Comparison of hIGF-1 gene transfection to the hBMSCs and human meniscal fibrochondrocytes.
BACKGROUND
Treatment strategies for meniscal injury are shifting from meniscectomy to repair, especially cell-based therapy. Delivering selected genes to donor cells can modify differentiation and proliferation. Efficiency of gene transfection and expression may relate to cell type.


METHODS
Full-length hIGF-1 cDNA was cloned into eukaryotic expression vector by PCR. Human BMSCs and meniscal fibrochondrocytes were isolated and cultured in vitro and hIGF-1 gene was transfected by FuGene 6. Expression of EGFP and hIGF-1 were determined. Biological activity of the hIGF-1 in medium was assessed by MTT chromatometry. Real-time quantitative PCR and Western blot were used to assess the expression of exogenous genes. Efficacy of gene transfection was detected by immunohistochemistry staining and flow cytometry.


RESULTS
Sequences of hIGF-1 were verified by sequence analysis. Expression of EGFP increased gradually and reached peak intensity 48 h after transfection. Transfection efficiency of BMSCs was higher than meniscal fibrochondrocytes. The population doubling time was decreased in both cell types. Peak concentration of hIGF-1 in the medium of BMSCs and meniscal cells was 32.5±4.8 ng/ml and 24.5±4.6 ng/ml, respectively. Secreted hIGF-1 possessed the ability to enhance proliferation of the cell line. Results of qPCR and Western blot confirmed the expression of hIGF-1. Type II collagen appeared within the cells, and percentage of cells in S stage was increased in both cell types after transfection.


CONCLUSIONS
hIGF-1 cDNA can be transfected into BMSCs and meniscal fibrochondrocytes, resulting in gene expression. Expression efficiency in BMSCs was higher than that in fibrochondrocytes.
Department of Joint Surgery, Affiliated Hospital of Qingdao University, Qingdao, Shandong, China (mainland).
Haining
H
Zhang
Department of Pharmacy, Affiliated Hospital of Qingdao University, Qingdao, Shandong, China (mainland).
Ping
P
Leng
Department of Trauma Surgery, Affiliated Yantai Yuhuangding Hospital of Qingdao University Medical College, Yantai, Shandong, China (mainland).
Tian
T
He
Department of Joint Surgery, Affiliated Hospital of Qingdao University, Qingdao, Shandong, China (mainland).
Yingzhen
Y
Wang
D015153:Blotting, Western; D048430:Cell Shape; D019902:Chondrocytes / Q000378:metabolism*; D005434:Flow Cytometry; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D008592:Menisci, Tibial / Q000166:cytology*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics; D014162:Transfection
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007334:Insulin-Like Growth Factor I