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背景:通过测序进行序列分析是发现基因突变的直接手段,转化生长因子β1(TGF-β1)作为细胞最主要的生长调控因子,参与多项生理和病理过程。该基因的突变对于所在细胞的生物学行为改变可能具有重要意义。目的:分析人成骨肉瘤细胞系中TGF-β1成熟肽基因序列,探讨TGF-β1基因突变存在的可能性及其机制,为肿瘤及骨软骨损伤的基因治疗提供新思路。设计:非随机非对照实验研究。地点和对象:实验在第四军医大学唐都医院解放军骨科中心骨肿瘤研究所完成,对象为人成骨肉瘤细胞系SOSP-9607(本室建立)。干预:提取人成骨肉瘤细胞系SOSP-9607中的总RNA,参照Genebank数据库中的已知序列设计合成引物,采用RT-PCR法扩增TGF-β1成熟肽基因,克隆入质粒pcDNA3.0进行序列测定和分析。主要观察指标:将多次测序所得的TGF-β1成熟肽基因序列与Genebank数据库中的已知序列进行比较,分析恒定差异。结果:发现人成骨肉瘤细胞系SOSP-9607中TGF-β1基因存在两处点突变,其中第1788位突变引起相应多肽的结构改变。结论:人成骨肉瘤细胞系SOSP-9607中存在TGF-β1基因突变,其可能在病理机制中起重要作用,具有强功能活性突变子的获得对于骨软骨损伤的修复有重要意义。
BACKGROUND: Sequence analysis by sequencing is a direct method for discovering gene mutations. Transforming growth factor β1 (TGF-β1) is the most important growth regulator of cells and participates in many physiological and pathological processes. Mutations in this gene may have important implications for changes in the biological behavior of the cells in which they are located. OBJECTIVE: To analyze the gene sequence of TGF-β1 mature peptide in human osteosarcoma cell line and investigate the possibility and mechanism of TGF-β1 gene mutation, providing new ideas for gene therapy of tumor and osteochondral lesions. Design: Non-randomized, non-controlled experimental study. Location and Object: The experiment was performed at the Institute of Bone Tumors, Orthopaedics Center, Tangdu Hospital, Fourth Military Medical University. The objective was human osteosarcoma cell line SOSP-9607 (established in this room). Intervention: Total RNA was extracted from human osteosarcoma cell line SOSP-9607. Synthetic primers were designed based on known sequences in the Genebank database. TGF-β1 mature peptide gene was amplified by RT-PCR and cloned into plasmid pcDNA3.0. Sequence determination and analysis. MAIN OUTCOME MEASURES: The TGF-β1 mature peptide gene sequence obtained from multiple sequencing was compared with known sequences in the Genebank database to analyze the constant difference. RESULTS: It was found that there were two point mutations in the TGF-β1 gene in human osteosarcoma cell line SOSP-9607, and the 1788 mutation caused a change in the structure of the corresponding polypeptide. CONCLUSION: There is a TGF-β1 gene mutation in human osteosarcoma cell line SOSP-9607, which may play an important role in pathological mechanism. The acquisition of a highly functional mutant has important significance for the repair of osteochondral injury.