Hypoxia-induced Mitogenic Factor Activated PI3k/Akt/mTOR And PKC/MAPKs Pathway To Mediated Proliferation Of Rat Pulmonary Artery Smooth Muscle Cell

Objective:The effects of different concentrations of HIMF can cause the proliferation of rat pulmonary artery smooth muscle cell proliferation,and the extent of whether there is a difference,and then verify whether the process is through the activation of PI3K/Akt/mTOR and PKC/MAPKs signaling pathway,and this process is regulated by Ca²⁺.Methods:?1?extraction and culture of rat pulmonary artery smooth muscle cells:successful rat pulmonary artery smooth muscle cells isolated by enzymatic method and the cell morphology was observed under light microscope,cells with fibrous actin?F-actin?were identified by cellular immunology method for marking.2-3 cells were cultured at 37 DEG C and incubated in 5%CO2 incubator.?2?the logarithmic growth phase of PASMCs cells of 2-3generation rats without FBS DMEM/F12 24h cell cycle synchronization detection,adding different concentrations of recombinant protein HIMF by EdU immunofluorescence(A group?0ng/ml?,B group?5 ng/ml?,group C?10 ng/ml?,D group?20 ng/ml?,E?40 ng/ml?after the proliferation of rat PASMCs.?3?the expression of Ca²⁺of rat pulmonary artery smooth muscle cells related to PI3K/Akt/mTOR and PKC/MAPKs signaling pathway were added to each factor:signal pathway inhibitors after adding 20ng/ml recombinant HIMF protein after 30 minutes,by Western blot?Western blot?method to detect the expression of phosphorylated downstream,and to detect the proliferation of the cells.Results:?1?with different concentrations of recombinant protein HIMF in rats after PASMCs can be found by EdU immunofluorescence staining,cell proliferation than the control group increased significantly,and in group D?20 ng/ml?reached the maximum level.?2?the addition of calcium ions and chelating agents EGTA and BAPTA,adding 20 ng/ml recombinant HIMF protein,downstream factor PI3K phosphorylation is only with recombinant protein group decreased;II joined the PI3K inhibitor LY94002,adding 20 ng/ml recombinant HIMF protein,downstream factor Akt phosphorylation is only adding recombinant protein also decreased the addition of the Akt inhibitor KRX-0401 after adding 20 ng/ml recombinant HIMF protein,downstream factor mTOR phosphorylation is only adding recombinant protein group decreased compared with the above groups only add 20ng/ml of recombinant HIMF protein,cell proliferation was decreased.?3?the addition of calcium ions and chelating agents EGTA and BAPTA,adding 20 ng/ml recombinant HIMF protein,downstream factor PKC phosphorylation is only with recombinant protein group decreased;II joined the PKC inhibitor LY317615,adding 20 ng/ml recombinant HIMF protein,downstream factor mapkS phosphorylation is only adding recombinant protein also decreased the above groups;and only add 20ng/ml of recombinant HIMF protein in cell proliferation were decreased.Conclusions:HIMF recombinant protein can induce the proliferation of rat smooth muscle cells,which may be activated by PI3K/Akt/mTOR signaling pathway,which is regulated by Ca2+.