Feasibility of SOX-9 eukaryotic expression plasmid in transfection of rabbit bone marrow stromal cells / 中国组织工程研究

ABSTRACT

BACKGROUND: SOX-9 plays an important role in occurrence and development of cartilaginous tissues,enhances agglutination of mesenchymal cells,has structural domains of transcriptional activation,and can directly regulate the transcription,OBJECTIVE: To construct pDC316-SOX-9 plasmid for transfection of rabbit bone marrow stromal cells (BMSCs) using SOX-9 gene,and to investigate the effects of SOX-9 gene on growth characteristics of BMSCs and product expression. DESIGN,TIME AND SETTING: The cell gene engineering in vitro experiment was performe,d at the Beijing Institute of Genome, Chinese Academy of Scienees from June 2006 to January 2007.MATERIALS: Eight SPF New Zealand rabbits aged 6 weeks were used for culture of BMSCs.METHODS: pDC316-SOX-9 plasmid was used for transfection of rabbit BMSCs by liposome mediated method,Cell transfection included a SOX-9 plasmid transfection group,a blank plasmid group and a blank control group (only treatment of liposome). MAIN OUTCOME MEASURES: Cell morphology; growth activity; The SOX-9 protein expression in rabbit BMSCs were detected by immunohistochemistry,hematoxylin-eosin staining,reserve transcriptase-polymcrase chain reaction (RT-PCR) and enzyme-labeled immunosorbent assay (ELISA). RESULTS: Some cell colonies were detected at 4 days after pDC316-SOX-9 plasmid transfection.Spindle-shaped cells were collected after clone amplification.Cells in the blank control group gradually died over time.Cell activities in the SOX-9 plasmid transfeetion group and the blank plasmid group significantly prolonged,reached a peak at 2 weeks of transfection,and then gradually decreased.At 6 days,BMSCs were yellow in the SOX-9 plasmid transfection group following immunohistochemistry,expressing SOX-9 protein.Hematoxyliu-eosin staining showed many dikaryocytes,rich cell proliferation,similar to chondroblasts.No SOX-9 protein expression and unproductive cell proliferation in the blank plasmid group.SOX-9 mRNA was detected in the SOX-9 plasmid transfection group,but SOX-9 mRNA was not detected in the blank plasmid group and blank control group.SOX-9 levels were significantly higher in the SOX-9 plasmid transfection group than in the blank plasmid group and blank control group at 24,48 and 72 hours,1,2 weeks (P＜ 0.01).CONCLUSION: Rabbit BMSCs were successfully transfected with pDC316-SOX-9 plasmid to enhance cell growth activity and to persistently stably secrete SOX-9 protein,resulting in the differentiation of BMSCs into cartilages.