他可莫司滴眼液治疗角膜碱烧伤的免疫学机制研究

发布时间：2018-03-17 08:10

本文选题：角膜　切入点：碱烧伤　出处：《南昌大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的:研究他可莫司(FK506)滴眼液治疗大鼠角膜碱烧伤的药物有效浓度及给药方案,评价其抑制角膜碱烧伤后免疫炎症反应,改善角膜微环境的效能,并探讨其作用机制。方法:NaOH滤纸片敷贴法制作大鼠角膜中度碱烧伤模型,96只wistar大鼠随机分为A、B、C、D四组,A、B、C组为治疗组,给予浓度0.05%、0.1%、0.5%FK506滴眼液治疗,4次/天,D组为阴性对照组。观察不同组别大鼠角膜透明性、基质融解程度以及新生血管形成情况。在不同时间点(第3,7,14,30d)处死大鼠取下角膜, HE染色分析角膜组织结构的变化,比较炎症细胞浸润程度的差异,免疫荧光法检测CD4/CD8阳性T细胞时空表达规律。结果:碱液滤纸片烧伤角膜后,角膜雾状水肿,烧伤区角膜上皮缺失,基质融解形成溃疡,角膜缘血管扩张,后期形成新生血管,角膜失去透明性。FK506治疗组的角膜在溃疡形成面积,角膜混浊等级以角膜新生血管化程度方面均明显好于对照组。①.角膜融解定量分析结果:烧伤后3d,A、B、C组角膜融解面积分别为(3.14±0.25)mm2、(2.65±0.28) mm2、(2.27±0.47) mm2,而对照组D组为(4.06±0.41) mm2,差异有统计学意义(P0.05),低浓度FK506治疗组与中、高浓度组相比亦存在统计学差异(P0.05),而0.1%与0.5%浓度组之间则无明显差别(P0.05); 7d时角膜融解面积缩小,分别为A组(1.82±0.51)mm2、B组(1.34±0.41) mm2、C组(0.90±0.39) mm2,与对照组D组(2.47±0.54) mm2,相比差异有统计学意义(P0.05)。②.角膜混浊定量分析结果:治疗组在烧伤后角膜混浊评分低于对照组(P0.05),其规律与角膜融解面积的变化曲线相一致,中高浓度组之间则无差异。③.角膜新生血管定量分析结果:治疗组在烧伤后3d角膜新生血管面积分别为A组(4.26±0.36)mm2、B组(3.88±0.33) mm2、C组(3.54±0.46) mm2均小于对照组D组(5.00±0.12) mm2,差异有显著意义(P0.01),A与B、C比较也有统计学意义(P0.05),而B与C比较无统计学意义(P0.05);此种差异性保持至第14d方逐渐减小。烧伤角膜HE染色检查显示FK506治疗组基质中炎症细胞浸润的数量明显少于对照组,计数结果显示3d时,A组(77.67±4.51)个/0.04mm2、B组(51.33±3.21)个/0.04mm2、C组(41.67±3.06)个/0.04mm2,而同期对照组D组(97.00±6.25)个/0.04mm2,差异均有统计学意义(P0.05)。免疫荧光提示治疗组中CD4阳性细胞的数量少于对照组,通过光密度分析,低浓度治疗组为中等强度荧光,中高浓度治疗组为低荧光,而对照组则表现为弥漫性强荧光。在整个角膜碱烧伤病理过程中,CD8+ T淋巴细胞的表达情况,给药组与对照组对比未见明显差异。结论:他可莫司(FK506)局部使用可明显下调角膜碱烧伤后T淋巴细胞介导的特异性免疫反应,同时也可间接减轻非特异性炎症反应,并且表现为浓度依赖性。FK506局部干预后可阻止角膜碱烧伤后基质融解程度,抑制角膜新生血管的生长,减轻角膜组织结构的破坏,改善角膜预后,从而为角膜碱烧伤急性期的治疗提供了新的免疫治疗模式。
[Abstract]:Aim: to study the effective concentration and administration of tacrolimus FK506) eye drops in the treatment of corneal alkali burns in rats, to evaluate the efficacy of tacrolimus FK506 eye drops in inhibiting the immune inflammation and improving the corneal microenvironment after alkali burns in rats, and to explore its mechanism. Methods 96 wistar rats with moderate alkali burn of cornea were randomly divided into four groups: group A (n = 4) and group C (n = 4). The group D was treated with FK506 eye drops 4 times per day as the negative control group. The corneal transparency was observed in different groups of rats. The degree of stromal fusion and the formation of neovascularization. At different time points (3 ~ 714 ~ 30 days), rats were killed to remove cornea, and HE staining was used to analyze the changes of corneal tissue structure, and to compare the degree of inflammatory cell infiltration. The temporal and spatial expression of CD4/CD8 positive T cells was detected by immunofluorescence assay. Results: after alkali filter paper burn cornea, corneal haze edema, corneal epithelium loss, matrix fusion ulcer, corneal limbal vasodilation and neovascularization were formed. Corneal loss of transparency. FK506 treatment group in corneal ulcer formation area, Corneal opacity grade in corneal neovascularization was significantly better than that in control group. 1. Quantitative analysis of corneal fusion: the area of corneal fusion in group C was 3.14 卤0.25 mm ~ 2 卤2.65 卤0.28 mm ~ (2) 卤0.47) mm ~ (2), respectively, compared with that in group D (4.06 卤0.41) mm ~ (2) (P < 0.05). Significance of P0.05, low concentration of FK506 treatment group and middle, There was also a statistical difference between the high concentration group and the high concentration group, but there was no significant difference between the 0.1% and 0.5% concentration groups, and the area of corneal fusion decreased at 7 days. The results of quantitative analysis of corneal opacification: the score of corneal opacification after burn in the treatment group was lower than that in the control group (P0.05), the rule and the corneal surface of the corneal fusion surface were significantly lower than that in the control group (P 0.05), compared with the control group D (2.47 卤0.54) mm 2. The corneal opacity score in group A was 1.82 卤0.51 卤0.41 卤0.41 卤0.41 mm ~ (2) respectively, and that in group C was 0.90 卤0.39 mm ~ (2) 2, which was significantly different from that in group D (P < 0.05). The change curve of the product is consistent, Results of quantitative analysis of corneal neovascularization: the area of corneal neovascularization in the treatment group was 4.26 卤0.36 mm ~ (-2) mm in group A on the 3rd day after burn, which was 3.88 卤0.33 mm ~ (2) mm2 in group B and 3.54 卤0.46) mm2 in group C, respectively, which was significantly lower than that in group D (5.00 卤0.12) mm. The difference between P0.01A and BHOC was statistically significant, but there was no significant difference between B and C, and the difference was gradually decreased until the 14th day. The number of inflammatory cells infiltrated in the matrix of FK506 treated group was obviously lower than that of control group by HE staining of burn cornea. The counting results showed that the number of CD4 positive cells in group A was 77.67 卤4.51) and that in group B was 51.33 卤3.21) / 0.04mm2m-2 on day 3, while that in group D was 97.00 卤6.25 / 0.04mm2.The number of CD4 positive cells in group B was lower than that in group B, and the number of positive cells in group B was lower than that in group B (P < 0.05). Medium intensity fluorescence was found in low concentration group, low fluorescence in medium and high concentration group, and diffuse hyperfluorescence in control group. The expression of CD8 T lymphocytes was observed in the whole pathological process of alkali burn of cornea. There was no significant difference between the treatment group and the control group. Conclusion: topical use of tacrolimus FK506 can significantly down-regulate the specific immune response mediated by T lymphocytes after alkali burn of cornea and indirectly attenuate the nonspecific inflammatory response. The results showed that local intervention with concentration dependent. FK506 could prevent the degree of stromal fusion after alkali burn, inhibit the growth of corneal neovascularization, alleviate the destruction of corneal tissue structure, and improve the prognosis of cornea. This provides a new immunotherapy model for the treatment of corneal alkali burn in acute stage.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R779.1

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