[Experimental study on differentiation of rat BMSCs to chondrocytes transfected by TGF-beta1 and IGF-1 gene alone and together].
OBJECTIVE
To investigate the secretion of target gene and differentiation of BMSCs transfected by TGF-beta1 and IGF-1 gene alone and together into chondrocytes and to provide a new method for culturing seed cells in cartilage tissue engineering.


METHODS
The plasmids pcDNA3.1-IGF-1 and pcDNA3.1-TGF-beta1 were amplified and extracted, then cut by enzymes, electrophoresed and analyzed its sequence. BMSCs of Wistar rats were separated and purificated by the density gradient centrifugation and adherent separation. The morphologic changes of primary and passaged cells were observed by inverted phase contrast microscope and cell surface markers were detected by immunofluorescence method. According to the transfect situation, the BMSCs were divided into 5 groups, the non-transfected group (Group A), the group transfected by empty vector (Group B), the group transfected by TGF-beta1 (Group C), the group transfected by IGF-1 (Group D) and the group transfected both by TGF-beta1 and IGF-1 (Group E). After being transfected, the cells were selected, then the proliferation activity was tested by MTT and expression levels were tested by RT-PCR and Western blot.


RESULTS
The result of electrophoresis showed that sequence of two bands of the target genes, IGF-1 and TGF-beta1, was identical with the sequence of GeneBank cDNA. A few adherent cells appeared after 24 hours culture, typical cluster formed on the forth or fifth days, and 80%-90% of the cells fused with each other on the ninth or tenth days. The morphology of the cells became similar after passaging. The immunofluorescence method showed that BMSCs were positive for CD29 and CD44, but negative for CD34 and CD45. A few cells died after 24 hours of transfection, cell clone formed at 3 weeks after selection, and the cells could be passaged at the forth week, most cells became polygonal. The boundary of some cells was obscure. The cells were round and their nucleus were asymmetry with the particles which were around the nucleus obviously. The absorbency values of the cells tested by MTT at the wavelength of 490 nm were 0.432 +/- 0.038 in group A, 0.428 +/- 0.041 in group B, 0.664 +/- 0.086 in group C, 0.655 +/- 0.045 in group D and 0.833 +/- 0.103 in group E. The differences between groups A, B and groups C, D, E were significant (P < 0.01). The differences between groups A and B or between C, D and E were not significant (P > 0.05). RT-PCR and Western blot was served to detect the expression of the target gene and protein. TGF-beta1 was the highest in group C, 0.9250 +/- 0.0220, 124.3417 +/- 2.9820, followed by group E, 0.7717 +/- 0.0120, 101.7667 +/- 1.2410 (P < 0.01); The expression of IGF-1 was the highest in group E, 1.0200 +/- 0.0260, 128.1717 +/- 9.1520, followed by group D, 0.4650 +/- 0.0420, 111.0450 +/- 6.2480 (P < 0.01). And the expression of collagen II was the highest in group E, 0.9800 +/- 0.0340, 120.3550 +/- 12.5500, followed by group C, 0.7200 +/- 0.0260, 72.2467 +/- 7.3640 (P < 0.01).


CONCLUSIONS
The repairment of cartilage defects by BMSCs transfected with TGF-beta1 and IGF-1 gene together has a good prospect and important significance of clinic application in cartilage tissue engineering.
Fu
Qin
Q
Department of Spine Surgery, 2nd Affiliated Hospital, China Medical University, Shenyang Liaoning, 110004, P.R. China. fuq@cmu2h.com
Yu
Dongdong
D
Wang
Yong
Y
Fu
Yonghui
Y
Gong
Shuyi
S
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology*; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D010957:Plasmids; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D023822:Tissue Engineering / Q000379:methods*; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000096:biosynthesis / Q000235:genetics*
D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; D007334:Insulin-Like Growth Factor I; D003094:Collagen