Effect of silencing myocardin gene expression on differentiation of mouse bone mesenchymal stem cells into smooth muscle-like cells induced by PDGF-BB.
- Author:
Guan HUANG
1
;
Mei XU
;
Jun YU
;
Han MENG
;
Xue CHEN
;
Yan LI
;
Qiu-rong RUAN
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Bone Marrow Cells; cytology; metabolism; Cell Differentiation; Cells, Cultured; Down-Regulation; Gene Silencing; Genetic Vectors; Male; Mesenchymal Stromal Cells; cytology; metabolism; Mice; Muscle, Smooth, Vascular; cytology; Myocytes, Smooth Muscle; cytology; metabolism; Myosin Heavy Chains; metabolism; Nuclear Proteins; biosynthesis; genetics; physiology; Plaque, Atherosclerotic; pathology; Plasmids; Platelet-Derived Growth Factor; pharmacology; Proto-Oncogene Proteins c-sis; RNA, Messenger; metabolism; RNA, Small Interfering; Trans-Activators; biosynthesis; genetics; physiology; Transfection
- From: Chinese Journal of Pathology 2009;38(2):117-120
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVEConstruction 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.
METHODSMouse 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.
RESULTSThe 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).
CONCLUSIONSubset 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.
