Wu Zhong-Jun, Yang Su-Fen, Zheng Shu-Sen, Shi De, Wu Wei-Wei, Li De-Wei
Organ Transplant Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.
Hepatobiliary Pancreat Dis Int. 2004 Nov;3(4):511-5.
Tissue-type plasminogen activator(tPA), which is highly efficient and specific in resolution of thrombus, could significantly improve the survival rate and life-quality of the patients with thrombosis. This study was designed to clone human tPA gene, construct eukaryotic expression plasmid pcDNA3.1(+)/tPA, and evaluate their biological activities.
The tPA gene was obtained from dead human heart tissue with reverse transcriptase-polymerase chain reaction (RT-PCR). It was cloned to eukaryotic expression plasmid pcDNA3.1(+) by recombination strategy. The eukaryotic expression plasmid pcDNA3.1(+)/tPA was identified by restriction enzyme digestion and sequenced. The pcDNA3.1(+)/tPA was transfected into vascular smooth muscle cell (VSMC) by using lipofection. The tPA expression level was detected by Northern blot and dot blot, and the protein biological activities of tPA were detected by the fibrin plate technique.
The tPA gene was cloned and pcDNA3.1(+)/tPA was constructed. The tPA expression levels of mRNA and protein were highly increased after pcDNA3.1(+)/tPA transfected into VSMC, and the expression of tPA protein showed evident biological activities.
The present study has laid a foundation for further animal experiment in treating thrombus in transplanted organ by tPA gene transfection.
组织型纤溶酶原激活剂（tPA）在血栓溶解方面具有高效性和特异性，可显著提高血栓形成患者的生存率和生活质量。本研究旨在克隆人tPA基因，构建真核表达质粒pcDNA3.1(+)/tPA，并评估其生物学活性。
采用逆转录聚合酶链反应（RT-PCR）从人死亡心脏组织中获取tPA基因。通过重组策略将其克隆至真核表达质粒pcDNA3.1(+)。通过限制性内切酶消化和测序鉴定真核表达质粒pcDNA3.1(+)/tPA。利用脂质体转染法将pcDNA3.1(+)/tPA转染至血管平滑肌细胞（VSMC）。通过Northern印迹和斑点印迹检测tPA表达水平，采用纤维蛋白平板技术检测tPA的蛋白生物学活性。
成功克隆tPA基因并构建了pcDNA3.1(+)/tPA。将pcDNA3.1(+)/tPA转染至VSMC后，tPA的mRNA和蛋白表达水平显著升高，且tPA蛋白表达显示出明显的生物学活性。
本研究为进一步通过tPA基因转染治疗移植器官血栓的动物实验奠定了基础。
