论文部分内容阅读
目的构建hBMP-7基因真核表达载体,观察其在兔脂肪源性干细胞(adipose-derived stem cells,ADSCs)的表达,并观察其对ADSCs向成骨细胞分化的影响。方法 3月龄清洁级健康日本大耳白兔,雌雄不限,体重3～4 kg。取兔皮下脂肪约5 mL,采用胶原酶消化离心贴壁培养法培养ADSCs,取第3代细胞进行实验;CD44、CD49d、CD106免疫荧光染色鉴定ADSCs。构建真核表达载体pcDNA3.1-hBMP-7,经鉴定后利用LipofectamineTM 2000介导转染ADSCs,免疫组织化学染色检测hBMP-7在ADSCs中的表达;ALP定量测定、Ⅰ型胶原免疫荧光检测hBMP-7基因转染对ADSCs向成骨细胞分化的影响。结果倒置相差显微镜观察示ADSCs呈梭形、多角形分布;表面抗原标志CD44、CD49d呈阳性表达,CD106呈阴性表达。成功构建pcDNA3.1-hBMP-7真核表达载体,并利用脂质体介导的方法成功导入ADSCs中,免疫组织化学染色提示hBMP-7能在ADSCs中表达。ALP定量测定示,转染后7、10、14 d pcDNA3.1-hBMP-7转染组(实验组)ALP活性明显高于pcDNA3.1空载体转染组(对照组),差异有统计意义(P<0.05);转染后7、14 d,实验组Ⅰ型胶原表达量均高于对照组(P<0.05)。结论构建的真核表达载体pcDNA3.1-hBMP-7可在兔ADSCs中表达,且转染后的ADSCs ALP定量测定、成骨标志物Ⅰ型胶原的表达均高于pcDNA3.1空载体转染细胞,为开展以干细胞为载体的局部基因治疗奠定了基础。
Objective To construct eukaryotic expression vector of hBMP-7 gene and observe the expression of hBMP-7 gene in adipose-derived stem cells (ADSCs) and to observe its effect on the differentiation of ADSCs into osteoblasts. Methods 3-month-old clean Japanese white rabbits, male or female, weighing 3 ~ 4 kg. About 5 mL of rabbit subcutaneous fat was obtained. ADSCs were cultured by collagenase digestion and culture in vitro. The third passage cells were used for the experiments. CD44, CD49d and CD106 immunofluorescence staining were used to identify ADSCs. The eukaryotic expression vector pcDNA3.1-hBMP-7 was constructed and transfected into ADSCs by LipofectamineTM 2000. The expression of hBMP-7 in ADSCs was detected by immunohistochemical staining. The expression of hBMP-7 was detected by ALP, -7 gene transfection on the differentiation of osteoblasts from ADSCs. Results Inverted phase contrast microscopy showed spindle-shaped and polygonal-shaped distribution of ADSCs. The surface antigen markers CD44 and CD49d were positive and CD106 was negative. The eukaryotic expression vector pcDNA3.1-hBMP-7 was successfully constructed and successfully transfected into ADSCs by liposome-mediated method. Immunohistochemical staining indicated that hBMP-7 was expressed in ADSCs. ALP assay showed that the ALP activity of pcDNA3.1-hBMP-7 transfection group (experimental group) was significantly higher than that of pcDNA3.1 empty vector transfection group (control group) at 7,10,14 d after transfection (P <0.05). At 7 and 14 days after transfection, the expression of type I collagen in the experimental group was significantly higher than that in the control group (P <0.05). CONCLUSION: The eukaryotic expression vector pcDNA3.1-hBMP-7 can be expressed in ADSCs of rabbits and the expression of ALP in ADSCs after transfection is higher than that of pcDNA3.1 empty vector Cells, laid the foundation for local gene therapy using stem cells as a carrier.