负载人骨形态发生蛋白-7基因的兔脂肪干细胞向成骨细胞分化的实验研究

目的 探讨人骨形态发生蛋白-7(hBMP-7)基因修饰对兔脂肪干细胞(ADSCs)成骨能力的影响. 方法 原代培养兔脂肪干细胞,免疫组织化学方法检测细胞表面抗原CD44、CD49d、CD106的表达,对细胞进行鉴定;阳离子脂质体介导hBMP-7基因转染ADSCs,绿色荧光蛋白表达观察转染效率、逆转录-聚合酶链反应(RT-PCR)检测目的基因hBMP-7的表达;ALP定量测定,Western blot检测Ⅰ型胶原、骨钙素的表达,以评价转基因ADSCs向成骨细胞分化的情况. 结果 从兔脂肪组织中分离出来的ADSCs CD44、CD49d表达呈阳性,CD106表达呈阴性.RT-PCR检测筛选后的ADSCs稳定表达hBMP-7.转染后7、10、14 d ALP定量测定转染组明显高于未转染组,差异有统计学意义(P<0.05);成骨标志物Ⅰ型胶原、骨钙素的表达转染组明显高于未转染组.结论 从脂肪组织中分离出的ADSCs是一种较好的组织工程种子细胞.hBMP-7重组质粒转染ADSCs后表达目的蛋白,并诱导ADSCs向成骨细胞分化.

Osteoblast-differentiation of rabbit adipose-derived stem cells by in vitro transfection of bone morphogenetic protein-7

Objective To explore the osteogenic potential of adipose-derived stem cells (ADSCs)when exposed to the cDNA of human bone morphogenetic protein-7 (hBMP-7) during osteoblast-differentiation in vitro. Methods The ADSCs were primarily cultured from rabbit fat tissue. CD44, CD49d and CD106 were detected and identified by immunohistochemistry. ADSCs were inducted with the method of stable transfection of pcDNA3. 1-GFP-hBMP-7 by Liprofectamine2000. The green fluorescent protein (GFP) expression was observed to access transfection efficiency of the transfected cells. RT-PCR was used to detect the expression of hBMP-7 gene in the induced ADSCs. The transform of ADSCs was assessed by detecting the ALP and expressions of collagen Ⅰ and osteocalcin. Results ADSCs were successfully isolated from rabbit adipose tissue. The isolated ADSCs expressed CD44 and CD49d but CD106 was absent. The pcDNA3. 1-GFP-hBMP-7 transfected ADSCs expressed hBMP-7. The intracellular ALP spectrophotometry in the hBMP-7 group was higher than in the control group after transfection. The expressions of collagen Ⅰ and osteocalcin in the hBMP-7 group were higher than in the control group. Conclusion ADSCs isolated from rabbit adipose tissue may be an excellent seeding cell for tissue engineering because they can differentiate into osteoblasts in vitro by hBMP-7 gene transfection.