title: [Primary culture and functional identification of distal pulmonary artery smooth muscle cells in mice].
abstract: Objective:To establish a method of isolation and primary culture of mice distal pulmonary artery smooth muscle cells (PASMCs) and identify the functional properties.Methods:PASMCs were harvested from the distal pulmonary artery (PA) tissue of mice by enzymatic digestion of collagenaseⅠand papain; and the growth characteristics were observed under inverted microscope and identified by Immunofluorescence technique. Effects on the intracellular calcium ion concentration of distal PASMCs were detected by Fura-2-AM fluorescent probe tracer under a fluorescence microscope in Krebs solution containing clopiazonic acid (CPA) and nifedipin (Nif).Results:PASMCs density reached approximately to 80% in a typical valley-peak-like shape after 6 days. Cell α-smooth muscle actin (α-SMA) immunofluorescence identified that 95% of the cultured cells were PASMCs. More than 95% PASMCs responded well to calcium-potassium Krebs solution (potassium ion concentration of 60 mmol/L) and showed a rapid increase in basal [Ca(2+) ](i) after 1 minute's perfusion (Δ[Ca(2+) ](i)>50), which demonstrated that the voltage-dependent calcium channels (VDCC) of distal PASMCs were in good function; after the perfusion of calcium Krebs, calcium-free/calcium-Krebs containing CPA and Nif, distal PASMCs showed two typical peaks, indicated the full function of store-operated calcium channel (SOCC) in distal PASMCs.Conclusion:This experiment successfully established a stable and reliable mice distal PASMCs model and the study of pulmonary vascular diseases could benefit from its higher purity and better functional condition.
authors.affiliation: Guangzhou Institute of Respiratory Diseases, State Key Laboratory of Respiratory Diseases, the First Affiliated Hospital, Guangzhou Medical University, Guangzhou 510120, China.
authors.forename: M C
authors.initials: MC
authors.lastname: Li
authors.forename: Y Q
authors.initials: YQ
authors.lastname: Chen
authors.forename: C T
authors.initials: CT
authors.lastname: Zhang
authors.forename: Q
authors.initials: Q
authors.lastname: Jiang
authors.forename: W J
authors.initials: WJ
authors.lastname: Lu
authors.forename: J
authors.initials: J
authors.lastname: Wang
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D002478:Cells, Cultured; D016257:Fura-2 / Q000031:analogs & derivatives; D008297:Male; D051379:Mice; D009131:Muscle, Smooth, Vascular; D032389:Myocytes, Smooth Muscle; D011651:Pulmonary Artery / Q000378:metabolism / Q000502:physiology*
chemical_list: D015220:Calcium Channels; C049925:fura-2-am; D002118:Calcium; D016257:Fura-2
keywords: Myocytes, smooth muscle; Primary culture; Store-operated calcium channel; Voltage-dependent calcium channel