| Background:With the development of the economy and the changes in the lifestyle, more attention has been paid to ischemic disease caused by arthro sclerosis such as coronary atherosclerotic heart disease and Peripheral Vascular Diseases and the myocardial infarction and occlusion of artery became a leading cause of mortality in modern society. Despite the great development of the intervention and surgical therapy, they showed to be not effective to a portion of patients, especially those with the severe artery disease or with other disease not suitable for surgery. Thus the new therapy could be of great advantage to these subjects. The previous study showed that, the collateral circulation produced in the ischemic region could improve the prognosis. Since the slow-process could not totally satisfy the need of the ischemic part, the therapeutic angiogenesis is used to increase the concentration of local angiogenic factors to promote the angiogenesis. Now many factors were regarded to be angiogenic and could be used in the therapy, such as VEGF and FGF.The study in the function of Thymosin?4 (T?4) has already showed the important part in angiogenesis. Both the in vivo and in vitro experiment showed the effect on proliferation, migration and the tube formation of the epithelial cells and the homing and differentiation of the epithelial progenitor cells to promote angiogenesis while the low-express could significantly affect the process. The VEGF, PI3K, and ERK signal pathways have been identified to be involved in the function of T?4, but the specific process was still unclear. Also there was none relative study on the relationship between the T?4 produced by the ischemic part and the development of the angiogenesis.Notch signal pathway is highly conserved in the development and plays an important part in the cell differentiation, proliferation, migration, arterio-venous differentiation and organofaction. The absence of Notch could significantly influence the process of angiogenesis, and lead to the decrease of the functional vascularization. in which the Notch may interact with the VEGF. Since the function of Notch signal pathway in the promotion of angiogenesis of T?4 was little known, more researches are needed.Objects:The research was aimed at studying the effect of the Notch signal pathway in the tube formation and migration of the HUVECs enhanced by T?4, and the relationship between T?4 and the neovascularization in vivo.Methods:In the in vitro experiment. HUVECs was used. First HUVECs were treated with the T?4 with different concentration (0,0.1,0.5, 1ug/ml) and different time (0,4H,8H, 12H,24H), then Western blot was used to detect the expression of cleaved-notch1 and notch4. DAPT-a notch inhibitor was used to inhibitor the expression of notch, and vascular endothelial growth factor (VEGF) and Vascular Endothelium Catherin (VE-Catherin) were detected. Then the tube formation was tested with the growth factor reduced matrigel to investigate the effect of Notch. The separated lymphocyte using the density gradient centrifugation was added to the upper compartment in 0.1 ml of free-serum medium, and 0.6 ml of free-serum medium was added to the lower compartment.6H later, the cells migrated to the lower compartment were collected and counted, and the transwell was used to detect the affect of the Notch in the effect of the T(34 on the cell-cell junction and the permeability of the HUVECs. We designed to synthesis the siRNA to interfere with the express of the Notch1 and Notch4 respectively, and then the siRNA was transfected into the HUVECs.24H later, tube formation in the growth factor reduced matrigel and transwell test were done again to study the difference between the two transfected groups. In the animal in vivo experiment, C57 wide type mice within 6-10 weeks were studied. We established the hind limb ischemia model with the ligation of the proximal portion of the right femoral artery and the superficial and deep branches, and the sham procedure was done as. the dissection of the femoral vessels without ligation. The blood was collected in the cardiac apex and the gastronomies muscle was harvested at the pre operation, post-1d, post-1w, and post-2w. The enzyme-linked immunospecific assay (Elisa) was used to detect the level of the T?4 in the serum, western blot to examine the expression of the VEGF, and immunohistochemisty to evaluate that of the CD31 as a marker of the neovessels. In the clinical study,275 consecutive subjects underwent coronary angiography were recruited and given the written informed content. All the subjects were divided into three groups according to the angiograms and collateral formation:the control group,85 subjects whose angiograms showed no visible diseased vessel without collateral circulation; poor collateral group,103 subjects with at least?1 coronary stenosis of?75% without visible collateral circulation; good collateral group,87 patients with at least?1 coronary stenosis of?75% with well-developed collateral circulation. Collateral score were recorded according to the Rentrop classification. Then the blood was collected before the angiography, and all the samples were examined by the Elisa for the level of the T?4.Results:After the treatment of Tp4 with a different concentration and time, the expression of the cleaved-notchl and notch4 was increased, obviously at the concentration of 500ng/ml at 24h, then when DAPT was used to inhibit the activity of Notch, the expression of VEGF detected by Western blot was significantly decreased with the treatment of T?4, whereas with the affect of T?4, the expression of the VE-cadherin was obviously reduced no matter with the DAPT or not. The tube formation test showed that with the treatment of T?4 in the matrigel without growth factor, the tube formation ability was enhanced and reduced with DAPT. In the transendothelial test, lymphocytes that could trans to the lower compartment were significantly reduced in the DAPT treated group and increased in the only T?4 group. The western blot was used to determine the expression of the Notchl and Notch4 of the HUVECs transfected with the siRNA, and the siRNA with the highest inhibition ratio was selected. After the transfection, the HUVECs grew in the matrigel and was treated with TP4, and the results showed that the transfected groups were with a obviously reduced ability of the tube formation compared with the negative control group added with T?4, but still enhanced compared with normal group and the two transfected groups showed no significant difference. At the same time, the transwell test also revealed that the transfected HUVECs was with a significant decrease in the number of the lymphocytic that could trans to the lower compartment compared with the only T?4 group. In the in vivo experiment in mice, the serum level of the T?4 detected by Elisa showed that the level of T?4 began to increase in the post-1d and to last to the post-1w with a high level, then the western blot detected the expression of the VEGF showed that the level of the VEGF of post-1d and post-lw were increased and of the pos-2w was significantly reduced compared with post-1d and post-1w but still increased compared with the normal group (p<0.05), and the immunohistochemisty showed that the CD31 in the post-1d and post-1w group significantly increased and in the post-2w was significantly decreased compared with these in the post-1d and post-1w, but still expressed. In the clinical study, the serum level of T?4 in the poor collateral group was significantly lower than that in the good(1024.50±516.92ng/ml,1373.00±1082.88 ng/ml), and the multiple ordinal logistic regression showed the level of T?4 was a strong independent association factor with the collateral Rentrop score(p=0.035).Conclusions:1. After the treatment of T?4 on the HUVECs, the expression of Notch1 and Notch4 was affected and both were increased.2. Both Notchl and Notch4 joined in the progress of the tube formation of the HUVECs induced by the T?4, and the effect decreased and still existed when the express of Notch was inhibited.3. Migration of the HUVECs enhanced by T?4 was significantly reduced when the Notch activity was blocked by the DAPT. VEGF and VE-cadherin was significantly reduced after the inhibition of Notch with the treatment of T?4.4. The neovascularization in the hind limb ischemia model was obviously correlated with the express of the T?4 and VEGF, and the effect of the vascularization of the may be related to the express of VEGF. |
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