| Disease of cardiovascular system is one of the diseases with the highest incidence on the world, and is the leading cause of death. The major treatment to it is the transplantation of blood vessel. There are 570,000 patients accepting transplantation of coronary artery alternative pathway blood vessel only in America. And a great deal of peripheral vascular transplantation is needed in operation of blood alternative pathway. Therefore the number of small caliber blood vessel graft is needed larger and larger. But the applying of small caliber blood vessel grafts in clinic is restricted evidently because thrombus is formed easily in these grafts at acute stage. At the present hotspot is constructed of blood vessel in the study of vascular tissue engineering, which the diameter is less than 6 millimeter. The most important problem needed to be solved is the construction of blood vessel graft in vitro, which can counteracted against the blood flow-shear stress and thrombogenics. It can be assured that the immunological rejection can not take place and the blood vessel is expedite after transportation of the grafts. In this thesis, seeded cell is human umbilical vein endothelial cell (HUVEC). Substrate materials are gelatin-layer and rabbit common carotid artery (RCCA) of which is removed from VEC by the method of supper low temperature. Step-increased flow-shear stress is loaded with growing seed cells on the substrate material. The object of this experiment is to research tolerated level of EC to flow-shear stress.Major content and result in this thesis:(1) HUVEC of umbilical cord of new baby from the gift of Chongqing hospital of Cancers is cultured in vitro by the improved digest method, and is. identified by morphology and immunohistochemistry. The result indicates that the ratio of Factor VIII related antigen positive cell is more than 95% in HUVEC, and it can be used in experiments.(2) RCCA is treated by super low temperature. The result shows that VEC of RCCA is removed completely, without the damage of the internal elastic membrane of RCCA.(3) Two kinds of flow culture model, i.e. pulsing laminar flow culture model and the system of perfuse-flow chamber, are constructed on the ground of traditional flow chamber.(4) The loading pattern is firstly designed, by the step increased flow-shear forceloaded successfully on HUVEC growing on gelatin substrate. The relation of adhesiveness of HUVECs with time and intensity of shear stress is studied. Results are as the following:(a) HUVECs fall off evidently when shear stress is less than 5 dyne/cm2. This means that tress at lower level flow-shears goes against adhesion of endothelial cells.(b) Adhesion of HUVEC on the gelatin substrate, when shear stress loaded is with step-increased, i.e. 5dyne/cm2 (5h) 7.5dyne/cm2 (10h) 10dyne/cm2 (24h), is stronger than that when shear stress loaded is always same, i.e. 7.5dyne/cm2or 10dyne/cm2.(c) Tolerance of HUVEC to shear stress is lower than physiological level (40 dyne/cm2) after they grow on the gelatin substrate.(5) HUVECs are planted in the blood lumen of RCCA in which endothelial cells are removed and continue to be cultivated at static state in vitro. After 3 days, it is found that HUVEC grow with single layer. HUVECs can normally grow on the surface of different kinds of blood vessel material in terms of the staining result and pictures of SEM.(6) HUVECs grow on lumen layer of RCCA are cultured by the method of habituated culture and the tolerated flow-shear stress was loaded with the step-increased flow-shear stress. Some results are found to:(a) The tolerated capability of HUVECs to flow-shear stress, growing on the substrate of RCCA, is much stronger than that of HUVECs, growing on the gelatin substrate, from 5dyne/cm to 30dyne/cm .(b) Adhesion of HUVECs on the gelatin substrate, loaded in step-increased, at 5dyne/cm2 (5h) 10dyne/cm2 (5h), 20dyne/cm2 (5h) and 30dyne/cm2(24h), respectively is stronger than that when shear stress loaded is always same, i.e. 5dyne/cm2 (5h),10dyne/cm2... |
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