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目的 重组TPA基因并构建体外表达模型 ,为缺血性心脏疾病的基因治疗及防止血管再狭窄奠定基础。方法 构建真核表达载体pcDNA3 1( +)TPA ,然后将pcDNA3 1( +)TPA转入中国苍鼠卵巢 (CHO)细胞 ,并观察外源性TPA表达情况。结果 真核表达载体pcDNA3 1( +)TPA在CHO细胞表达量好 ,发色底物法测得外源性TPA活性为 12 2 96IU/ 10 6细胞 / 2 4hr ,未转pcD NA3 1( +)TPA的CHO细胞测得为 3 176IU/ 10 6细胞 / 2 4hr ;酶联免疫实验 (ELASA)检测结果为 5 86 172ng/ 10 6细胞 / 2 4hr ,未转pcD NA3 1( +)TPA的CHO细胞测得为 9 60 8ng/ 10 6细胞 / 2 4hr ,达未转TPA基因组的 60倍。结论 pcDNA3 1( +)TPA转入CHO细胞后 ,外源性TPA基因获有效表达 ,为TPA临床基因治疗提供了理论依据
Objective To construct TPA gene and construct an expression model in vitro to lay a foundation for gene therapy of ischemic heart disease and prevent restenosis. Methods The eukaryotic expression vector pcDNA3 1 (+) TPA was constructed. Then pcDNA3 1 (+) TPA was transfected into Chinese ovariectomized CHO cells and the expression of exogenous TPA was observed. Results The eukaryotic expression vector pcDNA3 1 (+) TPA expressed well in CHO cells. The exogenous TPA activity was 12 2 96 IU / 10 6 cells / TPA CHO cells measured 3 176 IU / 106 cells / 24 hr; ELASA test results 5 86 172 ng / 106 cells / 24 hr CHO cells transfected without pcD NA3 1 (+) TPA Measured at 9 60 8ng / 10 6 cells / 2 4hr, up to 60 times the TPA genome. Conclusion The transfection of pcDNA3 1 (+) TPA into CHO cells effectively expresses the exogenous TPA gene and provides a theoretical basis for the clinical gene therapy of TPA