人腱细胞Smad2、Smad4基因MH2结构域的克隆和序列鉴定

Cloning and sequence analysis of MH2 domain of Smad2 and Smad4 in human tenocytes

目的从人腱细胞内克隆转化生长因子－β特异的细胞内信号转导基因Ｓｍａｄ２、４的功能性结构域－ＭＨ２结构域。 方法 原代培养人腱细胞，从培养的细胞中提取总 ＲＮＡ，逆转录合成单链 ｃＤＮＡ；设计引物进行 ＰＣＲ，扩增 Ｓｍａｄ２、４基 因ＭＨ２结构域的基因片段，将其定向插入ｐＧＥＭ－Ｔ载体，转化大肠杆菌ＤＨ５α。挑选阳性克隆并进行核苷酸序列测定 分析。结果测序结果与 Ｇｅｎｅｂａｎｋ中克隆的基因序列完全一致。结论从人腱细胞中克隆成功 Ｓｍａｄ２、４基因的 ＭＨ２结 构域，证实了 Ｓｍａｄ２、４基因在人腱细胞中的表达；提示人腱细胞内存在 ＳＭＡＤ２、４信号转导途径，ＴＧＦ－β对人腱细胞 分化的调节可能是通过ＳＭＡＤ２、４信号转导途径实现的。

Objective To clone MH2 domain of intercellular signal transduction gene Smad2 and Smad4 specific for transforming growth factor-β (TGF-β).Methods Total RNA was extracted from cultured primary human tenocytes for reverse transcription of the single-stranded cDNA.PCR with designed primers was performed to amplify the MH2 domain sections of Smad2 and Smad4, which were inserted into pGEM-T vector and the resulted plasmid was transformed into E.coli DH5a. The positive clones were screened and their double-stranded cDNA sequenced. Result The sequence of MH2 domain of Smad2 and Smad4 cloned from the tenocytes was identical with that from the Genebank. Conclusions MH2 domain cDNA of Smad2 and Smad4 was successfully cloned from human tenocytes, indicating the presence of signal transduction pathway which mediates the regulation of human tenocyte differentiation by TGF-β.