[Construction of expression vector for recombinant annexin II and characteristics of its fibrinolysis].
The study was designed to investigate annexin II resulting in molecular pathological mechanism of the primary fibrinolysis and establish annexin II vector model for further research on disturbance of coagulation. A target gene was amplified from human umbilical vein endothelial cells (HUVEC) by RT-PCR. Annexin II gene fragment was purified and ligated with molecular biological recombinant technology. The recombinant of plasmid annexin II was transfected into HL-60 cells and its distribution in the cell and structure characteristics of annexin II protein were evaluated by multi-photon excitation laser scanning microscope. By means of flow cytometry (FCM) and Werstern blot technique, the protein expression was qualitatively and quantitatively analyzed. Transfected cells were treated in vitro with annexin II antisense oligonucleotide (AS) targeting to the start site of annexin II cDNA. The results showed that the recombinant pZeoSV2(+)/ANN II was constructed successfully and expressed in HL-60 cells. The protein expression was distributed on the surface of cell by fluorescence assay. After transfection for 48 hours, the cells occurred higher level of expression. The level of the plasmin was significantly enhanced in the present of annexin II. The FCM and Western blot analysis showed the annexin II expression was similar both in transiently and stably transfected in HL-60 cells. Annexin II antisense oligonucletide and McAb significantly inhibited the activity of plasminogen. It was concluded that annexin II plays an important role in the fibrinotysis. Annexin II vector was defined as a expression tool for further studying fibrinolysis and coagulopathy in malignant disease.
Zhang
Xiao-Hui
XH
Department of Hematology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. zhangxh8080@hotmail.com
Zhou
Hua-Rong
HR
Hu
Yu
Y
Wei
Wen-Ning
WN
Yang
Lin-Hua
LH
Shen
Guan-Xin
GX
Qiao
Zhen-Hua
ZH
Song
Shan-Jun
SJ
D017306:Annexin A2 / Q000235:genetics* / Q000502:physiology; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology; D005342:Fibrinolysis; D005822:Genetic Vectors; D018922:HL-60 Cells; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis
D017306:Annexin A2; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins