The Preliminary Research On The Interaction Between STIM1 And Orai1 In Pulmonary Artery Smooth Muscle Cells

Objective: Our research is to find the location relationship and the direct interaction between STIM1 and Orai1 in primary cultured PASMCs from normal rats,that provides a theoretical basis for pulmonary arterial hypertension(PAH).Method: Obtain STIM1/Orai1 gene by PCR,then connect them with vector p LV-c-flag and p LV-c-HA,finally construct virus vectors p LV-c-flag-STIM1, p LV-c-HA-Orai1.Virus vectors infect rats PASMCs and then Western-blot detects protein expression;Observe store-operated calcium entry(SOCE)after treated with Ca2+ release agonist(CPA) by Calcium fluorescence measurement in PASMCs; Observe the location of flag,HA-tag by immunofluorescence combined with confocal laser technology.Then treated with CPA,compares the location before and after treatment.Observe the relationship and direct interaction between STIM1 and Orai1 by co-immunoprecipitation with STIM1,Orai1 antibody in rats PASMCs.Result: Recombinant vectors p LV-c-flag-STIM1, p LV-c-HA-Orai1 were constructed sucessfully by identification of restriction enzymes and sequence alignment; PASMCs were isolated from normal rats and stably cultured; Virus vectors infected primary cultured PASMCs,Western-blot showed expression of STIM1,Orai1 was statistically increased.The co-location of flag,HA-tag by immunofluorescence combined with confocal laser technology was obviously observed,that figured out STIM1 expressed in the cytoplasm.Compared before and after treatment with CPA, there were no significant differences.By the way of co-immunoprecipitation with STIM1,Orai1 antibody separately,destined protein was distinguished from protein complex by Western-blot,which proved that there was a direct interaction between STIM1 and Orai1 in rats PASMCs.Conclusion: There is a direct interaction between STIM1 and Orai1 in rats PASMCs...