The Effect And Mechanism Of Recombinant Adenovirus Carrying Pten Gene On Human Airway Smooth Cells Proliferation In Vitro

BackgroundBronchial asthma has been characterized by chronic and allergic airway inflammation,which induces airway remodeling over time.The hyperplasia of airway smooth muscle cells(ASMCs) pay an important part in the airway remodeling.Airway remodeling in asthma refers to the structural changes occurring in the airway wall.including epithelial cell hyperplasia and metaplasia,subepithelial fibrosis,muscle cell proliferation and angiogenesis,of which airway smooth muscle cells(airway smooth muscle cell,ASMC) is the main structural component.The biological characteristics of asthmatic ASMC changes greatly,including hyperplasia,hypertrophy,and migration,which can not only enhance the contractility of bronchi responsing to stimuli,thus increases airway hyperresponsiveness,but also promote the airway wall thickening,leading to an increase in the basic airway resistance and the occurrence of irreversible airflow obstruction.Homologous of tensin and auxiliary protein,phophatase and tensin homolog deleted on chromosone 10(PTEN) is a new tumor suppressor gene discovered in recent years,it is also the first tumor suppressor gene holding phosphatase activity found so far,having an important impact on cell growth,proliferation and differentiation,apoptosis,adhesion,migration and blood vessel growth,and many other biological behavior of cells.With study in-depth,researchers also found it also plays an important role in the non-neoplastic diseases,such as cardiac hypertrophy, hypertension,atherosclerosis,bronchial asthma etc..Previous research has confirmed that PTEN can inhibit the proliferation of tumor cells,cardiac and vascular smooth muscle cells through pathways of PI3K,FAK and ERK,which are also the main signal pathways mediating ASMC proliferation and migration.So we inferred that the overexpression of PTEN may have a similar impact on the proliferation of ASMC,and it can inhibit the proliferation of ASMC through pathways of PI3K,increased the ratio of the cells of G0/G1 and blocked the proceeding of cell cycle.ObjectivesTo find a new therapy for airway remodeling in asthma,we study the effect and the mechanism of recombinant adenovirus carrying pten gene on human airway smooth cells proliferation in vitro.Methords1,1.The trachea was get from of human who have an operation of pulmonary lobectomy,with their permission.2.The HASMCs from the trachea were primarily cultured by the tissue explants adherent method.Then,they were observed by inverted microscope and confirmed by immunocytochemical method withα-actin.3,Primary cultured airway smooth muscle cells were infected with the adenovirus containing PTEN.To ascertained the suitable infection programme andM.O.I.,We following up expression of GFP,we detected the infective efficiency of virus response to different M.O.I.,and the positive rate of GFP expression at 24h,48h and 72h after adenovirus infection.4,RT-PCR and Western Blot were used to detect the level of PTEN mRNA and protein in the infected cell to ensure PTEN overexpression as a result of adenovirus infection. 5.MTS/PMS method was used to detect the proliferation of HASMCs.6.Cell cycle progress of PI-staining cells was detected by flow cytometry.7.The expression levels of Akt and p-A kt proteinwas detected by Western Blot method.8.The expression levels of P21 mRNA was detected by RT-PCR method.9.All statistical tests were operated with the program SPSS13.0,all the data was expressed by(?)±S,One-Way ANOVA was applied in the comparison of more samples.We accepted statistical significant values of P＜0.05.Results1.The cellular morphology of HASMCs was observed by inverted microscope, showing fusiform shape.When they were near-confluent,peak-valley will be seen. Immunocytochemical study revealed strong expression ofα- actin.2.After Ad-PTEN and Ad-GFP infection,the positive rate of GFP expression in the HASMCs was growing up in M.O.I.-dependent and time-dependent manner;When M.O.I.increased up to 100,the percent of HASMCs expressing GFP exceeded 98%;There was no significant difference in the positive rate of GFP expression between group with MOI 100 and group with MOI 200.For each strata of M.O.I.,there was no more increase in the positive rate of GFP expression as the expression time exceeded 48h,there was no significant difference in the positive rate of GFP expression between group with expression time of 36h and group with expression time of 48h.So we took MOI 100 and 48h as appropriate standard to continue the subsequent study.3.RT-PCR and Westem Blot showed that infection of recombinant adenovirus carrying pten gene resulted in PTEN overexpression in the HASMCs.4.MTS/PMS were used to detect the proliferation of HASMCs.The absorbance (A490 value) of the Ad-PTEN transfected cells were lower than those in Ad-GFP group and the control group(P＜0.05),while there was no significant difference between Ad-GFP group and control group(P＞0.05).5.Flow cytometry was used to detect the effect of recombinant adenovirus carrying pten gene transfection on the cell cycle progress of HASMCs.The proportion of Ad-PTEN transfected cells in G0 / G1 phase was significantly greater than that of Ad-GFP group and the control group(P＜0.05),there was no significant difference between Ad-GFP group and the control group(P＞0.05).And there is no significant difference in the ratio of the cells of S and G2 / M between the groups(P＞0.05).6.The expression levels of Akt and p-A kt protein was detected by Western Blot method.Its results show that there is no significant difference in the the level of Akt expression between the groups(P＞0.05).While the level of p-Akt expression of Ad-PTEN group was lower than that of Ad-GFP group and the control group(P＜0.05),there was no significant difference between Ad-GFP group and the control group(P＞0.05).7.The expression levels of P21 was detected by RT-PCR method.Its results show that the level of P21 m RNA expression of Ad-PTEN group was higher than that of Ad-GFP group and the control group(P＜0.05),there was no significant difference between Ad-GFP group and the control group(P＞0.05).Conclusions1.Primary cultured airway smooth muscle cells were infected with the adenovirus containing PTEN,which can successfully transfected the HASMCs cultured in vitro, and PTEN was overexpressed in gene and protein level.2.The overexpression of PTEN can inhibit the proliferation of ASMCs in vitro, increased the ratio of the cells of G0/G1 and blocked the proceeding of cell cycle.3.Tthe overexpression of PTEN can inhibit pathways of PI3K/PKB/AKt.4.The overexpression of PTEN can increased the gene expression of P21.5.The overexpression of PTEN could efficiently inhibit the proliferation of HASMCs,pathways of PI3K/PKB / AKt and P21 maybe involved in the progress.