The Effects And Mechanism Of TIPE2on Expression Of TF In Human Umbilical Vein Endothelial Cell

BackgroundIn recent years, the incidence of the coronary heart disease, atherosclerosis, hypertension, hyperlipidemia and other cardiovascular disease rising gradually on a global scale, and the age of onset is obviously earlier, which is a serious danger to the mankind. Many studies suggested that the occurrence of cardiovascular disease is caused by the long time reaction of genetic factors and environmental factors. But the specific mechanism of cardiovascular disease still has been not fully elucidated.Numerous studies have shown that endothelial injury, inflammatory reaction and abnormal activation of the coagulation system could lead to the occurrence and development of cardiovascular disease. Vascular endothelial cells play an important role on maintaining the balance of intravascular and vascular tone and the preventing thrombosis. High blood glucose, insulin resistance and oxidative stress, inflammatory reaction and other factors can cause endothelial injury. The endothelial injury may aggravate the inflammation, and activate the coagulation system which could lead to thrombosis. In turn, the activation of coagulation system could further aggravate the inflammatory process, which leads to endothelial dysfunction and atherosclerosis. Tissue factor(TF) is the start activation factor of exogenous coagulation system, and a key hub of the coagulation-inflammation of the network. The abnormal expression of TF can lead to abnormal activation of the coagulation system, in turn, lead to the occurrence of blood clots, and aggravate inflammation process. Studies have shown that lipopolysaccharide (LPS) and tumor necrosis factor alpha inflammatory factor(TNF a) could induce the expression of TF on endothelial cells and monocytes through activating MAPK signal pathway and NF-kB, AP-1, Egr-1of transcription factors, etc. Besides TF, other inflammatory factor such as ICAM-1, IL-6also play an impotant role in promoting cardiovascular diseases. So that the repair of endothelial damage and inhibit the effect of TF are important targets for the therapy of cardiovascular diseases.TIPE2(tumor necrosis factor alpha induced protein and the like-2, TIPE2) belongs to the TNFAIP8(tumor necrosis factor alpha induced protein-8) family, mainly expressed in the lymphoid tissue and inflammatory tissue, is a newly inflammation negative regulatory factor, while could maintain the balance of endogenous immune and adaptive immune system. Studies showed that, TIPE2may regulate the response of macrophage on oxLDL mediated inflammatory reaction and the information of atherosclerotic plaque was increased in TIPE2deficient mice.In addition, TIPE2could inhibit the MAPK and NF-kB signal pathway, which decrease the activation of AP-1in macrophage treated with LPS.Althoug TIPE2is the negative regulatory factor of inflammation which could inhibit the MAPK pathway and the expression of TF is regulated by MAPK pathway, the relationship between TIPE2and TF is not clear. Whether TIPE2could regulate the expression of TF is still unknow.In recent years, Lentiviral Vectors has been a commonly gene transferation tools, which can effectively integrate the exogenous gene into the host chromosome, which could be expressed in the host for a long time. The transduction of Lentiviral Vectors was safe and high efficient, so that it become the current hot topics in the study of gene transfer vector.The preliminary data suggested that HUVEC could express little TIPE2protein, so that we could study the gene function of TIPE2through overexpression of TIPE2. Based on the above background, the purpose of this study was constructing the recombinant lentiviral vectors encoding human TIPE2gene and obeserved the effect of TIPE2Lentiviruses on the expression of TF and related mechanism in the HUVEC, thus to further understanding the mechanism of cardiovascular diseases and providing theoretical basis for the establishment of the prevention and treatment strategy of cardiovascular diseases.Part1The construction and identification of Recombinant Lentiviral Vectors encoding Human TIPE2GeneObjectiveTo establish the Recombinant Lentiviral Vectors encoding Human TIPE2gene and provide the platform for follow-up study about TIPE2gene function research.Methods1. The establishment of the TIPE2overexpression lentiviral vectors:specific primers targeting TIPE2was designed, PCR techniques was used for amplification of TIPE2. The purified PCR products was used for Homologous recombination with Linearized GV287plasmid vector after digestion by AgeI/AgeI enzyme. Then the connected product was used for bacterial conversion, coated board, picking, amplification cultivation cloning, extraction of plasmid, and then identification by Agarose gel electrophoresis and sequencing.2. The packaging of GV287-TIPE2lentiviral vectors:The GV287vectors and plasmid pHelper1.0, pHelper2.0were cotransfected to the293T cells to package the lentivirus particles.3. The mesurement of Virus particles concentration:the lentivirus particles were harvested and concentrated, the Real-time quantitative PCR was used to detect virus concentration.Results1. TIPE2gene and the GV287vector were successfully connected, sequencing results matched with the target sequence.2. TIPE2-GV287lentiviral vectors particles was successfully package, the concentration was2.00E+8TU/ml.Conclusions1. TIPE2gene overexpression lentivirus vectors GV287-TIPE2was successfully established2. GV287-TIPE2lentivirus vector could be used for the next part of the experimental study.Part2The effects and Mechanism of TIPE2Lentiviruses on Expression of Tissue factor in Human Umbilical Vein Endothelial CellObjectiveTo explore the relationship between TIPE2and TF for further understanding the mechanism of cardiovascular diseases and providing theoretical basis for the establishment of the prevention and treatment strategy of cardiovascular diseases.Methods1. Human umbilical vein endothelial cell line culture:cells were cultured at37℃in a humidified5%CO2incubator supported with high sugar DMEM medium containing10%fetal bovine serum.2. The TIPE2protein expression of HUVEC cells and THP-1cells were detected by western blot and compared each other.3. HUVEC cells were stimulated by LPS of different concentration and the expression of TIPE2and related inflammatory factors were detected. Group as follows:①CON②0.1μg/ml③0.25μg/ml④0.5μg/ml⑤1μg/ml; the mRNA level of TIPE2, TF and ICAM-1, IL-6were detected by real time quantitative PCR method(RT-qPCR); the protein level of TIPE2, TF were detected by western blot.4. HUVEC cells were treated by LPS at different time points and the expression of TIPE2and related inflammatory factors were detected:Group as follows:①CON②30min③1h④4h⑤8h⑥12h; the mRNA level of TIPE2,TF and ICAM-1,IL-6were detected by RT-qPCR method; the protein level of TIPE2, TF were detected by western blot.5. HUVEC cells were infected with lentiviral vectors. Based on the best infection index (MOI), HUVEC cells were infected with GV287-TIPE2lentiviral vectors and negative control lentiviral vectors separately, then the TIPE2protein level were detected by western blot.6. The proliferation of HUVEC cells after infected with lentiviral vectors were tested by CCK8.7. The effect of TIPE2overexpression on TF、ICAM-1、IL-6in HUVEC cells. Group as fllows:①NC②TIPE2③NC+LPS④TIPE2+LPS; TF、ICAM-1、IL-6mRNA level were detected by RT-qPCR method; the protein level of TIPE2, TF were detected by western blot.8. The p38MAKP, ERK1/2signal pathway were detected by western blot after TIPE2overexpression on HUVEC cells.Results1. There was little TIPE2protein expressed on HUVEC cells.2. HUVEC cells were treated with LPS of different final concentration. RT-qPCR detection results showed that the TIPE2mRNA levels of all groups treated with LPS were significantly lower than the control group, seperately (p<0.05); Western blot results showed that TIPE2protein levels of all groups treated with LPS were lower than the control group.3. HUVEC cells were treated with LPS at different time points. RT-qPCR detection results showed that the TIPE2mRNA levels of all groups treated with LPS were significantly lower than the control group, seperately (p<0.05); Western blot results showed that TIPE2protein levels of g all groups treated with LPS lower than the control group.4. HUVEC cells were treated with different final concentration of LPS. RT-qPCR detection results showed that the TF and IL-6mRNA levels of all groups treated with LPS were significantly higher than the control group, seperately (p<0.05); ICAM-1mRNA level of group0.5μg/ml and group lμg/ml were significantly higher than the control group, seperately (p<0.05); Western blot results showed that TF protein level of group0.25μg/ml,0.5μg/ml and group1μg/ml were higher than the control group.5. HUVEC cells were treated with LPS at different time points. RT-qPCR detection results showed that the TF mRNA levels of group4h、group8h were significantly higher than the control group, seperately (p<0.05), the ICAM-1mRNA levels of group4h, group8h, group12h were significantly higher than the control group, seperately (p<0.05); IL-6mRNA level of each group treated with LPS excepted group12h were significantly higher than the control group, seperately (p<0.05); Western blot results showed that TF protein level of all group treated with LPS were higher than the control group.6. HUVEC cells were successfully infected with TIPE2-GV287lentiviral vectors,Western blot results showed that the TIPE2protein level of experimental group increased significantly. 7. CCK8results showed that the cell growth rate of overexpression TIPE2group were higher than the group NC at48h and72h time points,separately.8.The mRNA basal level of TF、ICAM-1、IL-6of the overexpression TIPE2group were significantly lower than the group NC (p<0.05); When treated with LPS, the TF mRNA level of the group (TIPE2+LPS) was downregulated; but there was no effect on ICAM-1and IL-6when combinated with LPS stimulation.9. Western blot results showed that TIPE2could inhibits the TF expression with and without LPS stilmulation.10. Western blot results showed that compared with the group NC, the phosphorylation p38of the group TIPE2were decreased at15min,30min,60min time points when treated with LPS, while the phosphorylation ERK1/2were significantly increased.Conclusions1. HUVEC cells expressed little TIPE2protein, LPS could significantly decrease the expression of TIPE2.2. LPS could increase the expression of TF, ICAM-1and IL-6.3. HUVEC cells were successfully infected with Lentiviral vector GV287-TIPE2, the expression of TIPE2increased significantly.4. TIPE2could inhibit the expression of TF with or without LPS. TIPE2could only inhibit the basal level of ICAM-1and IL-6.5.TIPE2could regulate the expression of TF through regulating p38MAPK and ERK1/2signal pathway.