Abstract:

Objective To discuss the effect of over-expression of Dectin-1 gene on the function of the dendritic cells （DCs）， and to clarify the mechanism of immune function of Dectin-1 gene in inhibiting the maturation and activation of DCs and its immune protection on the heart allografts. Methods The bone marrow stem cells of the mice were induced to form DCs and then cultured and expanded in vitro. The Dectin-1 gene was transfected into the DCs by adenovirus vector with green fluorescent protein （GFP）， the immature DCs （imDCs） modified with the Dectin-1 gene were regarded as DC-Dectin-1 group， and DCs group （untransfected with virus）and DC-GFP group（transfected with GFP） were sep up.Immunofluorescence assay used to detect the adenovirus transfection after 24 h； Western blotting method was used to detect the expressions of Dectin-1 protein in DCs in various groups；flow cytometry and enzyme-linked immunosorbent assay （ELISA） were used to detect the phenotype， function， and levels of interleukin-10 （IL-10） and interleukin-12 （IL-12） in cell culture supernatant of DCs in various groups before and after lipopolysaccharide（LPS） stimulation.The model of abdominal heterotopic heart allograft in the allogeneic mice was established， which was divided into DCs group， DC-GFP group，and DC-Dectin-1 group. The DCs， DC-GFP，and DC-Dectin-1 were infused on the first， third and fifth days after transplantation， respectively.On the seventh day after transplantation， HE staining was used to observe the pathomorphology of heart allografts of the mice in various groups； TUNEL staining was used to observe the apoptosis of heart allografts of the mice in various groups；the median survival time （MST） of heart allografts of mice in various groups was observed after transplantation. Results The gene modified Ad5F35 vector could improve the transfection efficiency of Dectin-1 gene， and GFP could continue to be expressed in the DCs after transfection. Before LPS stimulation， the expression levels of CD40， CD80， CD86， and human major histocompatibility complex Ⅱ （MHC-Ⅱ） in the cells in DCs， DC-GFP and DC-Dectin-1 groups were low， which presenting the imDCs phenotype； compared with before LPS stimulation， the expression levels of CD40， CD80， CD86， and MHC-Ⅱ in the cells in DCs and DC-GFP groups were increased after LPS stimulation， which presenting the mature DC phenotype. However， the expression levels of CD40， CD80， CD86， and MHC-Ⅱ in the cells in DC-Dectin-1 group were low. Before LPS stimulation， there were no statistically significant differences in the levels of IL-10 and IL-12 in cell culture supernatant in various groups（P>0.05）； after LPS stimulation， compared with DCs and DC-GFP groups， the IL-10 level in the cell culture supernatant in DC-Dectin-1 group was significantly increased （P<0.05）， while the IL-12 level was significantly decreased （P<0.05）. On the seventh day after transplantation， compared with DCs group and DC-GFP group， the inflammatory cell infiltration in heart allografts tissue of the mice in DC-Dectin-1 group was decreased， the rejection was significantly alleviated， and the number of TUNEL positive apoptotic cells were significantly decreased. Compared with DCs and DC-GFP groups， the MST of heart allografts of the mice in DC-Dectin-1 group was significantly prolonged （P<0.01）. Conclusion Dectin-1 gene can inhibit the maturation and activation of the immature DCs under the action of LPS， weaken their antigen presenting function， prolong the survival time of heart allografts of the mice to a certain extent， and induce the formation of allografts immune tolerance.

Key words: Dectin-1, Gene transfection, Dendritic cells, Heart allografts, Immune tolerance