pcDNA3.1/ASIC1a真核表达载体的构建及其在佐剂性关节炎大鼠关节软骨细胞中的表达

Construction of pcDNA3.1/ASIC1a eukaryotic expression vector andits expression in articular cartilage cells of adjuvant arthritis rats

目的通过构建真核表达pcDNA3.1/ASIC1a质粒,转染佐剂性关节炎(AA)大鼠关节软骨细胞,建立酸敏感通道在关节软骨细胞中过表达的模型,观察ASIC1a mRNA及其蛋白的相对表达。方法通过PCR扩增目的基因ASIC1a,并用XbaⅠ和BamHⅠ进行双酶切,同时用这两种酶双酶切质粒pcDNA3.1,将其酶切产物按常规方法连接并转化入大肠杆菌,挑去菌落培养,提取质粒,进行酶切鉴定及测序,然后用Lipofectamine 2 000转染试剂盒将所构建质粒转染入关节软骨细胞,使用荧光定量PCR、RT-PCR和Westernblot法检测ASIC1a mRNA和蛋白表达,以鉴定模型建立成功与否。结果①通过对大鼠脑组织RNA的提取及逆转录,获得ASIC1a基因,并与载体连接,通过大肠杆菌培养,获得足够多的实验用转染质粒,经酶切鉴定包含目的基因,且酶切条带准确清晰。②Lipofectamine 2 000细胞转染后,获得稳定阳性克隆细胞,通过对ASIC1a mRNA及其蛋白相对表达量进行检测,表明转染后细胞mRNA及蛋白量表达均相对上升。结论成功构建了pcDNA3.1/ASIC1a重组表达载体,并用于观察酸敏感离子通道表达水平对关节软骨细胞代谢的影响。

Objective To construct eukaryotic expression vector of acid sensing ion channel-la （ASIC1 a）, and transfeet it into the articular cartilage cells of adjuvant arthritis （AA） rats to make the model of overexpression of ASIC1 a. Methods Through PCR, recyling and purification of gelatin, connecting with vectors, translation into agarose eleetrophoresis, competent bacteria, the collection of plasmid and the identification of double enzyme cutting to contrast the plasmid to be used for transfection. We used lipofectamine 2 000 transfection reagent to transfeet the plasmid into the articular cartilage cells, then screened out the masculine clone with G418. We also determined the relative expression of the mRNA and protein of ASICla by fluorescence quantitative PCR and immunocytoehem- istry respectively to identify whether the model of overexpression was constructed successfully. Results ①We obtained the gene of ASICla by the extraction and reverse transcription of the rats＇ brains and connected it with vec- tors, then we obtained enough plasmid to be used for later experiments in colon bacillus. The plasmid was identified by enzyme cutting where the purpose gene was included, and the electrophoretic stripes were accurate and distinct. ②After the lipofectamine 2 000 transfection,we had masculine clone cells steadily. After the determination of relative expression of the mRNA and protein of ASICla, we found that the two substances both rised relatively. Conclusion The model of overexpression is constructed successfully which can be used to observe the effect on metabolism of articular cartilage ceils of the acid sencing ion channels＇ expression.