[Change of IL-22R1 expression in human airway smooth muscle cells in response to different stimulating agents].

OBJECTIVE: To explore the effects of serum of asthmatic patients, dexamethasone, interleukin-4 (IL-4), interferon-gamma (IFN-γ) and transforming growth factor-ß (TGF-ß) on the expression of interleukin-22 receptor 1 (IL-22R1) mRNA and protein in HASMCs in vitro. METHODS: IL-22R1 mRNA and protein expressions in HASMCs treated with different stimulating agents were measured by real-time PCR and Western blotting, respectively. RESULTS: IL-22R1 mRNA and protein expressions in HASMCs were significantly increased after stimulation by serum from asthmatic patients, but decreased after co-stimulation with dexamethasone. IL-22R1 mRNA and protein expressions in the cells both increased after stimulation by IL-4, IFN-γ and TGF-ß. CONCLUSION: IL-22R1 in HASMCs might be involved in the pathogenesis of asthma, and the therapeutic effect of dexamethasone on asthma is mediated, at least partially, by IL-22R1. The effects of IFN-γ, IL-4, and TGF-ß on asthma may also be attributed to their actions on HASMCs.

[Alteration of PTEN gene expression affects the migration of human airway smooth muscle cells in vitro].

OBJECTIVE: To investigate the changes in human airway smooth muscle cell (HASMC) migration and related signaling pathway after interference with PTEN gene expression. METHOD: HASMCs were infected with an adenovirus vector and RNA interference vector of human PTEN gene to establish the cell model with PTEN gene over-expression (Ad-GFP-PTEN-HASMC) and one with PTEN gene silencing (Ad-shPTEN-HASMC), using Ad-GFP-infected and a blank cells as the negative controls and LY294002 as the positive control. Fluorescence microscopy and flow cytometric analysis were used to evaluate the transfection efficiency, and Western blotting was performed to examine the expression of PTEN and the activation of AKT and ERK1/2 signal pathway. Transwell assay and wound healing assay were used to assess the migration of HASMCs. RESULTS: The adenovirus over-expression vector and RNA interference vector significantly affected the expression of human PTEN gene. Up-regulation of PTEN gene resulted in a slow-down of the HASMC migration, an inhibition of PI3K/AKT signal pathway at the protein level but no changes in Ras-Raf-MEK1/2-ERK1/2 signal pathway. Down-regulated PTEN gene expression, however, was not associated with an enhancement of HASMC migration, but activated PI3K/AKT signal pathway and inhibited Ras-Raf-MEK1/2-ERK1/2 signal pathway. CONCLUSION: Upregulation of PTEN gene can effectively inhibit airway smooth muscle cell migration, the effect of which is probably mediated by the PI3K/AKT pathway.

[Effect of recombinant adenovirus carrying PTEN gene on the proliferation of human airway smooth muscle cells in vitro and the mechanisms].

OBJECTIVE: To observe the effect of exogenous phophatase and tensin homolog deleted on chromosone 10 (PTEN) gene transfer via recombinant adenoviruses on the proliferation of human airway smooth muscle cells (HASMCs) in vitro and investigate the possible mechanisms. METHODS: With a recombinant adenovirus vector containing PTEN (Ad-PTEN) constructed using the pAdxsi system, PTEN gene was transiently transfected into HASMCs and the transfection efficiency was determined by fluorescence microscope. RT-PCR and Western blotting were performed to detect the expression of PTEN mRNA and protein in the infected cells. MTS/PMS assay was used to analyze the proliferation of HASMCs, and the cell cycle changes of the transfected cells were evaluated by flow cytometry with PI staining. The expression levels of Akt and p-A kt proteins were detected by Western blotting, and P21 mRNA expression determined by RT-PCR. RESULTS: The recombinant adenovirus Ad-PTEN showed a wild-type PTEN gene transfer efficiency of 98% at the multiplicity of infection (MOI) of 100. RT-PCR and Western blotting showed that infection with the recombinant adenovirus resulted in PTEN overexpression in the HASMCs, causing also increased ratio of G(0)/G(1) cells and proliferation inhibition of the ASMCs. The overexpression of PTEN significantly decreased the expression level of p-Akt but increased P21 mRNA expression. CONCLUSION: The recombinant adenovirus containing PTEN can be successfully transfected into HASMCs cultured in vitro, resulting in PTEN overexpression at both the mRNA and protein levels. PTEN overexpression can efficiently inhibit the proliferation of HASMCs possibly through the PI3K/PKB/AKt and P21 pathways.

[Construction of a recombinant adenovirus vector containing PTEN and its expression in airway smooth muscle cells].

OBJECTIVE: To construct a recombinant adenovirus vector containing phosphatase and tensin homolog deleted on chromosome 10 (PTEN) using the pAdxsi system. METHODS: PTEN cDNA from plasmid pcDNA3-PTEN was cloned into the shuttle plasmid pShuttle-GFP-CMV. The shuttle vector was transformed into competent DH5alpha strain with the vector pAdxsi to achieve the homologous recombination. The recombinant construct was subsequently linearized with PacI and transfected into HEK293 cells via Lipofectamine 2000. The recombinant adenovirus particles were collected, and after titration, the recombinant adenovirus was traced by monitoring GFP expression under fluorescence microscope. The expression of PTEN mRNA and protein in the recombinant adenovirus vector and airway smooth muscle cells were detected by PCR and Western blotting, respectively. RESULTS: GFP was expressed in HEK293 cells infected by recombinant adenovirus, and the expression intensity increased gradually with the passage of time, with obvious cytopathic effect (CPE) noted in the cells. After 3 cycles of amplification, the titer of adenovirus containing PTEN reached an appropriate level. The viral titer of pAdxsi-GFP-PTEN was 2x10(10) pfu/ml, and PTEN mRNA expression was detected by PCR. The homologous protein expressed in the infected human airway smooth muscle cells significantly increased in comparison with that in the control cells. CONCLUSION: The recombinant adenovirus containing PTEN is constructed successfully, which provides an experimental basis for studying the role of PTEN gene in asthma therapy.