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+.