| BackgroundAstham is a chronic inflamatory of the airways in which many cells and celluar elements paly an important role.The chronic inflamation is associated with airway hyperresponsiveness.Athma is usually associated with widespread,but variable,airflow obstruction within the lung that is often reversible.If the inflamation continues,it will result to partly irreversible airflow obstruction which is caused by airway remodeling. Airway remodeling in asthma is a complex process that involves structural changes in virtually all tissues of the airway wall. The histologic changes to the airways caused by chronic inflamation that consist of epithelial proliferation and goblet cell differentiation, subepithelial fibrosis,human airway smooth muscle (HASM) growth, angiogenesis, gland hyperplasia and hypertrophy. Cytokines, chemokines, and growth factors from inflammatory cells and structural cells also contribute to remodeling. Human airway smooth muscle cells (HASMC) are wildly recognized that play an important role in airway remodeling in asthma. Unfortunately, the treatment of asthma now focus on the control of airway inflamation. Treatment and preventing airway remodeling is not paied much ateention to. More and more scietists choose HASMC as the new target to treat and prevent airway remodeling which become a new way to treat asthma.PTEN (phosphatase and tension homologue deleted on chromosome ten) gene, a tumor suppressor that first identified as a phosphatase, is located at chromosome 10q23.3 containing 9 exons,encoding a protein which consist of 403 amino acids. Researches have found that PTEN gene not only plays an important role in tumors in humans, such as endometrial cancer, ovarian cancer and colorectal cancer etc, but also in non-neoplastic diseases, such as cardiac hypertrophy, hypertension, atherosclerosis and bronchial asthma etc. PTEN protein can block phosphatidylinosito1 -3- kinase signaling pathway (PI3K/PKB/AKT) and mitogen-activated protein kinase (MAPK) signaling pathway (Ras-Raf-MEK1/2-ERKl/2) to inhibit proliferation and migration and induce apoptosis of tumor, cardiac muscle and vascular smooth muscle cells But whether PTEN are also paly same role on HASMC via the main signal pathways is unknown. We mainly discuss the effect of alteration of PTEN gene expression on the migration of HASMC in this paper.Methods:1. The bronchus samples were got from the Pulmonary lobectomy,chest surgery department of nanfang hospital, with permission of the lung cancer patients. Then HASMC was primarily cultured from the trachea of human in vitro with mature methods in our laboratory and a relative literature reference,which were identified by light inverted microscope and cell immunohistochemistry.2. Cells were divided into the following four groups:①interference group (Ad-GFP-shRNA-PTEN):HASMC were transfected with adenovirus-mediated shRNA interference vector of human PTEN gene in vitro at high multiplicity of infection (MOI of 100 PFU per cell);②over-expression group (Ad-GFP-PTEN): HASMC were transfected with adenovirus vector of human PTEN gene with MOI of 100 in vitro③negative control group (Ad-GFP):adenovirus-mediated green fluorescent protein(GFP) were transfected into human ASMCs with MOI of 100 in vitro;④blank control group (DMEM):DMEM were added to culture HASMC in vitro;3. Transwell assay and wound healing assay was used to detect the migration of HASMC with the change of PTEN gene expression by transfected with different adenovirus vector4. The change of protein expression in each group including PTEN, p-AKTser473, p-ERK1/2, Total-Akt, Total-ERK1/2 with the positive control was detected by Western blot.5. Data are expressed as mean±SEM. Statistical comparisons were analysed by using one-way ANOVA (LSD). Statistical significance was set at P<0.05. All statistical analysis was operated with SPSS 13.0 software.Using shofware image J to process pictures.Results:1. In light inverted microscope, HASMCs show a fusiform or polygon before confluence; fascicular form and typical "peak-and-valley" appearance after converging in some regional of cells. And the cultured cells were positive immunostaining for a-SM actin which distributed uniformly in the cytoplasm, and showed brown parallel filamentous structure.2. HASMC were transfected by adenovirus vector with different MOI (25,50,100,150,200)。The transfection efficiency varied from 70% to 90% by flow cytometric analysis. When MOI came to 100, transfection efficiency increased to 95%.So MOI of 100 were chosen to transfect cells. HASMC displayed fluorescent green light after transfected in 48 h under inverted fluorescence microscope.3. Expression of PTEN protein the interference group decreased after transfected with Ad-GFP-shRNA-PTEN, and expression of PTEN protein in over-expression group increased after transfected with Ad-GFP-PTEN; while negative control group and blank control group had no significant changed in PTEN protein expression.4. Compared to the two control group, expression of p-AKTser473 protein markedly increased in the interference group, whereas significantly reduced in over-expression group.p-ATK inhibitor LY294002 was used for positive control group. While four groups had no significant chang in total-Akt protein expression. Expression of p-ERK1/2 protein markedly decreased in the interference group, while no significant difference was detected in over-expression group. And four groups also had no significant change in total-ERK protein expression. p-ERK inhibitor U0126 was used for positive control group5.The result of transwell chamber analysis showed that the migration of HASMC was down-regulated by over-expression of PTEN compared with two control groups(P<0.05),but the ability of migration in the interference group was not significantly increased(P>0.05).The same result was found in wound healing assay. LY294002 was used for positive control group.Conclusions:1. Transwell assay and wound healing assay both show that over-expression of PTEN gene can inhibit the migration of HASMC compared with two control groups. So we conclude that PTEN gene maybe affect airway smooth muscle cell migration depending on regulating the PI3K/AKT pathway.2. Cross-talk was found with the intervention of PTEN gene:the contact between PI3K-Akt and Ras/Raf/MEK/ERK pathway. While interference the expression of PTEN induces phosphorylation and activation of Akt, that may inhibit the phosphorylation and activation of ERK1/2... |
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