[Differentiation of bone marrow mesenchymal stem cells into nucleus pulposus-like cells transfected by SOX9 eukaryotic expression vector in vitro].
OBJECTIVE
The biological treatment of intervertebral disc degeneration becomes a research hotspot in recent years. It is necessary to find an effective approach to induce bone marrow mesenchymal stem cells (BMSCs) differentiate to disc cells which could make application of cell transplantation as a treatment of intervertebral disc degeneration. To investigate the effects of the recombinant plasmid pcDNA3.1IE-SOX9Flag on differentiation of rabbit BMSCs into nucleus pulposus-like cells.


METHODS
The eukaryotic expression vector of pcDNA3.1IE-SOX9Flag was constructed. Rabbit BMSCs were isolated and cultured from one-month-old New Zealand white rabbits and were induced into osteogenetic cells in the osteogenesis supplement medium; and the cell surface markers were detected by flow cytometry. The cells at the 3rd passage were randomly divided into 3 groups: in transfected group, the cells were transfected with recombinant plasmid pcDNA3.1IE-SOX9Flag; in negative control group, the cells were transfected with plasmid pcDNA3.1; and in blank control group, the cells were treated with the media without recombinant plasmid. After selected by G418 for 7 days, the cells were harvested and RT-PCR was employed to assay SOX9 mRNA and collagen type II gene (Col2al) mRNA expressions in BMSCs. The expression of SOX9 protein was assayed by Western blot and collagen type II expression was also observed by immunohistochemical staining.


RESULTS
The SOX9 eukaryotic expression vector was constructed successfully. The BMSCs after 5 days of osteogenetic induction were positive for the alkaline phosphatase staining. What was more, CD44 expression was positive but CD34 and CD45 expressions were negative. The transfection efficiency was 34.32% +/- 1.75% at 72 hours after transfection. After 2 weeks of transfection, BMSCs turned to polygonal and elliptical. And the cell proliferation was gradually slow which was similar to the growth characteristic of nucleus pulposus cells. RT-PCR identification showed that SOX9 mRNA and Col2al mRNA expressions were positive in transfected group, and were negative in 2 control groups. Western blot detection showed that SOX9 protein expressed in transfected group but did not express in the control groups. At 2 weeks after transfection, the result of the immunohistochemical staining for collagen type II was positive in transfected group.


CONCLUSIONS
The recombinant plasmid pcDNA3.1IE-SOX9Flag can be successfully transfected into rabbit BMSCs, the transfected BMSCs can differentiate into nucleus pulposus-like cells, which lays a theoretical foundation for treatment of intervertebral disc degeneration with BMSCs transplantation.
Zhang
Qi
Q
Center of Orthopedic Surgery, Orthopedic Oncology Institute of Chinese PLA, Tangdu Hospital, Fourth Military Medical University, Xi'an Shaanxi, 710038, P R China.
Qian
Jixian
J
Wen
Yanhua
Y
Qiu
Xiuchun
X
Ma
Baoan
B
Fan
Qingyu
Q
D000818:Animals; D001853:Bone Marrow / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics*; D002478:Cells, Cultured; D005822:Genetic Vectors; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D055758:SOX9 Transcription Factor / Q000235:genetics*; D014162:Transfection
D012333:RNA, Messenger; D055758:SOX9 Transcription Factor