[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.


CONCLUSIONS
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.
Chen
Li-Li
LL
Department of Respiratory Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China. 2006chenlili@163.com
Luo
Ya-Ling
YL
Lai
Wen-Yan
WY
Xu
Jian
J
Zhong
Hao-Hai
HH
Zhang
Da-Cheng
DC
Ye
Han
H
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D001980:Bronchi / Q000166:cytology*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D009130:Muscle, Smooth / Q000166:cytology*; D051059:PTEN Phosphohydrolase / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D014162:Transfection
D012333:RNA, Messenger; D011994:Recombinant Proteins; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human