重组大鼠pAT_(153)+IGF-1基因单独转染及其与pcDNA_3+TGF-β_1基因共同转染兔软骨细胞的实验研究  

Effects of transfected recombined rat insulin-like growth factor 1 (pAT_(153)+IGF-1) with and without recombined rat transforming growth factor beta 1 (pcDNA_3+TGF-β_1) on rabbit chondrocytes ex vivo

作　　者：向川[1] 杜靖远[2] 翁习生[3] 卫小春[1] 

机构地区：[1]山西医科大学第二医院骨科,太原030001 [2]武汉华中科技大学同济医学院附属协和医院骨科 [3]中国医学科学院中国协和医科大学北京协和医院骨科

出　　处：《中华风湿病学杂志》2004年第11期649-653,i001,共6页Chinese Journal of Rheumatology

基　　金：国家自然科学基金面上资助项目(30371440)

摘　　要：目的通过对比重组大鼠胰岛素样生长因子1基因(pAT153+IGF-1)单独转染及其与重组大鼠转化生长因子β1基因(pcDNA3+TGF-β1)共转染兔软骨细胞后细胞增生及TGF-β1因子、IGF-1因子、Ⅱ型胶原含量的变化,探讨pAT153+IGF-1、pcDNA3+TGF-β1转染对兔软骨细胞分裂增生及分泌合成功能的影响,为将来可能的骨关节炎(OA)基因治疗提供理论基础。方法提取重组大鼠基因pAT153+IGF-1和pcDNA3+TGF-β1,将体外培养的兔膝关节软骨细胞分别用pAT153+IGF-1单转染、pAT153+IGF-1和pcDNA3+TGF-β1共转染,筛选阳性克隆后,对软骨细胞及其所分泌的TGF-β1、IGF-1、Ⅱ型胶原进行原位杂交、免疫组织化学、免疫荧光检测、流式细胞仪检测、3H标记的胸腺嘧啶脱氧核苷(3H-TdR)检测。结果基因转染组与空白组相比,TGF-β1、IGF-1、Ⅱ型胶原的含量均明显提高,DNA合成记数及细胞周期S期的比例也明显高于空白组(P<0.05);基因共转染组的上述指标较pAT153+IGF-1单转染组高,差异有显著性(P<0.05)。结论基因pAT153+IGF-1、pcDNA3+TGF-β1转染软骨细胞后,TGF-β1、IGF-1及Ⅱ型胶原含量显著增高,细胞增生及细胞数量明显增多,上述基因转染有助于软骨细胞活力的提高;Objective To investigate effects of transfected pAT153+IGF-1 with and withoutpcDNA3+TGF-β1 on rabbit chondrocytes proliferating and synthesizing of TGF-beta1, IGF-1 and collagen Ⅱ.Methods Monolayer cultures of rabbit articular chondrocytes were infected with recombinant plasmid pAT153 carrying genes encoding IGF-1, and were co-transfected by pcDNA3+TGF-beta1, pAT153+IGF-1. The synthesis of TGF-beta1, IGF-1, and type Ⅱ collagen was measured by in situ hybridization, immunohistochemistry,immunofluoroscopy, flow cytemeter and 3H-TdR radiolabeling. Results The expression of TGF-beta1,IGF-1 and type Ⅱ collagen was higher than control in two gene transfection groups (P<0.05). The co-transfection had higher TGF-beta1, IGF-1 and type Ⅱ collagen synthesis than that of recombinant pAT153+IGF-1(P<0.05). Conclusion Transferring of TGF-beta1 and IGF-1 genes to articular chondrocytes can greatly increase proliferation and synthesis of chondrocytes ex vivo. This result encourages further development of gene therapy for osteoarthritis. The co-transfection can provoke a more increased synthesis of TGF-beta1, IGF-1 and type Ⅱ collagen than pAT153+IGF-1, so it might be more useful in osteoarthritis therapy.

关 键 词：IGF-1 TGF-β1 软骨细胞 PCDNA3 Ⅱ型胶原 共转染 大鼠 双基 结论 理论基础 

分 类 号：R684[医药卫生—骨科学]