Effects Of HIMF On Hypoxia Induced Proliferation And Intracellular [Ca2+]i Of Rat Pulmonary Arterial Smooth Muscle Cells

?Objective?In this study,we cultured rat primary pulmonary artery smooth muscle cells(PASMCs)and treated with different time in hypoxia,to observe the effect of hypoxia on proliferation and intracellular [Ca2 +] i of rat primary PASMCs,as well as Hypoxia-induced mitogenic factor(HIMF)expressionin in different hypoxia treatment time,to investigate the effect and mechanism of HIMF on the proliferation of rat primary PASMCs.?Methods?(1)Extraction,culture and identification of rat primary pulmonary artery smooth muscle cells(PASMCs):The distal pulmonary arteries were isolated from the microscope by means of related microscopic devices(micro-shear and micro-tweezers).The primary PASMCs were isolated and digested by enzymatic digestion.The morphology of the cells was observed under light microscope,and the purity of the cells was identified by Smooth Muscle Cell Alpha Actin(?-SMA),Vimentin,and Factor VIII characteristic immunofluorescence staining.(2)To observe the effect of chronic hypoxia on the proliferation of rat primary PASMCs :Select the 2-3 generation cells with good growth status,divided them into different hypoxic time treatment groups(H24h?H48h?H60h?H72h),and place them into the three gas incubator(37 ?,4% O2,5% CO2,91% N2),to exposure at different time under hypoxia,we also set the normoxic control groups.Then,CCK-8,cell count and Edu method were used to observe the effect of different hypoxia time on the proliferation of rat primary PASMCs.(3)To investigate the effect of hypoxia at different time on the expression of HIMF in rat primary PASMCs:The effects of hypoxia on the expression of HIMF mRNA and protein in rat primary PASMCs were observed by real-time quantitative polymerase chain reaction(Q-PCR)and western blotting.(4)Determination of [Ca2 +] i concentration in rat primary PASMCs:Primary rat PASMCs were inoculated into a six-well plate slide and placed in a three-gas incubator at 37 ° C,4% O2,5% CO2,91%.Then treat them at the time point when the cell proliferation was the strongest in hypoxia,we also set up normoxic control group.The effect of intracellular [Ca2 +] i concentration in primary PASMCs was detected by cell dynamic fluorescence imaging system.(5)The Specific siRNA was used to silence the expression of HIMF in rat primary PASMCs,and then exposure them into the normal and the strongest proliferation time in hypoxia,to observe the effect of [Ca2 +] i concentration in rat primary PASMCs.(6)Construction of HIMF overexpressing lentiviral vector,transplantation of rat primary PASMCs with HIMF lentivirus,and then exposure them into the normal and the strongest proliferation time in hypoxia,to observe the effect of [Ca2 +] i concentration in rat primary PASMCs.?Results?1.The original PASMCs were isolated by enzymatic digestion.The morphology of the cells was observed by light microscopy.Immunofluorescence showed that ?-SMA was positive for fluorescence staining,while the results of Vimentin and Factor VIII staining were negative and the cell purity More than 98%.2.The proliferation activity of primary PASMCs in normoxic control group and chronic hypoxia(4% O2,5% CO2,91% N2)group increased with the prolongation of culture time,at 60 h after hypoxia,the proliferation of primary PASMCs in rats was significantly higher than that in normoxic control group.3.The levels of HIMF mRNA and protein in primary PASMCs of all the normoxic control groups were low level,hypoxia can induce the expression of HIMF mRNA and protein in rat PASMCs in a time-dependent manner.The expression of HIMF mRNA and protein in H48 h,H60h and H72 h groups was significantly higher than that in normoxia group.4.The level of stored calcium influx(SOCE)in rat primary PASMCs was significantly higher than that in normoxic control group(N60h)under chronic hypoxia(60h,4% O2).5.When silence the expression of HIMF gene in rat primary PASMCs with specific siRNA,and then treated with chronic hypoxia(4% O2,5% CO2,91% N2)for 60 h,the level of SOCE in PASMCs was significantly decreased compared with the chronic hypoxia group(H60h).After silencing HIMF gene and treated with normoxia for 60 h,there was no significant change in SOCE level in PASMCs compared with normal control group(N60h).6.Lentivirus overexpressing rat HIMF gene in primary PASMCs and treated with normoxia for 60 h,the level of SOCE in PASMCs was significantly increased compared with the normoxic control group(N60h).Overexpression of HIMF gene and treated with chronic hypoxia(4% O2,5% CO2,91% N2)for 60 h,there was no significant change in SOCE level in PASMCs compared with chronic hypoxia control group(H60h).?Conclusion?1.Hypoxia can up-regulate the expression of HIMF in rat primary PASMCs and promote the proliferation of PASMCs.2.Hypoxia can increase the level of stored calcium influx(SOCE)in rat primary PASMCs.3.HIMF may play an important role in the process of hypoxia-induced increase in SOCE levels in rat primary PASMCs.