胸腺素β4对兔角膜新生血管形成的影响

发布时间：2018-08-27 10:41

【摘要】：角膜无血管是角膜透明和正常视力的重要保证。虽然角膜新生血管对角膜损伤修复起到一定作用,但是长期慢性的角膜血管新生会形成角膜翳,破坏角膜正常微环境,使眼前节免疫赦免减弱或消失,影响视力健康。寻求有效治疗角膜新生血管的药物具有重大意义,胸腺素β4具有多重生物学功能,能预防细胞凋亡,减少炎症发生,有利于血管生成,促进角膜痊愈,但其对角膜新生血管的作用却一直没有定论,还尚有争议。因此,我们设计体外与体内的样本随机对照实验探究胸腺素β4对角膜新生血管形成的影响,为胸腺素β4对角膜新生血管作用的研究提供理论依据。体外原代培养兔的脐静脉内皮细胞,当第三代兔脐静脉内皮细胞生长至连续的单层状态,按一定密度传代至96孔板,分别按0ng/mL,10ng/mL、100ng/mL、1000ng/mL浓度加入胸腺素β4继续培养,于24h后分别加入cck-8试剂孵育1h,用酶标仪检测吸光度值,用于测定细胞增殖度。体内通过角膜囊袋法建立兔角膜新生血管模型,采用角膜内植入琼脂糖包裹碱性成纤维生长因子缓释明胶海绵小粒诱导兔角膜新生血管的形成。实验动物按胸腺素β4的浓度分为0.1μg/μL、1μg/μL、10μg/gL三个药物治疗组和PBS对照组,实验组每天两次角膜表面应用Tβ4滴眼治疗,对照组每天两次角膜表面滴加PBS,并观察新生血管生长情况,计算抑制率和消退率。实验结果表明,体外细胞增殖实验中样本浓度为100ng/mL和1000ng/mL的胸腺素β4对兔脐静脉内皮细胞有显著性增殖效果；体内模型实验中药物浓度为1μg／μL的胸腺素β4的抑制率最高,效果最好,10μg/μL的胸腺素β4早期抑制率较1μg/μL的胸腺素p4低,后期则较高。与PBS对照组相比,三个浓度的胸腺素β4药物治疗组都对角膜新生血管的形成有显著的抑制作用,但0.1μg/μL的胸腺素β4治疗组只是抑制了角膜血管生长,而1μg/μL的胸腺素β4治疗组和10μg/μL的胸腺素β4治疗组既抑制了角膜新生血管的生长,又显著地促进了角膜新生血管的退化。可见,胸腺素β4在体外细胞环境中具有促进血管内皮细胞增殖的效果,但在动物机体的大环境中,胸腺素β4对角膜新生血管具有抑制作用。
[Abstract]:Cornea without blood vessel is an important guarantee of corneal transparency and normal vision. Although corneal neovascularization plays an important role in the repair of corneal injury, chronic corneal neovascularization will form corneal pannus, destroy the normal microenvironment of cornea, weaken or disappear immune immunity of anterior segment, and affect the health of visual acuity. It is of great significance to seek effective drugs for the treatment of corneal neovascularization. Thymosin 尾 4 has many biological functions, which can prevent cell apoptosis, reduce inflammation, facilitate angiogenesis and promote corneal healing. However, its role in corneal neovascularization has been inconclusive and controversial. Therefore, we designed random controlled experiments in vitro and in vivo to explore the effect of thymosin 尾 4 on corneal neovascularization, and to provide a theoretical basis for the study of the effect of thymosin 尾 4 on corneal neovascularization. Primary cultured rabbit umbilical vein endothelial cells were cultured in vitro. When the third generation of rabbit umbilical vein endothelial cells grew to a continuous monolayer state and were subcultured to 96 well plates according to a certain density, Thymosin 尾 4 was added to Thymosin 尾 4 at the concentration of 0 ng / mL 10 ng / m L ~ (-1) L ~ (-1) ~ 100 ng 路m ~ (-1) 路L ~ (-1) ~ (-1) ~ 100 ng / mL, respectively. After 24 hours incubation with cck-8 reagent for 1 hour, the absorbance value was detected by enzyme marker, and the cell proliferation was measured. Rabbit corneal neovascularization model was established in vivo by the method of corneal capsule bag. Rabbit corneal neovascularization was induced by implantation of agarose encapsulated basic fibroblast growth factor (BFGF) sustained release gelatin sponge (BFGF) into rabbit corneal neovascularization model. The experimental animals were divided into three groups according to the concentration of thymosin 尾 _ 4 (0.1 渭 g / 渭 L ~ (-1) 渭 g / 渭 L ~ (10) 渭 g/gL). The experimental group was treated with T 尾 _ 4 eye drops twice a day, the control group was treated with PBS, twice a day and the growth of neovascularization was observed. The inhibition rate and extinction rate were calculated. The results showed that Thymosin 尾 4 with concentration of 100ng/mL and 1000ng/mL had significant effect on proliferation of rabbit umbilical vein endothelial cells in vitro, and thymosin 尾 4 with concentration of 1 渭 g / 渭 L had the highest inhibitory rate in vivo. The early inhibitory rate of 10 渭 g / 渭 L thymosin 尾 4 was lower than that of 1 渭 g / 渭 L thymosin p4, and the later stage was higher. Compared with PBS control group, three concentration of thymosin 尾 4 drug treatment group all had significant inhibition on corneal neovascularization, but 0.1 渭 g / 渭 L thymosin 尾 4 treatment group only inhibited corneal angiogenesis. However, 1 渭 g / 渭 L thymosin 尾 4 treatment group and 10 渭 g / 渭 L thymosin 尾 4 treatment group not only inhibited corneal neovascularization growth, but also significantly promoted corneal neovascularization degeneration. It can be seen that thymosin 尾 4 can promote the proliferation of vascular endothelial cells in vitro, but thymosin 尾 4 can inhibit corneal neovascularization in the environment of animal body.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R772.2;S865.19

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