Studies On The Expression Of DGKα And PKC In The Brain And Cerebellar In The Pathological Process Of Fracture Healing

When fracture occurs, local injection of nerve growth factor (NGF) can promote fracture healing. Diacylglycerol kinase (DGK) is a important intracellular second messenger and is involved in a variety of pathophysiological cellular responses.To explore the mechanism of DGKa accelerating the process of fracture healing and providing new method for clinical cure of fracture, we detected the expression, intracellular localization and distribution of DGKa and protein kinase C (PKC) in brainstem and cerebellar in rat model of fracture.In this study, fracture model were made by SD rats, and normal SD rats were subjected to sham operation. Dividing the rat bone fracture agglutination into three stages according to the agglutination of fracture staging methods and bone fracture topographic histology change:nonage, after bone fracture one～three days; metaphase, after four～fourteen days; anaphase, after fifteen days. On the day number3,7,14,24and42, one control group rat and five model group rats were put to death. Semiquantitative RT-PCR was used to detect expression of DGKa and PKC mRNA in brainstem and cerebellar. Immunohistochemical staining and immunofluorescent double-staining were used to detect the expression, regional distribution and cellular localization of DGKa and PKC protein.The results showed as follows:inflammatory infiltration was mainly pathological changes in brainstem and cerebellar of fracture rats at nonage and metaphase, the neurons in brainstem and cerebellar had no obvious morphological changes during the three stages. In brainstem, the expression level of DGKa and PKC mRNA on the day number3,7and14of model group was higher than control group (P<0.05). The expression level of DGKa mRNA in cerebellar was higher than control group at3,7,14and24(P<0.05). The expression change of PKC mRNA between model group and control group has no statistic differences (P>0.05) in cerebellar. Immunohistochemical staining showed that in the brainstem of model and control group on each fracture day, the fibrous structures expressed DGKa positive reaction and DGKa immunoreactive cells were not found in the brainstem of control group. DGKa immunoreactive cells appeared in the brainstem of model group on3day and the perinuclear area of cytoplasm was positive. On day7and14, the positive reaction translocated to the cytoplasm, and day24and42, there was no DGKa immunoreactive cells can be found. Immunofluorescent double-labeling staining showed DGKa immunoreactive cells in the brainstem were neurons. In the brainstem of model and control group, only the fibrous structures were PKC positive. PKC expressed in cytoplasm in model group on day3,7,14,24and42, PKC positive cells were not found. Immunofluorescent double-labeling staining showed DGKa and PKC co-expressed in cells. In the cerebellar of model and control group, the fibrous structures expressed DGKa positive reaction. Pyriform neurons showed positive in cytoplasm of model group on day3,7,14and24, in the control group and model group on42day, the DGKa immunoreactive pyriform neurons were not found. In the cerebellar of model and control group, the PKC positive expressed in fibrous structures but not in pyriform neurons.In summary, there are different expressions and distributions of DGKa and PKC in brainstem and cerebellar in the pathological process of fracture healing. The higher expression of DGKa mRNA in nonage and metaphase suggested DGKa might take part in the pathological process at the two phases. The DGKa positive reaction in the brainstem and cerebellar suggested that the neurons of brainstem and pyriform neurons of cerebellar might take part in the pathological process. The co-expression of DGKa and PKC in brainstem suggested that DGKa may play its role through DAG-PKC signaling pathway.