[Experimental research on human insulin-like growth factor I gene transfect the cultured bone marrow mesenchymal stem cells].
OBJECTIVE
To investigate the effectiveness of human insulin-like growth factor I (hIGF-I) gene transferred into the cultured goat bone marrow mesenchymal stem cells with liposome, and find a new method of cell-mediated gene therapy.


METHODS
Bone marrow was extracted from adult goats and cultured in vitro by monolayer. Then the recombinant eukaryotic expression plasmid pIRES2-EGFP-hIGF was transfected into cells by FuGene 6. After transfection, the marker gene coding enhanced green fluorescent protein (EGFP) was observed at different time points. The hIGF concentration in the supernatant fluids was measured by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry stain of hIGF was performed to detect the target protein. Besides, reverse transcription polymerase chain reaction and flow cytometry were also adopted in order to find out the changes of cells after transfection.


RESULTS
Bone marrow stem cells were all in the long shuttle-like shape and adhered to the disk. The expression of EGFP was first found at 4 h after transfection. The amount and intensity of EGFP increased gradually during the period of detection and got to the peak degree at 72 h, after that decreased slowly. EGFP was also seen in the second generation cells, but the intensity was relatively faint. The IGF-I concentration secreted into the supernatant was in accordance with the EGFP observed with the peak concentration at 34.75 ng/ml. The outcome of RT-PCR and immunohistochemistry was positive. The morphology of many stem cells was changed into triangular or irregular forms under the circumstance of the secreted hIGF-I. Percentage of stem cells in the S stage increased after transfection.


CONCLUSIONS
The hIGF-I gene can be transfected efficiently and safely into BMSCs by FuGene 6, and the hIGF-I protein can be secreted into the supernatant in a relatively high level during a long period, therefore accelerate the proliferation and differentiation of the transfected cells.
Zhang
Hai-Ning
HN
Department of Orthopaedics, the Affiliated Ninth People's Hospital of Shanghai Second Medical University, Shanghai 200011, China. zhhaining@hotmail.com
Hou
Xiao-Kui
XK
Tang
Ting-Ting
TT
Leng
Ping
P
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002454:Cell Differentiation; D005822:Genetic Vectors; D006041:Goats; D006801:Humans; D066298:In Vitro Techniques; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010957:Plasmids / Q000235:genetics*; D014162:Transfection
D007334:Insulin-Like Growth Factor I