摘要
分离、培养仔猪关节软骨细胞,从中提取总RNA,用RT-PCR法扩增出TGF-β的cDNA,与pM D 18-T载体相连,转化JM 109大肠杆菌,提取质粒,鉴定所扩增片断为目的片断后,培养仔猪关节软骨细胞,并在培养液中添加不同水平的铜,分别在0、12、24、48 h培养结束时,收集细胞,提取细胞总RNA。采用RT-PCR法扩增出TGF-β的cDNA,以-βactin为内参,用凝胶分析系统对扩增出的cDNA进行扫描分析,以TGF-β的cDNA的光密度值与-βactin的cD-NA的光密度值之比作为TGF-βmRNA基因表达的相对水平。结果表明,细胞培养液中添加不同水平的铜能促进软骨细胞中TGF-βmRNA基因表达,其中以31.2μm o l/L效果最佳。
To measure the expression of TGF-β gene mRNA in cartilage cells, the quantitative reverse transcription-polymerase chain reaction（RT-PCR） was constructed. Cartilage cells were cultured with 15% blood serum media supplemented with 0, 15.6, 31.2,62.5 μmol/L Cu in 90 mm culture plate. The total RNA in cartilage cells was extracted, TGF-β-cDNA was amplified and sequenced, and the expression of TGF-β gene mRNA was measured by quantitative RT-PCR. The results were as follows., sequence of the cloned TGF-β gene was 99.4% identical to that in GenBank. The expression of TGF-β gene mRNA increased in culture media added with final concentration of 15.6, 31.2,62.5 μmol/L Cu. In this study,the optimal copper concentration and optimal culture times for the expression of TGF-β gene mRNA were 31.2 μmol/L and 48 h, respectively.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2005年第5期508-510,共3页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(39970567
30170713)
