Construction of Eukaryotic Expression Plasmid of hTGF-β3 and Its Inducing Effect on Differentiation of Precartilaginous Stem Cells into Chondroblasts
10.1007/sl1596-011-0484-2
- Author:
YOU HONGBO
1
;
CHEN ANMIN
;
LIU TIE
;
WANG MAOPENG
;
ZHANG GUOLIANG
Author Information
1. Department of Orthopaedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan430030, China
- Keywords:
TGF-β;
plasmid;
stem cell;
gene transfection;
cartilagenesis
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2011;31(4):524-529
- CountryChina
- Language:Chinese
-
Abstract:
This study examined the construction of eukaryotic expression plasmid of human transforming growth factor-β3 (hTGF-β3) and its inducing effect on the differentiation of precartilaginous stem cells (PSCs) into chondroblasts.hTGF-β3 gene was amplified by using polymerase chain reaction (PCR) and then inserted into the eukaryotic expression plasmid pcDNA3.1 to construct the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3.Rat PSCs were isolated and purified by employing an immunomagnetic cell sorting system.pcDNA3.1(+)-hTGF-β3 was transfected into purified PSCs with the use of linear polyamines.The expression of TGF-β3 and cartilage-specific extracellular matrix (ECM)components was detected after transfection by real-time quantitative PCR,ELISA,immunochemistry and Western blotting,respectively.The results showed that the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3 was successfully established as identified by enzyme digestion and DNA sequencing.Real-time quantitative PCR and ELISA revealed that hTGF-β3 was strongly expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs.Real-time quantitative PCR,immunochemistry and Western blotting showed that the cartilage-specific ECM markers,i.e.,cartilage oligomeric matrix protein (COMP),Aggrecan,collagen type Ⅹ and Ⅱ were intensely expressed in the pcDNA3.1(+)-hTGF-β3-transfected cells.It was concluded that hTGF-β3 could be stably expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs and induce the differentiation of PSCs into chondroblasts.
