人纤溶酶原激活物抑制物1转基因小鼠的建立

　　摘　要：目的建立人纤溶酶原激活物抑制物1(PAI-1)转基因小鼠。方法利用构建的高效真核表达载体pCAGGS—PAI-1，制备注射用DNA。以显微注射法将目的DNA注入小鼠受精卵，再将受精卵移植到受体母鼠，制备转基因小鼠。经鼠尾组织DNA提取、PCR筛选、Southern杂交、DNA序列测定对转基因小鼠进行鉴定。结果对获得的29只小鼠经PCR筛选、Southern杂交分析、DNA序列测定，得到了2只整合了人PAI-1cDNA的转基因小鼠。在刚出生的转基因小鼠尾部及后肢发现出血性改变，免疫组织化学观察到在，肾脏局部PAI-1表达稍有增高。结论本研究中，我们建立了人PAI-1基因转基因小鼠，为进一步研究PAI-1在肾脏疾病中调控细胞外基质降解的机制提供了动物模型，为研究利用抗凝、促纤溶疗法治疗慢性肾炎奠定了基础。

Construction of mice transgenic for human plasminogen-activator inhibitor type 1

LIU Shuwen ,CHEN Xiangmei ,Ye Yizhou

　　(Division of Nephrology,General Hospital of PLA,Key Laboratory and Kidney Center of PLA,Beijing 100853,China)

　　Abstract：Objective To construct mice transgenic for human plasminogen-activator inhibitor type 1 (PAI-1),and to probe the role and mechanism of PAI-1 in the synthesis and degradation of extracellular matrix in kidney diseases.Methods The eukaryotic expression plasmid pCAGGS-PAI-1 was digested with Pvu Ⅰ and Hind Ⅲ,recovered using agarose-gel electrophoresis,and then purified by using CsCl density-gradient ultracentrifugation.DNA was microinjected into male pronuclei of fertilized mouse eggs,the injected mouse eggs were then transplanted into the oviduct of pseudopregnant female mice.The offsprings were screened by using polymerase-chain reaction,and then identified by Southern-blot analysis and DNA sequencing.Results Two of twenty-nine survived offsprings were identified to be mice transgenic for human PAI-1.The mice transgenic showed subcutaneous hemorrhage at the end of the tail and hind limb.Immunohistochemical examination showed slightly elevated PAI-1 expression in the kidney.Conclusion Mice transgenic for human PAI-1 were constructed,providing an animal model for further investigations of the role and mechanism of PAI-1 in the pathogenesis and progression of kidney diseases.

　　Keywords：Plasminogen-activator inhibitor type 1;Transgenic mice

　　参考文献：

　　［1］Vassalli JD,Sappino AP,Belin D.The plasminogen activator/plasmin system.J Clin Ivest,1991,88:1067-1072.

　　［2］Sprengers ED,Kluft C.Plasminogen activator inhibitors.Blood,1987,69:381.

　　［3］Waltz DA,Sailor LZ,Chapman HA.Cytokines induce urokinase-dependent adhesion of human myeloid cells.J Clin Invest,1993,91:1541-1552.

　　［4］Gyetko MR,Todd RF,Wilkinson CC,et al.The urokinase receptor is required for human monocytc chemotaxis in vitro.J Clin Invest,1994,93:1380-1387.

　　［5］Dano K,Andreasen PA,Grondahl HG,et al.Plasminogen activator,tissue degradation and cancer.Adv Cancer Res,1985,91:139-266.

　　［6］Liotta LA.Cancer cell invasion metastasis.Sci Am,1992,266:54-59.

　　［7］Erickson LA,Fici GJ,Lund JE,et al.Development of venous occlusions in mice transgenic for the plasminogen activator inhibitor-1 gene.Nature,1990,346:74-76.

　　［8］Hogen B,Beddington R,Lacy E.Manipulating the mouse embryo.A laboratory mannual.2nd ed.Cold Spring Harbor Laboratory Press,1994.

　　［9］颜子颖,王海林译.精编分子生物学实验指南.北京:科学出版社,1998.