兔干眼模型的建立及FK506治疗干眼的疗效及作用机制研究

发布时间：2018-11-06 10:32

【摘要】： 第一部分皮下注射氢溴酸东莨菪碱建立兔干眼模型的实验研究目的:探讨兔皮下注射氢溴酸东莨菪碱,建立干眼模型的特点和眼表病理损害机制。方法:健康新西兰大白兔18只,随机分为三组,每组6只。实验组皮下注射氢溴酸东莨菪碱,其中低剂量组(L,1.0mg/次)、高剂量组(H, 2.0mg/次),对照组注射等量生理盐水。注射时间点为每天8点11点、14点和18点,连续30天。用药后0、7,14,21,30d对实验组及对照组行泪液分泌试验、泪膜破裂时间(BUT)、角膜荧光素染色,印迹细胞检查。第30d空气栓塞法处死实验动物,取角膜、结膜及泪腺组织行HE染色,免疫组化检测FAS,BCL-2及NFκB)的表达水平。结果:酚红棉线法测得高剂量(H)组造模前泪液分泌量为16.25±2.299mm,第21d时下降为6.75±1.982mm,对照组为16.50±2.619mm,差异具有统计学意义(p0.05),同期低剂量组(L)为15.00±2.390mm,与对照组相比无明显差异。H组第3d出现BUT缩短,L组则第7d发生BUT的变化,此后BUT值均低于5s,对照组BUT大于20s。实验组均出现角膜荧光素染色阳性,H组出现时间早(3d),持续时间长。L组和H组PAS染色与对照组相比,杯状细胞形态紊乱、核淡染,部分核固缩,L组和H组之间无明显差异。HE染色显示H组泪腺周围见散在淋巴细胞和腺腔凋亡性改变,角膜上皮呈重度瘤样增生基质层变性伴水肿,结膜杯状细胞反应性增多,固有层见淋巴细胞浸润数量增加。L组角膜浅层上皮轻度增生,细胞排列稍紊乱,结膜和泪腺组织病理改变轻微。电镜观察高剂量组泪腺腺管管腔壁绒毛较对照组明显减少,角膜表层微绒毛较对照组减少,上皮细胞空泡变,细胞核固缩,结膜表面微绒毛减少,上皮细胞呈坏死凋亡改变,低剂量未见明显显微改变。凋亡抑制因子BCL-2在高剂量模型组泪腺、角膜和结膜组织中极少表达,对照组各组织均呈高度阳性表达,两组差异显著p0.001;促凋亡因子Fas在模型组泪腺组织中呈中度表达,在角膜和结膜组织中呈中高度表达。炎症转录因子NFκB在模型组兔眼泪腺腺泡细胞胞浆和胞核轻度表达,腺腔管壁细胞胞核和胞浆中重度表达,角膜组织上皮细胞和基底细胞胞浆、胞核以及结膜上皮细胞中NFκB呈中度阳性表达,对照组各组织NFκB极少表达,两组差异有统计学意义(p0.001)。结论:兔皮下注射氢溴酸东莨菪碱,4次/天,2.0mg/次,可有效维持药物外周浓度,抑制泪腺的分泌,引起角结膜上皮细胞的损伤以及角结膜上皮和泪腺组织细胞凋亡因子和炎症因子阳性表达增加,成功建立以泪液缺乏为主,凋亡和炎症反应共同参与的干眼动物模型。第二部分FK506对氢溴酸东莨菪碱至兔干眼模型的疗效目的:观察FK506滴眼液干预兔干眼模型的疗效及其作用机制方法:健康新西兰大白兔30只,,随即分为实验组18只和对照组12只。实验组采用上述方法每天8点11点、14点和18点皮下注射氢溴酸东莨菪碱注射液2.0mg/次,并从第四天开始左眼点0.05%FK506滴眼液,4次/天,右眼不做处理,对照组皮下注射生理盐水。实验第0,3,7,14,21,35d对实验组及对照组行泪液分泌试验、泪膜破裂时间(BUT)、角膜荧光素染色检查。分别在第7,14,和35d采取空气栓塞法分别随即处死模型组5只兔子,取双眼角膜、结膜和泪腺组织,PCR定量分析IL-1β、TNF-α和MMP-9等炎性因子在各组织的表达。第35d采用印记细胞学方法取双眼角结膜上皮细胞行免疫组化检测NFκB表达水平的差异。结果:造模后3,7天实验兔双侧眼均角膜荧光素染色强阳性。第14d开始FK506治疗眼角膜荧光素染色评分低于未用药眼,至第35d,差异进一步增大具有统计学意义(P0.01)。FK506治疗组眼的角结膜上皮NFκB表达显著低于对照眼,不同时间点治疗组结膜组织中IL-1β、TNF-α和MMP-9表达水平低于对照组,两者差异有统计学意义(p0.05)。14天时双侧泪腺MMP-9水平造模后与对照组相比呈现增高,但用药干预眼与未治疗眼之间无明显差异。结论:FK506通过抑制炎症信号分子NFκB的激活,降低角膜,结膜组织中IL-1β、TNF-α和MMP-9等炎症因子的水平,有效地抑制眼表免疫性炎症,进而减轻干眼对眼表组织的损害。
[Abstract]:The experimental study on the establishment of rabbit model of dry eye by subcutaneous injection of the first part by subcutaneous injection of hydrobromic acid Objective: To study the characteristics of the dry eye model and the pathology of ocular surface in rabbits by subcutaneous injection of hydrobromic acid. Damage mechanism. Methods: 18 healthy New Zealand white rabbits, randomized The experimental group was divided into three groups, each group of 6 rats. The experimental group was injected with hydrobromic acid, the lower dose group (L, 1. 0mg/ times) and the high dose group (H, 2. 0mg/ time), and the control group. The same amount of saline was injected into the group. The injection time point was 8: 11, 14, and In the experimental group and the control group, the tear secretion test, the tear film rupture time (BUT) and the corneal fluorescein were measured at 18: 0, 7, 14, 21, and 30 days after the administration. The cells were stained and blotted. The experimental animals were sacrificed by the method of air embolism on the 30th day. The cells of the cornea, conjunctiva and lacrimal gland were stained with HE, and the FAS, BCL-2 and N were detected by immunohistochemistry. Results: The amount of tear secretion in the high-dose (H) group was 16.25-2.299mm in the high-dose (H) group and 6.75-1.982mm in the control group and 16.50-2.619mm in the control group (P 0.05), while the low-dose group (L) was 15.00-2.390 in the same period. There was no significant difference between the control group and the control group. In the H group, the BUT was shortened in the third day, and the changes of the BUT occurred in the group L, and the BUT value was lower after that. In the control group, the BUT of the control group was more than 20s. In the experimental group, the staining of the corneal fluorescein was positive, and the H group showed a positive staining. The present time was early (3d) and the duration was long. The PAS staining of the L and H groups was compared with that of the control group. There was no significant difference between the group L and the H group. HE staining showed that there was a change in the apoptosis of the lymphocytes and the gland cavity around the lacrimal gland of the H group, the changes of the stromal layer with severe tumor-like hyperplasia and edema in the corneal epithelium, and the increase of the reactivity of the conjunctiva cup-like cells. Inherent layer was found to increase in the number of infiltration of lymphocytes. The superficial epithelium of the L group was slightly hyperplastic and the cells were arranged slightly. The pathological changes of the conjunctiva and the lacrimal gland were mild. The microvilli in the lumen of the lacrimal gland of the high-dose group were significantly reduced in the high-dose group, the microvilli in the surface of the cornea decreased, the vacuolation of the epithelial cells was reduced, the cell nucleus was fixed, the microvilli on the surface of the conjunctiva were reduced, and the epithelial cells were necrotic. The apoptosis-inhibiting factor BCL-2 was expressed in the lacrimal gland, the cornea and the conjunctival tissue of the high-dose model group, and the control group was highly positive, and the difference between the two groups was significantly higher than that of the control group, and the difference between the two groups was significantly higher than that of the control group. The expression of the inflammatory transcription factor NF-B in the cytoplasm and the nucleus of the cell and the cytoplasm of the cell and the cytoplasm of the cell, the cytoplasm, the nucleus and the conjunctival epithelium of the epithelial cell and the basal cell of the rabbit's lacrimal gland in the model group were observed in the model group. NF-B in the control group was expressed moderately, and NF-B in the control group was very rarely expressed, and the difference between the two groups Conclusion: The peripheral concentration of the drug can be effectively maintained, the secretion of the lacrimal gland, the damage to the conjunctival epithelial cells and the conjunctival epithelium and the lacrimal gland can be effectively maintained. The positive expression of the cell apoptosis factor and the inflammatory factor increased, and the success was based on the lack of tear. A dry-eye animal model that is co-involved in the reaction of apoptosis and inflammation. The effect of two-part of FK506 on the dry-eye model in the eastern and eastern part of the rabbit Objective: To observe the effect and mechanism of FK506 eye drops in the dry eye model of rabbits Methods: Thirty-one healthy New Zealand white rabbits were divided into experimental group and control group. 5% FK506 eye drops, 4 times/ day, right eye not treated, control group subcutaneous injection of physiological saline. Experiment Days 0, 3, 7, 14, 21, 35d In the group and control group, the tear secretion test, the tear film rupture time (BUT) and the corneal fluorescein staining were performed. 5 rabbits were respectively sacrificed in the model group at the 7th, 14th, and 35d respectively, and the two eyes of the cornea, the conjunctiva and the lacrimal gland tissue and the PC were taken. R. Quantitative analysis of the expression of IL-1, TNF-1 and MMP-9 in the tissues. A Cytological Approach to the Expression of NF-B in the Conjunctival Epithelium of the Binocular Conjunctival Epithelial Cells The results showed that in 3 and 7 days after the model, the two-side eyes of the experimental rabbits were stained with fluorescein. At the same time, the staining score of the eye-cornea fluorescein was lower than that of the non-treated eyes at the beginning of the 14th day, and the difference between the two groups was statistically significant (P0.01). The FK506 treatment group The expression of IL-1, TNF-1 and MMP-9 in the conjunctival tissue of the treatment group was lower than that of the control group. Conclusion: FK506 can reduce the level of IL-1, TNF and TNF in the cornea and conjunctival tissue by inhibiting the activation of NF-B in the inflammatory signal.
【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】：R777

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