The Effect Of Silenceing CCR7Gene On Dendritic Cells Maturation In Vitro

Objective and Significance: In recent years, many studies have shown that thepathogenesis of coronary heart disease involved the immune and inflammatoryresponse， in which the most important antigen-presenting cells dendritic cells(Dendritic Cells DCs) plays an important role. Chemokine receptor CCR7is lowexpression in immature dendritic cells (imDCs) surface, while it is high expression inmature dendritic cell surface. So we conjecture that CCR7might have a closerelationship in DCs maturation. Besides, during the process of the immune response,imDC mainly play a role in the uptake of antigens, and mature DCs present thisantigens to T lymphocytes, this process cause the immune and inflammatory responseeventually. Where CCR7can regulate of DCs maturation is not clear. In this study,we isolated and cultured rat bone marrow-derived dendritic cells (BMDCs) in vitro,and further identified the BMDCs by morphological cell phenotype. Then we builtthe chemokine receptor CCR7RNA interference lentiviral vector, and transfected itinto imDCs, detected CCR7mRNA and protein expression by RT-PCR andWestern Blot respectively six days later. Meanwhile detected macrophageinflammatory protein1a (MIP-1a) and monocyte chemoattractant protein-1(MCP-1)content in cell culture supernatants by ELISA six days later. Many relevant studieshave shown that the secretion of MIP-1a is gradually decreased, while the secretion ofMCP-1is gradually increased in culture supernatants along with the maturation ofDCs. The aim of the study is that exploring the effect of silencing CCR7gene ondendritic cells maturation in vitro. The result might provide new ideas for thetreatment of atherosclerotic heart disease, even cardiovascular disease through inhib-ition of DCs mature.Methods: Isolation of rat bone marrow-derived dendritic cells in vitro: removedthe femur and tibia in two legs of150-180g male or female SD rat, collected bonemarrow cells by rinsing the bone marrow cavity to with5ml RPMI-1640supplementwith10%FCS, DC differentiation is induced by5ng/ml granulocyte-macrophagecolony stimulating factor (GM-CSF) and5ng/ml interleukin-4(IL-4), were collected on day6, and differentiated by2.5-5ng/ml GM-CSF,12days later, we acquired themature dendritic cells. Collected the BMDCs in day6, the day9, the day12, and theday14respectively, identified the BMDCs by flow cytometry. The RNA interferencelentiviral vector was packed by Genechem company, we acquired four shRNA targetsand the titers were detected by the company. CCR7mRNA and CCR7proteinexpression by RT-PCR and Western Blot was detected to confirm the best siRNAtarget. Our study were divided into three groups: the experimental group DCs weretransfected with the CCR7-GFP-LV in day6, while the negative control group DCswere transfected with NC-GFP-LV, and blank control group was normal culturedDCs. Six days after the stable transfection, detected the CCR7mRNA and proteinexpression in the three groups by RT-PCR and Western Blot respectively, detectedthe MIP-1a and MCP-1content in culture supernatants of these three groups，anddetected the growth and proliferation of DCs by MTT assay.Results: In day6to day9, the dendritic cells cultured in vitro were semi-suspended state, round, non-dendritic.12days later, the dendritic cells were at asemi-suspended semi-adherent state, round or oval, part of the cells showsed dendriticprotrusions. Flow cytometry analysis showed that in early stage of incubation, theexpression of CD86、MHC-II were low and moderate respectively, while in the latestage of incubation, the expression of CD86and MHC-II were all increased.TheELISA results showed thar the content of MIP-1a was gradually decreased, while thecontent of MCP-1was increased in culture supernatants along with the prolongedincubation. We confirmed the best siRNA target was CCR7-RNAi-LV3#by RT-PCRand Western Blot assay, and the concentrated virus titer of6×108TU/ml. Six daysafter CCR7shRNA lentivirus transfection,70-75%of BMDCs expressed GFP. CCR7mRNA expression level in experimental group was (26.85±0.03)%, which wassignificantly lower than the blank control group (93.01±0.01)%and the negativecontrol group (83.69±0.01)%(P<0.05). CCR7protein expression level inexperimental group was (47.86±0.02)%, which was significantly lower than theblank control group (93.50±0.05)%and the negative control group (91.35±0.02)%(P<0.05). The ELISA results showed that the MIP-1a levels in the experimentalgroup was (30.70±0.50)pg/ml, which was significantly higher than those in the blank control group (25.33±1.74)pg/ml and the negative control group (26.95±0.22)pg/ml(P<0.05). The MCP-1levels in the experimental group was (25.50±0.95)pg/ml, which was significantly lower than those in the blank control group(31.06±1.41)pg/ml and the negative control group (31.44±1.95)pg/ml(P<0.05). TheMTT results showed that the OD value of the experimental group was significantlylower than those in the blank and the negative control groups when the CCR7wassilenced(P<0.05).Conclusions: BMDCs of CCR7lentiviral vector was successfully constructed,transfecting CCR7shRNA lentiviral vector into DCs could down regulate the CCR7mRNA and protein expression levels. Meanwhile the content of MIP-1a in the cellculture supernatant was increased significantly, and the content of MCP-1in the cellculture supernatant was decreased significantly. The growth and proliferation of DCswere significantly silenced. Silencing CCR7gene expression could inhibit DCsmaturation. This was the preliminary work for the next step to induce immunetolerance of DCs to associated antigen (such as oxidized LDL, ox-LDL), thereforethis study probably provide a new target for the treatment of coronary heart disease.