Construction Of PEGFP-N3-KLF2 Eukaryotic Vector And Impact On LPS-induced TF Expression In THP-1

Acute arterial thrombosis is a major cause of myocardial infarction and cerebral infarction,which are the most common cause of death in developed countries. Tissue factor (TF) is a physiological hemostasis and pathological thrombosis of the initiatior, which was paid more and more attention in the role of thrombosis. As TF source cells of atherosclerotic plaques, the precursor of foam cells is. The mechanism of TF expression and regulation in mononuclear cells has become one of the hot of clinical medicine.Many studies of domestic and foreign show that patients with such as unstable angina, acute coronary syndrome, hypertension and hyperlipidemia cardiovascular and cerebrovascular diseases, whose mononuclear cells in blood are in the high TF expression level.Also found KLF2 play a role in anti-thrombosis and protection endothelial and the expression of KLF2 of mononuclear cells in patients with acute coronary syndrome was significantly reduced. Therefore, this study aimed to construct pEGFP-N3-KLF2 overexpression vector, and to observe the effect of on KLF2 overexpression vector-transfected THP-1 cells expressing TF induced by LPS. Provides a wealth of new experimental data for the coagulation theory.Objective:To construct pEGFP-N3-KLF2 overexpression vector, and to observe the effect of on KLF2 overexpression vector-transfected THP-1 cells expressing TF induced by LPS.Methods:Obtained klf2 gene sequence from the NCBI. Primers were designed with premier5.0 software, PCR amplification the clone of klf2. Which was electrophoresed correctly. After purified, ligand the purified product with pEGFP-N3 vector. Pick monoclonal bacteria and abstract plasmid. Final ly, tested by PCR and double digestion, when the two results are correct, send pEGFP-N3-KLF2 fresh broth to sequencing. Mononuclear cells will be divided into 5 groups:①normal cell group,②cells transfected with empty plasmid group,③cells transfected with overexpression plasmid group,④LPS group,⑤LPS+transfected with overexpression plasmid group. Cells Were after cultured 2 h,12 h,24 h, 48 h and 72 h, and the detected TF protein expression by flow cytometry at different time points in each group.Results:The positive clone screening and sequencing results showed that pE-GFP-N3-KLF2 overexpression vector was constructed successfully. Observed in the experiment 2 h to 72 h, the normal cells TF expression at different time points consistent between the difference observation points was no significant difference; compareed with the normal control group, TF expression in empty vector transfected group and the overexpression plasmid was also no significant difference (P> 0.05). TF expression in LPS group significantly increased starting from 12 h, reach the peak in 48 h, compared with the control group, the difference was statistically significant (p<0.01), and LPS+ overexpression of transfected group was significantly lower TF expression than LPS group at the same time points, the difference was significant (P<0.01).CONCLUSION:①Constructed plasmid With green fluorescent protein landmark pEGFP-N3 vector. With the result of sequencing pEGFP-N3-KLF2,KLF2 overexpression plasmid was constructed successfully.②LPS significantly stimulated expression of TF in THP-1, but this effect was nhibited by transfection of overexpression plasmid of KLF2.