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目的 建立稳定表达人组织型纤溶酶原激活剂(tissue-type plasminogen activator,t-PA)的细胞系。方法将t-PA的反义DNA(t-PA cDNA)连接在真核表达载体pcDNA3.1(+)的Kpn I和Xba I酶切位点上,构建成重组质粒pcDNA3.1(+)t PA。用FuGENE6介导法转入人类AGZY83-a细胞系中,经G418筛选形成阳性克隆,接种于培养基中培养,检测表达产物。结果 各种酶切图谱及筛选后单克隆形成,证明tPA cDNA已正确组装在载体上,培养液中rt-PA的含量高于对照组且可持续12周,表明pcDNA3.1(+)tPA已可在AGZY83-a细胞中得到表达。结论 转染tPA cDNA的AGZY83-a细胞已可稳定表达人类tPA,为血栓性疾病的基因治疗奠定了一定的基础。
Objective To establish a cell line stably expressing human tissue-type plasminogen activator (t-PA). Methods The t-PA antisense DNA (t-PA cDNA) was ligated into the Kpn I and Xba I restriction sites of the eukaryotic expression vector pcDNA3.1 (+) to construct a recombinant plasmid pcDNA3.1 (+) t PA. The recombinant plasmids were transfected into human AGZY83-a cell lines by FuGENE6-mediated method and screened by G418 to form positive clones. The positive clones were inoculated into the medium and cultured, and the expression products were detected. Results After digestion and cloning, the tPA cDNA was correctly assembled on the vector. The content of rt-PA in the culture medium was higher than that of the control group for 12 weeks, indicating that pcDNA3.1 (+) tPA It can be expressed in AGZY83-a cells. CONCLUSION AGTY83-a cells transfected with tPA cDNA can stably express human tPA, which has laid a foundation for the gene therapy of thrombotic diseases.