[Effect of silencing myocardin gene expression on differentiation of mouse bone mesenchymal stem cells into smooth muscle-like cells induced by PDGF-BB].
OBJECTIVE
Construction of a small interfering RNA (siRNA) eukaryotic expression vector specific to mouse myocardin gene and study on the role of myocardin-siRNA on differentiation of mouse bone mesenchymal stem cells (MSCs) into smooth muscle-like cells induced by PDGF-BB in vitro.


METHODS
Mouse MSCs were isolated from bone marrow and cultured with 50 mg/L PDGF-BB and fetal bovine serum (20%). Specific myocardin-siRNA sequence was cloned into a plasmid pGenesil-1.0 vector, which contained U6 promoter. The recombinant plasmid and control plasmid were transfected into MSCs which had been cultured with PDGF-BB for 6 days beforehand. The expression of myocardin mRNA was detected by RT-PCR 48 hours after the transfection. Immunohistochemistry was used to detect the SM-MHC and to identify the smooth muscle-like cells.


RESULTS
The recombinant plasmids carrying myocardin-siRNA sequences were constructed successfully and the myocardin mRNA was reduced 42.86% by pGen-myo-shRNA in comparing with that of the controls (P<0.01); and the expression of SM-MHC protein was down-regulated (P<0.01).


CONCLUSIONS
Subset of mouse MSCs have the potential to differentiate into smooth muscle-like cells, a possible cell source responsible for atherosclerotic plaque formation, and myocardin expression may play an important role during this process.
Huang
Guan
G
Institute of Pathology, Affiliated Tongji Hospital, Tongji Medical College, Huazhong Science and Technology University, Wuhan 430030, China.
Xu
Mei
M
Yu
Jun
J
Meng
Han
H
Chen
Xue
X
Li
Yan
Y
Ruan
Qiu-rong
QR
D000818:Animals; D000077214:Becaplermin; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D015536:Down-Regulation; D020868:Gene Silencing; D005822:Genetic Vectors; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000378:metabolism; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D058226:Plaque, Atherosclerotic / Q000473:pathology; D010957:Plasmids; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering; D015534:Trans-Activators / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D014162:Transfection; D000099251:Myocardin
D000077214:Becaplermin; D018995:Myosin Heavy Chains; D009687:Nuclear Proteins; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D015534:Trans-Activators; D000099251:Myocardin