| BACKGROUND:Pathological angiogenesis is a kind of expression in many eye diseases, for example:corneal neovascularization(CNV), age-related macular degeneration (AMD), retina new blood vessels(RNV), diabetic retinopathy (DR), Neovascular glaucoma (NVG).In European and American countries, neovascularization has become the primary factor which lead to blindness. China as a developing country, CNV which is because of ocular injury has become a common eye diseases, accounting for about10%of the corneal diseases. And the consequence of the chemical corneal injury of corneal alkali burns is particularly serious, which now is become one of the most prominent problems that results to blindness. Some diseases have a close relation to CNV:such as infection, ocular trauma, corneal chemical injury, eye disease immunity, and so on, Which caused by alkali in burn is closely related to the vision.Although the growth of CNV is to the benefit of cleaning the infected, wounding healing, inhibiting corneal dissolution, repairing tissues, the normal corneal is smooth, transparent and have no blood vessels. However it is easy to produce plasma leakage, corneal edema, when the structure and function of cornea is imperfect, which would change the cornea "immune exemption" permissions and influence cornea transplant and let the light through the cornea, and would cause visual function decline.As the development of science and technology, as well as further studies to the CNV, a large number of experimental studies had found that the many factors involved in formation of CNV:1. Vascular endothelial growth factor(VEGF):VEGF is found by Ferrara in1989that is specific to promote mitotic original of the endothelial cells, which need specific receptor-mediated acts on the vascular endothelial cells, playing a role in promoting differentiation and chemotaxis to the vascular endothelial cells, and also promoting the proliferation and migration.Besides it can increase capillary permeability, so as to help the growth of new blood vessels and stromal cells. Thus it has an important sign-ificance of VEGF in promoting angiogenesis, maintaining blood vessels form and integrity.2. Fibroblast growth factor (FGF):FGF is a kind of promote fibroblast growth factor and exist widely in many organizations, which is found by Gospodarowicz in1974. Its biological activities need receptor-mediated, and the alkaline FGF biological is the strongest, which the main function is to the angiogenesis, and to promote vascular smooth muscle cells and endothelial cell proliferation and participate in tissue repair and inflammatory reaction.3. Interleukin (IL):Interleukin is a group of cell factors which is mainly produced by monocytes, which can play a part in the macrophage’s and lymphocytes’ proliferation,activation and differentiation, and that must also be need some specific receptors-mediated. Normally, the corneal micro environment is maintained by IL and other cell factors; But some experiments have found that IL-8expressed highly in the cornea after it was injury, which can promote angiogenesis.4. Transforming growth factor beta (TGF-β):TGF-β plays a role in cell proliferation, differentiation and apoptosis, which via the receptor signaling pathway is discovered by DeLarco in1978. Though TGF-β is found that it’s expression is on the low level to maintain normal corneal form in the normal cornea, some study found that TGF-β was high expression in the corneal new blood vessels from the cornea epithelial tissue after chemical injury. In other words, TGF-β played a role in promoting the formation of new blood vessels of cornea.5.Matrix metalloproteinases(MMPs); MMPs are a group of endogenous proteolytic enzymes relyed on Zn2+,which is mainly synthetized and secreted by fibroblasts and macrophages, endothelial cell. the main biological functions is to degradated extracellular matrix effective components, adjust film gelling, play a part in embryonic development directly or indirectly. It is suppressed in the normal tissue. Studies showed that MMPs was in a strong expression when the cornea produced new blood vessels,and was also found more in epimatrix of peripheral vascular in the new blood vessels membrane.A variety of treatment methods and treatment drug emerged nowadays, which due to the new study of mechanism research, animal experiment and clinical research about CNV.1. Against vascular endothelial growth factor drugs:Including VEFG monoclonal antibodies, VEGFR tyrosine kinase inhibitor, against vascular endothelial growth factor antibodies, etc. Bevacizumab is Used widely, which through a way of preventing VEGF from integrating to the VEGFR1and VEGFR2receptor on the surface of the endothelial cells, inhibiting the phenomenon of blood-vessels-leakaging and new blood vessels, so that to make the biological activities of endogenous VEGF failured is the main mechanism of it. And all of the VEGF a subtype are sociable to the Bevacizumab. The VEGFR tyrosine kinase domain that is activated by receptor-mediated is the starting point of the VEGF signal transduction, from which the angiogenesis procedure can be prevented effetively.2. CNV-inhibitor; numerous studies showed that the increasing of the angiogenic growth factors broke the balance between promote or restrain the formation of blood vessels of cornea, while the increasing of the angiogenic inhibitor can strengthen antiangiogenic power and rebuild the balance resulting new blood vessels disappear. Among them endostatin is the most important angiogenic inhibitor that is a kind of endogenous endothelial cell proliferation potent inhibitor which is discovered by O’Relly. Endostatin specifically acts on endothelial cells, especially microvascular endothelial cells which can inhibit migration, induce the apoptosis, and attain to inhibit the formation of blood vessels.3.glucocorticoid:it have significant anti-inflammatory and immune suppression effect which can reduce the inflammatory exudation, restrain fiber cell hyperplasia and the formation of new blood vessels. Studies have found that its restraining effection to the new blood vessels mainly rely on inhibiting endothelial cell transitions and vascularization.5. angiostatin Kl-4:it is a fibrinolytic enzyme degradation of the clips that has strong inhibit neovascularization which via restraining the vascular endothelial cells in proliferation station to induce endothelial cell apoptosis to reach, and no influence on the rest of the endot-helial cells. In brief, although treatment methods for stopping cornea new blood vessels are various, there is still no curative effect of the therapeutic method more exact and practical.The purpose of the research is what in order to determining the best concentration of the angiostatin for treatment CNV and how to preparing angiostatin solution。Objective1、To discusse the effects of the Angiostatin solution for treatment CNV in rat after alkali burning;2、The influence of Angiostatin solution to the expression CD31of CNV in rat after alkali burning is discussed. And comparing the growth inhibitory effect of different concentrations of Angiostatin solution to treatment CNV in rat after alkali burning;Method1、Culturing HUVECs in vitro was divided i into three groups (group A:control group; group B:16mg/L angiostatin; group C:32mg/L angiostatin)) to observe the expression of the VEGF and the proliferation of HUVECs was examined by MTT、RT-PCR and Western Bolt;2、The model of CNV after alkali burning in rats has been used;24rats were devided into4groups randomly, control team uses Saline, A team uses25ug/mL AS solution, B team uses50ug/mL AS solution, C team uses25ug/mL AS solution, qid The treatment continue2weeks; and the levofloxacin eye drops was used routine treatment, qid。To observe the expression of the CD31and the area of CNV was examined by MTT、RT-PCR and Western Bolt in each group.Results 1^The experiment was show which the growth inhibitory effect of Angiostatin to the HUVEC by culture experiments in vitro:angiostatin could significantly inhibit the growth of HUVECs in a dose-time-dependent manner (P<0.05)2^The area of CNV in rat were different, and group A、B、C were under group control at the indicated time points (P<0.05), but group C was obvious less than other three groups (P<0.05); After two weeks, there are amount of angiogenesis and chronic inflammatory cell in the all lays of corneal for control group rats; and the treatment groups more less than control group in different degrees.3、The expression of corneal CD31mRNA in the test groups was less than the control group’s, and it had negative correlation with concentration of AS in the test groups; The expression of CD31at each group was less than the control group (P<0.05),and the growth of CNV had negative correlation with concentration of AS.Conclusion1、It is show that angiostatin could significantly inhibit the growth of HUVECs in a dose-time-dependent manner by culture in vitro.2、The growth of CNV is dependent on the endothelial cell migration, growth, etc, when AS acting on VEGF targets, it can inhibit the growth of CNV by inhibit endothelial cell migration, generation and restructuring. AS could inhibit BRPs and endothelial cells aggregated,and make BRPs and endothelial cells lose contact, when the endothelial cells lost the sustaining of BRPs, once lost the stimulation of VEGF cell apoptosis would happen, and eventually lead to new blood vessels degradation. there is negative correlation between the inhibitive effect of CNV and concentration of AS:The higher AS concentration was, the lower the CNV3、Angiostatin is a cell factor generated when body is normal, as well as the advantage of its high efficiency, selective inhibition etc, there is a lot of potential to treat CNV disease, which is to lay a basis on using in clinical. |
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