Effects Of NGF On The Maturation,Differentiation And Function Of Murine Bone Marrow-derived Dendritic Cells

Objective To observe the influences of NGF on the maturation, differentiation and function of murine Bone Marrow-derived Dendritic Cells in vitro.Methods The first part, bone marrow cells of femur and tibia from conventional healthy C57BL/6mice were isolated. Cell samples were divided into no exogenous cytokine control group (negative control)、 NGFgroup(20ng/mL、50ng/mL、100ng/mg、150ng/mL)、GM-CSF+IL-4group(positive control、GM-CSF+IL-4+NGF group. Mononu-clear cells obtained from bone marrow cells were cultured with different stimulation in the way of adherent method and a half quantitative change of culture medium after day. Then, the morphology changes of cells were observed under inverted microscope and the surface markers were also analyzed by Flow cytometry (FCM) on day7. Compare CD11c+dendritic cells percentage and number、CD8a+/CD8a-subtype and the percentage of CD80+、CD86+and MHCⅡ+in CD11c+cells population of each group. The second part, immature DCs were generated from bone marrow precursors cultured with GM-CSF and IL-4by the CD11c positive immunomagnetic cell sorting. Then, the cells were stimulated with four different culture media containing PBS(negative control), NGF, LPS (positive control) and NGF+LPS for24hours. Flow cytometry was used to investigate the expression of CD8、CD86、MHCⅡ. Supernatants’ cytokine IL-12、IL-10、IL-6concentration were detected by enzyme-labeled immuno-sorbent assay (ELISA).Results In the concentration gradient of NGF,50ng/mL group had the highest percentage of CD11c+dendritic cells. CD11c+dendritic cell percentage and number of NGF group were more than the negative group, and lower than the positive control group. But, there is no difference between positive control group and the NGF+GM-CSF+IL-4group, and each groups are based CD8a-DCs.In CD11c+dendritic cells as an analysis group, there was no significant difference in the percentage of CD80、MHCⅡ and CD86expression between NGF group and the positive control group, so was the positive control group and the NGF+GM-CSF+IL-4group. In all selected cells as an analysis group, The percentage of CD80and MHCⅡ expression of NGF group were more than the negative group, and lower than the positive control group. But, there is no difference between positive control group and the NGF+GM-CSF+IL-4group. In the course of immature DCs to mature DCs development, compared to the LPS group, the expression of CD80、CD86、MHCⅡ, and the secretion of IL-12、IL-10、IL-6were up-regulated in the NGF+LPS group. NGF stimulation alone had no effect on it.Conclusion NGF not only can lead to bone marrow mononuclear cells into dendritic cells directed differentiation with CD8a-DCs-based, but also can further up-regulate LPS-induced DCs maturation and cytok-ine secretion.