The Influence Of Selenoprotein Selk To Free Ca²⁺ Level In Cell Cytosol And The Migrational Ability In Mouse Microglias And Monocytes

Microglia is the ’macrophage’ in the brain, it plays an important role in the occuring of alzheimer’s disease （AD）.Selenoprotein SelK is one of the selenoproteins expressed highly in the brain of mice. Recent research demonstrates that the activated macrophages predominantly express the uncleaved SelK and the resting macrophages and other cells predominantly exprss cleaved SelK. Complete SelK can increase the release of Ca²⁺ from endoplasmic reticulum in macrophages thus increase the ability of phagocytosis and migration of macrophages. Whether SelK can also exist in two states in the activated and resting microglias and whether the existing state of SelK can influence the intracellular free calcium level, phagocytosis ability and migration ability of the cells, and whether some of these related to the onset of Alzheimer’s disease is one of the important scientific problems needs to be solved. In this paper, to resolve the problems, the SelK in the microglias of mouse was knocked down or over expressed to study the relationship of SelK expression and the level of intracellular free calcium, the ability of migration. Previous studies show that the bone marrow-derived monocytes are more efficient in the cleaning up of Aβ protein, so the effects of SelK on monocytes were also studied in the sametime.We constructed the lentiviral vector pLKO.1-mSelK-ShRNA for the purpose of selenoprotein gene knocked down in the above two cells. The original pLKO.1-TRC cloning vector has a 1.9kb stuffer that was released by digestion with Agel and EcoRI. ShRNA oligos were cloned into the Agel and EcoRI sites in place of the stuffer. The transfection in the HEK-293T cells with the pLKO.1 ShRNA plasmid, psPAX2 packaging plasmid and pMD2.G envelope plasmid was conducted to construct the lentiviral vector pLKO.1-mSelK-ShRNA. The mRNA expression of mSelK in lentiviral infected microglia cell strain BV2 and monocyte cell strain Raw264.7 were studied with RT-PCR and qPCR. The expression of mSelK mRNA in BV2 and RAW264.7 cells was significantly reduced than the control group. The mRNA expression level droped to 0.09 times （RAW264.7 cells） and 0.21 times （BV2 cells） when calculated by 2-ΔΔCt method, and it could be concluded that the knocking down effect of lentiviral was extremely obvious. It was also found that the infection of lentivirals did not affect the cell viability and would not cause the activation of BV2 or RAW 264.7.The lentiviral vector pLKO.1-mSelK-ShRNA was used to infect BV2 cells and RAW264.7 cells, for the purpose of investigating the influence of the knocking down of mSelK on free Ca²⁺ level in cell cytosol and the migrational ability of resting microglias and bone marrow-derived monicytes. The Ca²⁺ probe Fluo-3 AM staining and flow cytometrywas used to analysis the changes of cytosolic free Ca²⁺ level of these cells. It was showed that, in the two cells, the cytosolic free Ca²⁺ level of knocking down group was significantly reduced. The cell migrational ability of BV2 and RAW264.7 cells was detected by transwell chamber assay. It was shown that the cell migrational ability of the cells, which mSelK was knocking down, was significant reduced compared with non-infected cells and the cells transfected with empty lentiviral vector.Then, on the basis of adenoviral vector pHBAd-RFP, we construct the recombinant adenoviral vector of mSelK, the pHBAd-RFP-mSelK, for the next research. Selenoprotein mSelK could be over expresstion in the two cells verified by western blot. It was also verified that the adenovirus infection have no significant impacts on the activation of the transfected cells and the cell viability. The cytosolic free Ca²⁺ level was detected by the flow cytometry instrument and the cell migrational ability was assessed by transwell chambers. It was showed that,when selenoprotein was overexpressed, the cytosolic free Ca²⁺ level was significantly increased. It was also shown that the cell migrational ability of the cells over expressing mSelK was significant increased, compared with non-infected and cells transfected with empty vectors.Using the mSelk adenoviral overexpressed vector, pHBAd-RFP-mSelK, to infect microglias for the purpose of increase the the number of mSelK genes in these cells.And then, western blot was used to verify the cleave stage and expression level of mSelK in the activated microglias and the resting microglias. The results showed that, when the cells from resting turn to be activated, the mSelK in the cells became full length state from cleave state. It was shown that, mSelK mainly played a role in the process of cell activation.By the above experimental results the following conclusions could be drawn: Selenoprotein mSelK could control the release of Ca²⁺ from endoplasmic reticulum and then increase the migrational ability of microglias and bone marrow-derived monicytes. When mSelK expression was significantly decreased in microglia and monocytes, the level of the level of Ca²⁺ from endoplasmic reticulum was significantly decreased and the migration ability was also significantly decreased. When mSelK expression was significantly increased in microglia and monocytes, the release of Ca²⁺ from endoplasmic reticulum was significantly increased and the migration ability was also significantly increased. The resting microglias predominantly exprss cleaved SelK, and the activated microglias, in the time of requiring large amount of Ca²⁺ to migrate and phagocyte, predominantly express the uncleaved SelK and expression level of mSelK was significantly increased.This is very beneficial for the prevetion and cure of Alzheimer’s disease.