• 1. Department of Oral and Maxillofacial Surgery, Jining Hospital of Stomatology, Jining Shandong, 232000, P.R.China;
  • 2. Department of Oral Prosthodontics, Jining Hospital of Stomatology, Jining Shandong, 232000, P.R.China;
  • 3. Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University;
  • 4. Department of Oral and Maxillofacial Surgery, Dental Dispensary of Shanghai Minhang;
FANGDianji, Email: dianjifang@aliyun.com
Export PDF Favorites Scan Get Citation

Objective To investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein 9 (BMP-9) and erythropoietin (EPO) genes co-transfection on osteogenic differentiation of adipose-derived stem cells (ADSCs) in vitro. Methods The inguinal adipose tissue was harvested from 4-month-old New Zealand rabbits, ADSCs were isolated with enzyme digestion and adherence method, and multipotent differentiation capacity was identified. The 3rd generation ADSCs were divided into 5 groups: normal cells (group A), empty plasmid control group (group B), BMP-9 or EPO recombinant adenovirus transfected cells (groups C and D), BMP-9 and EPO recombinant adenovirus co-transfected cells (group E). The inverted phase contrast microscope was used to observe the cell growth at 7 days; the expression of cell fluorescence was observed under a fluorescence microscope at 14 days, and viral transfection efficiency was calculated at 48 hours; Western blot was used to detect the expressions of BMP-9 and EPO proteins at 14 days. The expression of alkaline phosphatase (ALP) activity was detected at 3, 7, and 14 days after osteogenic induction, and alizarin red staining was used to detect calcium nodules formation and real-time fluorescence quantitative PCR to detect the expressions of osteopontin (OPN) and osteocalcin (OCN) at 3 weeks. Results At 7 days after transfected, some cells showed oval, round, and irregular shape under the inverted phase contrast microscope in groups A and B; a few fusiform cells were observed in groups C and D; oval cells increased obviously, and there were only few round cells in group E. The fluorescence microscope observation showed that BMP-9 and EPO, BMP-9/EPO recombinant adenovirus could stably transfected ADSCs, with transfection efficiency of 80%-93%. The expressions of BMP-9 and EPO proteins significantly higher in group E than the other groups by Western blot (P < 0.05). The ALP activity significantly increased in group E when compared with that in the other groups at 3, 7, and 14 days after osteogenic induction (P < 0.05); the number of calcium nodules in group E was significantly more than that in the other groups (P < 0.05). Real-time fluorescence quantitative PCR showed that OPN and OCN genes expressions were significantly higher in group E than other groups (P < 0.05), and in groups C and D than groups A and B (P < 0.05). Conclusion Recombinant adenovirus-mediated BMP-9 and EPO genes can transfect ADSCs, which can stably express in ADSCs, BMP-9/EPO genes co-transfection can more promote the expressions of osteoblast-related genes and protein than non-transfected and single gene transfection.

Citation: ZHANGGuangde, SUChengshuai, JINXia, YANGShimao, FANGDianji, GUOYanwei. RECOMBINANT ADENOVIRUS-MEDIATED BONE MORPHOGENETIC PROTEIN 9 AND ERYTHROPOIETIN GENES CO-TRANSFECTION IN PROMOTING OSTEOGENIC DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(3): 272-278. doi: 10.7507/1002-1892.20160055 Copy

  • Previous Article

    EFFECTS OF BONE MARROW MESENCHYMAL STEM CELLS TRANSPLANTATION FOR TREATING RAT SPINAL CORD INJURY AND CYTOKINE EXPRESSION AT INJURY SITES
  • Next Article

    EFFECT OF Hey1 EXPRESSION ON OSTEOGENIC DIFFERENTIATION AND PROLIFERATION OF C3H10T1/2 CELLS INDUCED BY BONE MORPHOGENETIC PROTEIN 9