VEGF对犬PDR形成及玻璃体内TGF-β1表达的影响

发布时间：2018-08-12 13:42

【摘要】：目的:(1)通过荧光素眼底血管造影和视网膜眼底血管内皮细胞CD105免疫组化来检测3组试验犬增殖性糖尿病视网膜病变的成模率,探究VEGF对犬PDR形成的作用。(2)通过相对荧光定量PCR方法测定3组试验犬玻璃体内TGF-β1 m RNA相对表达量,探究VEGF对玻璃体内TGF-β1表达的影响。方法:(1)选取身体健康状况大致相同的中华田园犬,建造糖尿病模型犬,随机分成S1和S2两组,每组各5只。其中S1组双眼玻璃体腔内注射VEGF(0.2 ug/kg);S2组双眼玻璃体腔内注射等量生理盐水;另外随机挑选5只健康犬双眼玻璃体腔内注射等量生理盐水,作为空白对照,设为S3组。(2)玻璃体腔内注射后,连续8 w,每周对3组试验犬进行荧光素眼底血管造影双眼检查,观察并记录各组试验犬眼底血管渗漏情况,在玻璃体腔内注射后第8 w,采集各组试验犬左眼相同部位的视网膜组织,制成病理切片,进行CD105免疫组织化学染色,显微镜下观察记录各组免疫组织化学染色情况。(3)玻璃体腔内注射后第8 w,采集各组犬左眼玻璃体2 m L,移入离心管中液氮保存。通过相对荧光定量PCR的方法测定记录各组试验犬玻璃体内TGF-β1m RNA相对表达量。(4)最后通过Spass分析软件进行数据分析。试验结果:(1)荧光素眼底血管造影检查显示,S1组在第5 w时,1只试验犬眼底血管开始出现轻微渗漏,到第8 w时,S1组试验犬眼底血管均出现荧光渗漏。而S2与S3组,在8 w时间内,眼底血管均未出现荧光渗漏。(2)通过光学显微镜镜观察各组试验犬视网膜石蜡切片,S1组血管内皮细胞CD105免疫组化染色均为阳性,CD105阳性表达率为100%;S2与S3组血管内皮细胞CD105免疫组化染色均为阴性,CD105阳性表达率为0%。(3)通过相对荧光定量PCR的方法测定出:S1组玻璃体内TGF-β1m RNA的表达量极显著高于S2和S3组,是S2组的3.180倍(p0.01),S3组的10.657倍(p0.01)。S2组玻璃体内TGF-β1m RNA的表达量显著高于S3组,是S3组的3.351倍(p0.05)。说明S1组玻璃体内TGF-β1 m RNA表达量最高,S2组次之,S3组最低。结论:(1)在糖尿病模型犬的基础上,外源性的玻璃体注射VEGF能够增大眼底视网膜血管壁的通透性,加速血管的渗漏,可以促进犬增殖性糖尿病视网膜病变的形成。(2)VEGF可以显著提高玻璃体内TGF-β1 m RNA的表达量,有正向调控作用,加快了视网膜新生血管形成。
[Abstract]:Objective: (1) to detect the model forming rate of proliferative diabetic retinopathy in three groups by fluorescein fundus angiography and CD105 immunohistochemistry. To investigate the effect of VEGF on the formation of PDR in dogs. (2) to determine the relative expression of TGF- 尾 1 m RNA in vitreous of three groups of dogs by relative fluorescence quantitative PCR, and to explore the effect of VEGF on the expression of TGF- 尾 1 in vitreous. Methods: (1) Chinese idyllic dogs with similar health status were selected to construct diabetic model dogs. The dogs were randomly divided into S1 and S2 groups with 5 dogs in each group. In S1 group, VEGF (0.2 ug/kg) and S2 group were injected intravitreous with the same amount of saline, and 5 healthy dogs were randomly selected to inject the same amount of saline into the binocular vitreous cavity as the blank control. (2) after intravitreal injection, three groups of dogs were examined with fluorescein fundus angiography every week for 8 weeks, and the fundus vascular leakage was observed and recorded in each group. At the 8th week after intravitreal injection, the retinal tissues in the same part of the left eye of the dogs in each group were collected and made into pathological sections. CD105 immunohistochemical staining was performed. The immunohistochemical staining of each group was observed under microscope. (3) at the 8th week after intravitreous injection, the vitreous body of the left eye of each group was collected and transplanted into the centrifuge tube to preserve liquid nitrogen. The relative expression of TGF- 尾 1m RNA in vitreous of experimental dogs was measured by relative fluorescence quantitative PCR. (4) finally, the data were analyzed by Spass software. Results: (1) fundus fluorescein angiography showed that the fundus vessels of 1 dog in the S _ 1 group began to leak slightly at the 5th week, and the fundus vessels of all the dogs in the S _ 1 group showed fluorescence leakage at the 8th week. In S2 and S3 groups, within 8 weeks, No fluorescein leakage was found in the fundus vessels. (2) the positive rate of CD105 expression of vascular endothelial cells was 100% in S _ 2 and S _ 3 groups by optical microscopy in S _ 1 group and S _ 1 group. The positive expression rate of CD105 was 0. (3) the expression of TGF- 尾 1m RNA in vitreous of group 1 was significantly higher than that in group S2 and S3 by relative fluorescence quantitative PCR. The expression of TGF- 尾 1m RNA in vitreous of S2 group was 10.657 times as much as that of S3 group (p0.01). The expression of TGF- 尾 1m RNA in S3 group was significantly higher than that in S3 group and 3.351 times higher than that in S3 group (p0.05). The results showed that the expression of TGF- 尾 1 m RNA was the highest in S1 group and the lowest in S2 group, followed by S3 group. Conclusion: (1) on the basis of diabetic model dogs, exogenous vitreous injection of VEGF can increase the permeability of retinal vascular wall and accelerate the leakage of blood vessels. (2) VEGF can significantly increase the expression of TGF- 尾 1 m RNA in vitreous body, which has positive regulation effect and accelerate the formation of retinal neovascularization.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.292

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