Roles of the ERK1/2 and PI3K/PKB signaling pathways in regulating the expression of extracellular matrix genes in rat pulmonary artery smooth muscle cells

Abstract

Roles of the ERK1/2 and PI3K/PKB signaling pathways in regulating the expression of extracellular matrix genes in rat pulmonary artery smooth muscle cells The pulmonary artery was then extracted, the fibrous tunica externa dissected and the runica intima stripped. The myogenous tissues of the vascular middle layer of the artery were then cut into 1 mm³ tissue blocks, and seeded into the culture flasks with the density as 1cm -2 . After 2 h wall-adherent cultivation at 37°C and 5% CO 2 , M199 medium containing 10% fetal bovine serum (FBS) was added. A week later, cells had grown outwards from the tissue blocks; when the growth covered 70% of the area of the base of the vessel, the culture was passaged. The 3rd-generation cells were taken for slice culturing and an immunohistochemical method was performed to detect the expression of cellular α-SMA and to confirm the purity of the PASMCs.The PASMCs were then divided into five groups and cultured in M199 medium containing different additional components, and for 48 h and 72 h. The control group was cultivated in M199 medium with 10% FBS. Indeed, in this study, the control group is negative control. The CTGF group with 10% FBS + 50 ng/ml CTGF (PeproTech, USA), the CP group with 10% FBS + 50 ng/ml CTGF + 20 μmol/L PD98059 (Sigma, USA), the CL group with 10% FBS + 50 ng/ml CTGF + 10 μmol/L LY294002 (Sigma, USA) and the CPL group with 10% FBS + 50 ng/ml CTGF + 20 μmol/L PD98059 + 10 μmol/L LY294002. RT-PCRFor the RT-PCR, the cDNA sequences of rat collagen III, fibronectin and β-actin were downloaded from NCBI gene database, then the Primer Premier 5 software was used to design appropriate PCR primers. The primer sequences were in the Table 1.

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