased.Compared with patients with gradeⅡ,the expression of these indexes increased in patients with gradeⅢandⅣ.Compared with patients with gradeⅢ,the expression of these indexes increased in patients with gradeⅣ.Compared with the normal control group,the plasma levels of IL-1βin patients with gradeⅡ,ⅢandⅣwere higher than those in patients with gradeⅡ,ⅢandⅣ(P<0.05).The expression of these indexes in patients with gradeⅢandⅣwas higher than that in patients with gradeⅢ(P<0.05).Conclusion:the results suggest that NLRP3-ASC-Caspase-1 signal pathway may cause chronic inflammation in patients with heart failure and play a role in the progression of chronic heart failure.

Culture of Porcine Peripheral Blood Monocyte-derived Dendritic Cells in vitro

[Objective] This study aimed to establish an in vitro culture model for porcine peripheral blood monocyte-derived dendritic cells （MoDCs）. [Method] Fresh peripheral blood mononuclear cells （PBMCs） were separated from pig, and precursor dendritic cells were obtained by adherence method. The dendritic cells were treated by recombinant porcine granulocyte-monocyte colony stimulating factor （rpGM-CSF） and recombinant porcine interleukin-4 （rplL-4） together, and lipopolysaccharide （LPS） respectively. The cells in different time periods were collected. The morphology of the collected cells was observed by scanning electron microscopy; the expression of surface molecules and phagocytic ability to FITC-dextran were detected by flow cy- tometry; and the stimulating ability for allogeneic T cells was detected by mixed lymphocyte reaction. [Result] The DCs suffering maturation induction in vitro showed typical dendritic morphology; compared with those of DCs untreated by LPS, the cell surface expression of CDla, CD80, CD86, SLAII and CD172a of DCs treated by LPS was significantly increased, the phagocytic ability was reduced slightly, and the stimulating ability for allogeneic T cells was enhanced to some extent. [Conclusion] An in vitro culture method was successfully established for porcine MoDCs in this study, laying a foundation for further study on the role of porcine MoDCs in immunoregulation and anti-virus infection.