[Inhibition on phenotypic transformation of detrusor smooth muscle cells after bladder outlet obstruction by E2F decoy strategy].
OBJECTIVE
To investigate the inhibitory effect of E2F decoy strategy on the phenotypic transformation of detrusor smooth muscle cells (DSMCs) so as to verify the effect of the E2F decoy strategy in improvement of the bladder function after bladder outlet obstruction (BOO).


METHODS
Rat DSMCs were cultured, underwent cyclic mechanical stretch so as to establish BOO model, and then were divided into 3 groups: E2F-ODN decoy group [transfected with E2F-decoy ODN tagged with carboxy-fluorescein (FAM), a at its 3' end], Mis-decoy group (transfected with mismatch E2F-decoy ODN), and control group (without transfection). Inverted fluorescence microscopy was used to detect the green fluorescence of FAM in the successfully transfected cells. The proliferation of the cells was observed by MTT method. RT-PCR was used to examine the mRNA expression of proliferating cell nuclear antigen (PCNA). Western blotting was used to detect the protein expression of PCNA and cdk2 kinase.


RESULTS
FAM-labeled E2F-ODN was detected stably in the DSMCs of the E2F-ODN decoy group 24 hours after transfection. The proliferation of the DSMCs of the E2F-ODN decoy group was decreased significantly compared with the mismatch E2F-ODN decoy and control groups (both P < 0.01). The mRNA expression of PCNA, protein expression of PCNA and cdk2 kinase of the E2F-ODN decoy group were all significantly lower than those of the other 2 groups (all P < 0.01).


CONCLUSIONS
E2F-decoy ODN can be transfected and stably expressed in DSMCs. The phenotypic transformation of DSMCs can be successfully inhibited by E2F decoy strategy, which clarifies the potential role of structural stability-based method on improvement of bladder function recovery after BOO.
Gong
Yu
Y
Department of Urology, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China. gong-david@hotmail.com
Du
Chuan-jun
CJ
Ding
Li-li
LL
Song
Bo
B
Jin
Xi-yu
XY
D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism; D050684:E2F Transcription Factors / Q000235:genetics* / Q000502:physiology; D005452:Fluoresceins / Q000737:chemistry; D005786:Gene Expression Regulation; D008856:Microscopy, Fluorescence / Q000379:methods; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics*; D010641:Phenotype; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013314:Stress, Mechanical; D014162:Transfection; D001743:Urinary Bladder / Q000166:cytology / Q000378:metabolism; D001748:Urinary Bladder Neck Obstruction / Q000235:genetics / Q000378:metabolism / Q000473:pathology
D050684:E2F Transcription Factors; D005452:Fluoresceins; D009841:Oligonucleotides; D018809:Proliferating Cell Nuclear Antigen; C024098:6-carboxyfluorescein; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2