| Objective:Diacylglycerol kinase (DGK) is an important intracellular second messenger and isinvolved in a variety of pathophysiological cellular responses. To explore the functions of DGKα,β,γin EAE and provide new method for clinical cure of demyelination disease, we detected theexpression, intracellular localization and distribution of DGKα,β,γin brainstem and spinal cordin rat model of experimental autoimmune encephalomyelitis (EAE).Methods:EAE were induced in Wistar rats by guinea pig spinal cord homogenate. SemiquantitativeRT-PCR was used to detect the expression of DGKα,β,γmRNA in brainstem and spinal cord.Immunohistochemical staining and immunofluorescent double-labeling staining were used todetect the expression, regional distribution and cellular localization of DGKαand DGKβprotein.Results:1. Inflammatory infiltration was mainly pathological changes in brainstem of EAE rats. Inspinal cord, inflammatory infiltration and demyelination were the main pathological changes.The neurons in brainstem and spinal cord had no obvious morphological changes.2. In brainstem and spinal cord, the expression level of DGKαmRNA in each EAE groupwas lower than normal control group. The expression level was decreased in the 1-2 point groupand increased in the 3 point group. The expression level of 4 point group was between 1-2 pointgroup and 3 point group.The expression of DGKβmRNA was highest in both brainstem and spinal cord of 4 pointgroup, was lowest in brainstem of 3 point group and spinal cord of 1-2 point group.In brainstem, the expression of DGKγmRNA was lowest of 1-2 point group and increasedobviously in 3 point group. In 4 point group DGKγmRNA expression level was between thenormal group and 1-2 point group. In spinal cord the expression level of DGKγmRNA of eachgroup had no significant difference.3. Immunohistochemical staining showed that only the fibrous structures were DGKαpositive in normal group, 1 and 2 point groups of brainstem. DGKαimmunoreactive cellsappeared in 3 points group and the perinuclear area of cytoplasm was positive. In spinal cord, the fibers of white matter and gray matter were DGKαpositive. The expression of DGKαin motorneurons was week in 2 point group and slight stronger in 3 and 4 point groups.Immunofluorescent double-labeling staining showed DGKαimmunoreactive cells were neuronsand oligodendrocytes.In brainstem of normal and each EAE groups, only the fibrous structures were DGKβpositive and the DGKβimmunoreactive cells were not found. In spinal cord of normal group,only the white matter and the fibers of gray matter were DGKβpositive. DGKβexpressedweekly in nucleus of motor neurons in 1 and 2 point groups, in cytoplasm of motor neurons in 3and 4 point groups. Immunofluorescent double-labeling staining showed DGKβexpressed inmyelin but not in neurons.In brainstem of normal group and each EAE groups, only the fibrous structures were PKCpositive. PKC expressed in cytoplasm in 3 point group. In spinal cord of normal and each EAEgroups, only the white matter and the fibers of gray matter were PKC positive and the PKCimmunoreactive cells were not found. Immunofluorescent double-labeling staining showedDGKαand PKC co-expressed in cells and fibers.ConclusiConclusion:1. There are different expressions and distributions of DGKα,β,γin brainstem and spinalcord during the EAE pathological process..2. The higher expression of DGKαmRNA in 3 points group suggested DGKαmight takepart the pathological changes of EAE in this phase. The expression of DGKαin nucleus andperinuclear area of cytoplasm suggested that the regulation of DGKαon diacylglycerol might beenhanced in the rough endoplasmic reticulum or ribosomes.3. The higher expression of DGKβmRNA in the 4 points group and the translocation ofDGKβin motor neuron suggested that DGKβmight take part in the pathological changes ofEAE in this phase, and promote the myelin phagocytosis.4. The co-expression of PKC and DGKαin cells and fibers suggested that DGKαmay playits role through DAG-PKC signaling pathway.
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