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  • Title: [Expression of human bone morphogenetic protein 7 gene in adipose-derived stem cells and its effects on osteogenic phenotype].
    Author: Ren Y, Han C, Jia Y, Yin H, Li S.
    Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2011 Jul; 25(7):848-53. PubMed ID: 21818953.
    Abstract:
    OBJECTIVE: To construct the eukaryotic expression vector of human bone morphogenetic protein 7 (hBMP-7) gene so as to observe its expression in rabbit adipose-derived stem cells (ADSCs) and its effects on osteogenic phenotype. METHODS: Several healthy 3-month-old Japanese rabbits of clean grade were chosen, female or male and weighing 3-4 kg. ADSCs were isolated and cultured with collagenase digestion, then were detected and identified by CD44, CD49d, and CD106 immunofluorescence staining. The eukaryotic expression vector of hBMP-7 gene (pcDNA3.1-hBMP-7) was constructed, which was transfected into rabbit ADSCs (3rd passage) by Lipofectamine 2000 after identified, then the expression of hBMP-7 in transfected ADSCs was detected. The alkaline phosphatase (ALP) level and the collagen type I expression were detected by intracellular ALP spectrophotometry and immunofluorescence, respectively to assess the effect of hBMP-7 gene on the osteoblastic differentiation of ADSCs. RESULTS: ADSCs mostly presented fusiform and polygon shape with positive expressions of CD44 and CD49d and negative expression of CD106. The eukaryotic expression vector of pcDNA3.1-hBMP-7 gene was successfully constructed and the expression of hBMP-7 was confirmed in ADSCs by immunohistochemical staining. The intracellular ALP quantitative detection showed that the activity of ALP was significantly higher in pcNDA3.1-hBMP-7 transfected group (experimental group) than in pcDNA3.1 transfected group (control group) at 7, 10, and 14 days after transfection (P < 0.05). The expression of collagen type I was higher in experimental group than in control group at 7 and 14 days after transfection (P < 0.05). CONCLUSION: The eukaryotic expression vector of pcDNA3.1-hBMP-7 gene is successfully constructed, which can express in ADSCs. The expressions of collagen type I and ALP in experimental group are higher than those in control group, which lays a basis for the local gene therapy of skeletal regeneration.
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