| Proliferation of pulmonary artery smooth muscle cells (PASMCs) is an important contributor to the vascular remodeling that occurs in chronic hypoxic pulmonary hypertension. Different growth factors, cytokine and proinflammatory mediators play important role in the vascular remodeling, in which they convey extracelluar signal to the nucleus by interacting with receptors on the cell surface, then regulate expression of target genes and contribute to cell differentiation, growth and proliferation. Now it is less known about negative regulator of cytokine-induced signaling pathway, which acts in vascular smooth muscle cells (VSMCs) growth.Recent studies have confirmed that the expression of negative regulators of cytokine signaling (SOCSs) could be induced in response to stimulation by a variety of cytokines, and its overexpression in cells results in inhibition of cytokine-mediated signaling pathway. This study was therefore undertaken to observe the expression of SOCS1 and SOCS3 gene, the most potent members of SOCS family, in rat PASMCs under normoxic or hypoxia conditions; By transfecting SOCS3 expression vector into PASMCs, we examine the effect of SOCS3 protein on proliferation and expression of cell-oncogenes(c-onc) in rat PASMCs under normoxic or hypoxia conditions. Methods1. After PASMCs were primarily cultured, expression of SOCS1 mRNA was detected by RT-PCR under normoxic or hypoxia conditions (2.5% O2); expressions of SOCS3 mRNA and protein were performed by RT-PCR, immunocytochemistry and Western blot under normoxic or hypoxia conditions.2. SOCS3 expression vectors and screening vectors were cotransfected into the PASMCs with lipofectamine 2000 in vitro. Positive cell clones were screened with G418. RT-PCR and immunocytochemistry were used to analyze the expression of SOCS3 mRNA and protein in PASMCs before and after transfection.3. The PASMCs transfected with SOCS3 expression vectors and the control cells were cultured under normoxic and hypoxia conditions respectively. The changes of cell proliferation were observed by flow cytometric DNA analysis and 3H-TdR incorporation.4. Expression of STATS mRNA and protein in PASMCs before and after transfection were detected by RT-PCR and Western blot under normoxic and hypoxia conditions; expression of c-myc, c-fos, c-jun mRNA before and after transfection were detected by RT-PCR under normoxic and hypoxia conditions respectively.Results1. Rat PASMCs primarily cultured were confirmed by immunocytochemistry using cx-SM-actin antibody. Expression of SOCS1 mRNA in PASMCs was not detected by RT-PCR under normoxic and hypoxia conditions, and expressions of SOCS3 mRNA and protein were not detected either by RT-PCR and Western blot under normoxic conditions respectively. When PASMCs were cultured under hypoxia conditions, the SOCS3 mRNA was detected at 2 h of hypoxia and reached maximal level at 6 h, declined at 12 h and disappeared at 24 h. The change of SOCS3 protein expression induced by hypoxia was similar to that of SOCS3 mRNA. Immunocytochemistry was used to confirm SOCS3 protein only to be in cytoplasmic distribution of rat PASMCs.2. The positive cell clones cotransfected with protein expression vectors and screening vectors were obtained after being screened with 150g/ml G418. The strong expressions of SOCS3 mRNA and protein were confirmed by RT-PCR and immunocytochemistry in cells transfected with SOCS3 gene.3. Under hypoxia, normal PASMCs in G0/G1 phase decreased, those in G2/M+S phase and H-TdR incorporation increased significantly from 6 h in a time-dependent manner. Cell cycle in cells transfected with SOCS3 gene began to have the same changes at 24 h exposed to hypoxia. 3H-TdR incorporation in transfected with SOCS3 gene began to increase from 6 h. In contrast to the same hour in normal PASMCs exposed to hypoxia, cells transfected with SOCS3 gene at G0/G1, increased, cells in S+G2/M and 3H-TdR incorporation decreased significantly.4. In normal PASMCs and SOCS3 gene-transfected cells, the expression of STAT3 mRNA increased after... |
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