| Purpose:DR. is the most common chronic complication of diabetes, and Important blinding eye disease. About10%patients has Serious visual impairment in15years duration of diabetes patients,and2%patientis become a blind, Diabetic retinopathy (DR) is one of the most important performance in diabetic microangiopathy,To be specific is a change of fundus lesions.BRB damage, and new blood vessels to form. The late new blood vessels film contraction force cause retinal detachment The pathogenesis of DR is very complex. Has not been fully elucidated now..But any pathological changes in nature are the disorder of dynamic balance in the body, The same to the formation of new blood vessels. Vascular stimulation factors increase and inhibition factors to reduce the balance of both disorders. The formation of the so-called "angiogenic switch".VEGF Is to promote new blood vessels in the body of one of the main factors. In recent years in the study of the pathogenesis and treatment of DR widely attention.Newly discovered vascular inhibition, outside the body, the experiment has proved that specific inhibition of the vascular endothelial cell proliferation and migration, thus inhibiting angiogenesis, vascular inhibition of tumor angiogenesis research has a large number of reports, many experiments showed that vascular endothelial growth factor (VEGF) in hypoxia related new retinal angiogenesis plays a key role. Therefore, to clarify the relationship between diabetes and retinal VEGF, As inhibiting angiogenesis mechanism is discussed, and for the prevention and treatment of retinal neovascular diseases to find effective means, is bound to lay the solid foundation for its clinical application, and produce great social and economic benefits.Process:Experimental Animals and GroupingExperimental animal model:Selected80healthy SD rats, Adaptive feeding1week and free drinking and eating. Weigh up all rats before the trial, and examinedall eyes of rats no pathological changes.After tail vein blood glucose meter inspecting blood sugar were within normal range。Then fast12h, gi ve STZ by intraperitoneal injection,dose of60mg/kg,to establish diabetes mo del. Every1week testing blood GLU levels. GLU is higher than350mg/dl that a diabetic rats model induced by success. Building successful72only,ran domly divided into two groups:group A as model experimental group, Grou p B as the model control group.Each group of36; In normal SD rats,36, giv e the same volume of citrate buffer solution by intraperitoneal injection.to be group C,as normal control.Intravitreal injection10g· L-1pentobarbital sodium with50mg· kg-1intraperitoneal inject on rats after anesthesia, Beautydoris mydr-iatic, AyrKain surface anesthesia, after the experiment drip3g· L-1ThyriteErbitux drops to pr-event infection.After the needle with27mark punch,injected with Angiostatin according to7.5ug/ul by microsyringe on left temporal corneal limbus after1mm of rats, and make sure no lens damage. Give Angiostatin to group A rats by this method,and give the same volume of PBS solution to group B, C rats.After2weeks each rat inject medicine once a day,,note medicine four consecutive days.Retinal tissue slice preparation and observationAfter the continuous administration12days,33days and61days,remove eyeball of each12rats respectively, fixed in4%paraformaldehyde, quickly,re- move front section part after2hours, take cotton wool in the vitreous cavityI n order to prevent the retinal detachment occurs, continue to fixed for24hour s, dehydrated alcohol gradient,. Transparent after paraffin embedding, sectioning, thickness is4microns, put water dewaxing to slice in logwood dye in aqueo us solution withlO min, ammonia differentiation in a few seconds; after water flushing1h, eosin dye solution dyed5min, tap water rinse. Sealing piece a fter the dehydration of transparent.The rat retinal VEGF protein detectionIn12days,33days and61days respectively in each rat retina, use anti VEGF antibody for Western blot analysis, and for each group of equivalent total protein50mu (g), the degeneration, electrophoresis, DianZhuan film, plus anti VEGF polyclonal antibody (1:1000) incubation, using indirect enzyme-linked method development. The same samples after elution treatment plus resistance to beta actin (beta actin) antibody incubation, development. Will have been developed signal using optical density analysis semi-quantitative analysis, and use the beta actin standardization.Result1. The establishment of the diabetic rats modelBefore and after building diabetic model rats, observe weekly blood sugar and weight changes. Building after STZ module in rats appear diabetes "three more andone little"(drink, food, urine, and weight loss) symptoms, Weight w eight than the normal control group rats decreased obviously, Their blood suga r levels significantly compared with normal control group (P<0.01). STZ mo dule of blood sugar levels rising trend in1～4weeks,5～7weeks in a rela tively stable period.2. Model rats retinal tissue biopsiesHE dyed visible layers of retina cells neatly, group C cell morphology is normal, no obvious pathological changes. Different periods of A and group B both retinal tissue, cell tissue edema, especially around the veins, disordered ar rangement of cells. Dyeing group A visible at different periods of retinal edem a is lighter group B, cells arranged relatively neatly.3. Vascular inhibition effects on model rats retinal VEGF expressionVEGF levels in group B were significantly higher than those of group A, groupA and B is higher than that group C, with the increase of the time showed a trend of decline after rising first; Group A retinal VEGF levels with the increase of the time also showed A trend of decline after rising first, and higher than that of group C (33days except). Different times group C retinal VEGF level has not changed.ConclusionBefore it is generally believed that the earliest of diabetic retinopathy lesions is to appear at the end of the duration of diabetes, retinal microvascular lesions. In recent years, however, research shows that, the early stages of diabetic retinal pathological changes have taken place in it.This experiment selects the male SD rats by intraperitoneal injection of65mg/kg body mass STZ, close to human induced in the rat model of type1DM, the formation of early diabetic retinopathy lesions, building the success rate of90%, visible STZ rats model of safe, reliable, convenient, feasible, high success rate.This experiment through the retina HE staining, found that STZ diabetic rats12days in the rat retina HE dyeing not obvious change was observed in each layer. Diabetes33days, the visible pathological changes in the retina, the organization degree of edema. Along with the course of the disease extended to61days, the layers of retina pathological change gradually aggravate, also suggests that early in the duration of diabetes, retinal histological lesions of each layer is not obvious, and the layers of retina tissue pathological changes happened gradually with progression. In addition, the injection of the PBS group B in the rat retina inner and outer nuclear layer separation, kernel layer cells arranged disorder, lower density, reduced Numbers of ganglion cells, and injected vein group A obvious inhibition of vascular endothelial cells proliferation and tissue edema conditions improveTo sum up, the study on the difference of retinal VEGF expression detection, it is concluded that injection of blood vessels inhibition can obviously reduce the expression level of VEGF in STZ diabetic rats retina, but still slightly higher than the control group, confirmed the high expression of VEGF in diabetic retina, and that blood vessels inhibition in the treatment of DR has potential application valu. |
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