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

doi:10.3877/cma.j.issn.2096-0263.2019.02.003

Abstract

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

Dawei Li, Yi Sun, Cuiping Jiang, Wenbin Cong, Haining Zhang. Research on human umbilical cord bloodmesenchymal stem cells transfected with the gene of human bone morphogenetic protein-2[J]. Chinese Journal of Geriatric Orthopaedics and Rehabilitation(Electronic Edition), 2019, 05(02): 75-81.

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Figures/Tables 5

References 29

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组别	12 h	24 h	36 h	48 h	60 h	72 h	84 h	96 h
对照组	128.5±1.9	212.4±2.1	321.6±2.0	448.5±3.1	400.1±2.9	384.3±1.4	375.8±1.4	366.2±3.0
实验组	787.1±1.2	1215.7±4.9	1948.3±1.6	3006.0±4.6	2664.9±7.6	2367.3±8.4	2196.0±4.6	1995.3±5.2
t值	662.562	418.266	1429.045	1027.186	622.344	522.479	849.123	609.180
P值	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01

组别	12 h	24 h	36 h	48 h	60 h	72 h	84 h	96 h
对照组	128.5±1.9	212.4±2.1	321.6±2.0	448.5±3.1	400.1±2.9	384.3±1.4	375.8±1.4	366.2±3.0
实验组	787.1±1.2	1215.7±4.9	1948.3±1.6	3006.0±4.6	2664.9±7.6	2367.3±8.4	2196.0±4.6	1995.3±5.2
t值	662.562	418.266	1429.045	1027.186	622.344	522.479	849.123	609.180
P值	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01

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