The study on modification of human umbilical cord blood mesenchymal cells by hIGF-1 gene

Abstract

Objective To study the modification of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) by hIGF-1 gene, and provide good seed cells for tissue engineering repair of articular cartilage. Methods hUCB-MSCs were isolated and cultured by density gradient centrifugation and cell adherence method, and cell identification was performed by flow cytometry. The eukaryotic expression vector, pIRES2-EGFP-hIGF-I, containing the full length of hIGF-1 gene was transfected into hUCB-MSCs via X-tremen HP. After transfection, the expression of fluorescent protein was detected by reverse fluorescence microscope and the transfection efficiency was calculated. ELISA was used to detect the content of hIGF-1 in the cell supernatant after transfection, and the immunofluorescence and RT-PCR were used to detect hIGF-1 in the hUCB-MSCs. The expression of the typeⅡ collagen was detected by immunohistochemistry. Results Flow cytometry showed that the hUCB-MSCs expressed CD90, CD105 and CD146 positively, and CD34, CD45, Anti-HLA-DR negatively. hIGF-1 gene was introduced into hUCB-MSCs with X-treme GENE HP DNA transfection reagent, and the transfection efficiency is (29+8)%. The hIGF-1 protein concentration in the supernatants determined by ELISA had significant difference between transfection group and control group (P<0.01). ELISA result showed that the hIGF-1 protein concentration in the supernatants determined after transfection at high level [(35±0.4)ng/ml] at 48 h point. The expression of hIGF-1 was detected by immunofluorescence and RT-PCR identification. Immunohistochemistry showed positive expression of type Ⅱcollagen after transfection. Conclusions hIGF-1 gene can modify human umbilical cord blood mesenchymal stem cells through X-tremen HP mediated modification, and promote the expression and secretion of hIGF-1 and type Ⅱ collagen. Key words: Insulin-like growth factor 1; Liposomes; Gene transfection; Mesenchymal stem cells