BMP7基因克隆及其在兔关节软骨细胞中的表达

目的：克隆骨形态发生蛋白BMP7基因并检测其在体外培养的兔关节软骨细胞中的表达。方法：从体外培养的幼兔膝关节软骨细胞中提取总RNA；按GenBank BMP7基因序列化学合成2条引物，采用RT-PCR方法得到BMP7基因；将BMP7基因片段插入到真核表达载体pcDNA 3.1中，构建pcDNA3.1-BMP7真核表达载体质粒；利用双酶切、PCR及核苷酸序列分析鉴定BMP7目的基因；将重组pcDNA3.1-BMP7真核表达载体质粒转染家兔关节软骨细胞，分别采用原位杂交、PCR、Western blotting法测定BMP-7表达。结果：克隆得到的BMP7基因核苷酸序列长约1 300 bp，构建的重组pcDNA 3.1-BMP7真核表达载体质粒目的基因BMP-7序列与GenBank报道的BMP-7基因（No. BC008584)一致，转染了BMP7基因的体外培养的成年家兔膝关节软骨细胞表达了BMP7蛋白。结论：成功构建表达BMP7蛋白的转基因关节软骨细胞，其可用于细胞移植治疗或作为种子细胞构建组织工程软骨修复关节软骨缺损。

Cloning of BMP7 gene and its expression in articular chondrocytes of rabbits

Objective To clone BMP7 gene in vitro, and determine the expression of BMP7 gene in articular chondrocytes of rabbits cultivated in vitro. Methods The total RNA was acquired from young rabbit particular cartilage , the BMP7 gene was inserted into pcDNA-3.1 to construct eukaryotic expression vector plasmid of pcDNA3. 1-BMP7; the target gene-BMP7 was identified respectively by PCR analysis, restriction enounces analysis and nucleotide sequencing; the plasmid was transfected into adult rabbit knee articular chondrocytes, then the expression of BMP7 was detected by in situ hybridization, PCR and Western blotting. Results The cloned BMP7 gene was about 1 300 bp, had the same length and sequence with those reported in GenBank. The expression of BMP7 gene in adult rabbit articular chondrocytes was determined. Conclusion The articular chondrocytes of adult rabbits transfected with pcDNA3.1-BMP7 are constructed sucessfully, and the chondrocytes can express the target gene BMP7 stably. The transfected chondrocytes can be used in cellular transplantation therapy or in constructing the tissue engineerinig cartilage to repair the articular cartilage defect.