Research on human umbilical cord bloodmesenchymal stem cells transfected with the gene of human bone morphogenetic protein-2

Abstract

Objective To gain mesenchymal stem cells from humanumbilical cord blood and transfect the recombinant plasmid pIRES2-EGFP-hBMP-2 into human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) with liposome and to induce hUCB-MSCs osteoblastization. Methods Separated cells from human umbilical cord blood with density gradient centrifugation, and hUCB-MSCs were gained with adherent method, using flow cytometry to identify the surface markers of hUCB-MSCs. Transfected recombinate plasmid pIRES2-EGFP-hBMP-2 into the third generation hUCB-MSCs with X-treme GENE HP DNA Transfection reagent, then detected the intensity of EGFP. Collected the medium at different time after transfection and the hBMP-2 content in the medium was measured by ELISA. Use immunofluorescent to locate the existence of hBMP-2 within the cells and RT-PCR techniques to measure the transcription of the hBMP-2 gene. Two weeks after transfection, immunohistochemistry were used to detect the type Ⅱ collagen for discovering the possible changes of hUCB-MSCs. Results HUCB-MSCs could be isolated from blood in umbilical cord by density gradient centrifugation and the different ability of adherence. Flow cytometry showed that the hUCB-MSCs positively expressed CD90, CD105 and CD146, and did not express CD34, CD45 and Anti-HLA-DR. The recombinant plasmid pIRES2-EGFP-hBMP-2 was successfully fused into umbilical cord blood mesenchymal stem cells, and the fusion rate was (27.7±7.6)%. The ELISA test compared the expression of hBMP-2 between the experimental group and the control group, P<0.01 showed a statistically significant difference. RT-PCR results showed that the hBMP-2 gene was stably transcribed and the immunofluorescently labeled hBMP-2 protein showed red fluorescence. Immunohistochemical staining of type Ⅱ collagen showed that some cells were browned. RT-PCR results showed that the expression of CRLT1 in umbilical cord blood mesenchymal stem cells transfected with BMP-2 was higher than that in untransfected umbilical cord blood mesenchymal stem cells and human synovial fibrosis group (P<0.05). Conclusions Recombinat plasmid pIRES2-EGFP-BMP-2 coated with X-treme CENE can be successfully transfected into hUCB-MSCs. Both the marker gene and objectivegene can be transcribed and expressed in hUCB-MSCs, and cells can synthesis hBMP-2, which stimulates the hUCB-MSCs'differentiation towards to chondrocytes. Key words: Recombinant human bone morphogenetic protein-2; Mesenchymal stem cells; Gene editing; Liposomes