一种新的干眼病模型—高渗盐水点眼小鼠干眼病模型的建立与评估

发布时间：2018-04-20 10:43

本文选题：干眼病 + 泪液　；参考：《第三军医大学》2010年硕士论文

【摘要】： 背景与目的干眼病(dry eye disease)又称角结膜干燥症(keratoconjunctivitis sicca)或者泪液功能不全综合征(dysfunctional tear syndrome),是指任何原因引起的泪液质和量的异常或动力学异常,导致泪膜稳定性下降,并伴有眼部不适,导致眼表组织病变为特征的疾病总称,是最常见的眼表疾病。干眼病严重影响患者的生活质量。干眼病发病机制复杂,多种因素均可以导致干眼病。美国眼科研究所干眼病研究小组将干眼病分为泪液生成不足型(包括Sj?gren’s综合症和非Sj?gren’s综合症泪液分泌不足)和泪液蒸发过强型。目前虽然有许多种干眼病模型,但是至今尚无一种动物模型可以准确模仿出该病频繁发生,逐渐恶化,机制复杂的特点。虽然干眼病诱发因素众多,机制复杂,但是该病发生的共同最终通路是泪膜不稳定,泪液中电解质浓度升高导致泪液渗透压升高。泪液的高渗透压是干眼病发病机制中的关键因素。研究表明,正常人泪液渗透压是302±9.7mOsm/L,干眼病患者泪液的渗透压是326.9±22.1mOsm/L。用316mOsm/L作为干眼病的诊断指标,其总体准确度优于其它任何一项干眼病诊断指标。本研究通过用高渗盐水给小鼠点眼,提高泪液的渗透压,制作泪液高渗透压性干眼病模型,观察是否可以引起和干眼病类似的体征和组织病理学损害,初步探讨泪液高渗透压在干眼病发病中的作用。方法 1.动物的选择与分组6～8周龄雌性BALB/c小鼠60只,用随机数字表法分为空白组(20只)、对照组(20只)、实验组(20只),双眼取用。对照组采用308mOsm/L氯化钠溶液点眼,每天5次,实验组采用500 mOsm/L氯化钠溶液点眼,每天5次,空白组不点眼。 2. SchirmerⅠ实验在裂隙灯显微镜下,用显微镊夹住酚红棉线,置于BALB/c小鼠眼睛的外眦部,60秒后取出,测量酚红棉线湿润的长度。 3.角膜荧光素染色和评分使用微量加液器在BALB/c小鼠下方结膜囊内滴入1μl 5%荧光素钠溶液,3分钟后在裂隙灯显微镜下采用钴蓝光检查并照相记录和评分。 4.角膜虎红染色使用生理盐水浸湿的虎红试纸,将浸湿的部分轻轻接触BALB/c小鼠的角膜。在裂隙灯显微镜下照相和记录。 5.泪液蕨类实验用毛细玻璃管采集BALB/c小鼠下穹隆部泪液,涂在洁净的玻片上,室温干燥,48小时内在光学显微镜下进行蕨类结晶图像分析,并照相记录。 6.角膜上皮和结膜上皮的组织学观察采用HE染色在光镜下观察角膜上皮形态,用图像分析软件测量中央角膜上皮厚度。取上方穹窿部结膜,采用PAS染色在光镜下观察上方穹窿部结膜上皮的形态。计算每个高倍视野下结膜杯状细胞的数量。 7.扫描电子显微镜观察在第42天,用扫描电子显微镜观察角膜上皮的形态。 8.统计学方法采用Kruskal-Wallis H检验比较同一时间点三组BALB/c小鼠的角膜荧光素染色评分。采用Wilcoxon秩和检验比较同一组不同时间点角膜荧光素染色评分。采用完全随机设计资料的方差分析比较同一时间点三组的角膜上皮厚度、泪液分泌量、结膜杯状细胞数量。采用t检验比较同一组不同时间点的角膜上皮厚度,泪液分泌量,结膜杯状细胞数量。结果 1. SchirmerⅠ实验第0天,空白组、对照组、实验组的酚红棉线浸湿长度未见统计学差异(P0.05)。从第7天开始,实验组酚红棉线浸湿长度和第0天比较明显减少(P0.01)。第7天酚红棉线浸湿长度较第0天下降了28%,第42天较第0天下降66%。 2.角膜荧光素染色和评分第0天,空白组、实验组、对照组均有BALB/c小鼠角膜少许点状着色,角膜评分各组未见统计学差异(P0.05)。实验组BALB/c小鼠随着实验的进行,角膜中央点状着色增加,逐渐出现片状着色,病变累及角膜各个象限,以角膜中央为重。从第7天起,实验组角膜荧光素染色评分和第0天相比明显升高(P0.01)。实验组第7天角膜荧光素染色评分较第0天升高了75%。 3.角膜虎红染色第0天,空白组、实验组、对照组均有3只BALB/c小鼠角膜虎红染色可见少量点状着色。实验组BALB/c小鼠从第7天开始,角膜点状着色逐渐增加,并且出现片状着色,累计角膜4个象限,以中央和鼻侧角膜为重。 4.泪液蕨类实验在各个检查时间点,取空白组、对照组、实验组BALB/c小鼠泪液,在光学显微镜下进行泪液蕨类结晶图像分析。空白组、对照组在各个检查时间点均可观察到形态良好的蕨类物形成。实验组BALB/c小鼠泪液结晶中的蕨类物从第7天开始逐渐减少,蕨类物之间的连接中断。第28天以后显微镜下仅能观察到稀少的小蕨类物,甚至完全消失。 5.角膜上皮HE染色的光镜观察和角膜上皮厚度测量空白组和对照组BALB/c小鼠角膜HE染色光镜观察可见上皮分层良好,分4～5层,基底部细胞排列呈柱状,靠近角膜表面逐渐变成鳞状上皮细胞。实验组BALB/c小鼠随实验进展基底层细胞部分缺失,上皮细胞分层紊乱,细胞层数减少,出现空泡。第0天和第7天,空白组、对照组、实验组BALB/c小鼠的角膜上皮层厚度未见统计学差异(P0.05)。从第14天起,实验组较对照组和空白组角膜上皮层厚度明显减少(P 0.05)。 6.结膜上皮PAS染色光镜观察和杯状细胞计数空白组和对照组BALB/c小鼠的结膜上皮细胞排列整齐,杯状细胞形态完整,分布在上皮细胞间,主要分布在穹隆部。实验组BALB/c小鼠随实验进展,上皮细胞排列紊乱,甚至出现缺失,杯状细胞形态不一致,数量减少。在第42天部分BALB/c小鼠上方穹隆部杯状细胞完全缺失。 7.扫描电子显微镜观察在第42天,用扫描电子显微镜观察空白组、对照组、实验组BALB/c小鼠角膜上皮细胞。可见空白组和对照组上皮细胞形态呈多边形,细胞间紧密连接,细胞表面满布微绒毛。实验组BALB/c小鼠角膜表面微绒毛丢失,角膜表层上皮细胞破坏。结论 1.用高渗盐水给BALB/c小鼠点眼可以在小鼠角膜和结膜引起与干眼病患者类似的体征和组织病理学变化。 2.用高渗盐水给BALB/c小鼠点眼是一个简单的,可靠,重复性好的模型,这个模型可以运用于干眼病发病机制和药物治疗的研究。 3.泪液高渗透压可能在干眼病发病机制中起重要作用。
[Abstract]:Background and purpose
Dry eye disease, also known as keratoconjunctivitis sicca (keratoconjunctivitis sicca) or tear dysfunction syndrome (dysfunctional tear syndrome), refers to any abnormal or kinetic abnormality in the quality and quantity of tears caused by any cause, resulting in a decline in the stability of the tear film, accompanied by eye discomfort, which is characterized by ocular tissue lesions. Disease is the most common ocular surface disease. Dry eye disease seriously affects the quality of life of patients.
Dry eye disease is complicated and many factors can lead to dry eye disease. Dry eye disease research team in the American Institute of Ophthalmology divides dry eye disease into inadequacy of tear formation (including Sj? Gren 's syndrome and non Sj gren' s syndrome) and excessive tear vaporization. No animal model can accurately mimic the frequent occurrence, deterioration, and complex mechanism of the disease. Although the causes of dry eye disease are numerous and the mechanism is complex, the common ultimate pathway of the disease is the instability of tear film, the increase of electrolyte concentration in tears leads to the increase of tear osmotic pressure. The hypertonic pressure of tear is the onset of dry eye disease. The key factors in the mechanism.
The study showed that the lacrimal osmotic pressure of normal people was 302 + 9.7mOsm/L. The osmotic pressure of the tears in the patients with dry eye disease was 326.9 + 22.1mOsm/L. with 316mOsm/L as the diagnostic index of dry eye disease. The overall accuracy was better than any other diagnosis index of dry eye disease.
In this study, the mice were given high osmotic saline to increase the osmotic pressure of tears and to make a model of high osmotic pressure dry eye disease. It was observed whether it could cause similar signs and histopathological damage to dry eye disease, and the use of high osmotic pressure in the pathogenesis of dry eye disease was preliminarily discussed.
Method
1. the selection of 1. animals and 60 female 6~8 weeks old female mice were divided into the blank group (20 rats), the control group (20), the experimental group (20) and the eyes of the control group. The control group adopted the 308mOsm/L Sodium Chloride Solution eye, 5 times a day, and the experimental group used 500 mOsm/L eyes, 5 times a day, and the blank group was not nod.
2. Schirmer I experiment, under a slit lamp microscope, used microscopic tweezers to clamp the phenolic red cotton thread and placed it in the outer canthus in the eyes of BALB/c mice. After 60 seconds, the wetting length of the phenolic red cotton thread was measured.
3. fluorescein staining and score were used to drop 1 L 5% fluorescein sodium solution in the conjunctival sac of BALB/c mice. After 3 minutes, cobalt blue light was used under the slit lamp microscope and recorded and scored.
4. corneal tigers red stained with saline soaked tiger red test paper, the wetted part of the cornea was touched lightly in the BALB/c mice. Under the slit lamp microscope, the film was photographed and recorded.
5. the lacrimal fern experiment was used to collect the tear of the lower dome of BALB/c mice with capillary glass tube, smear it on the clean glass, dry at room temperature, and analyze the crystal image of fern under the optical microscope for 48 hours and record it.
6. the histological observation of corneal epithelium and conjunctiva epithelium was observed by HE staining under light microscopy. The thickness of the central corneal epithelium was measured with image analysis software. The conjunctiva of the upper fornix was taken and the conjunctival epithelium in the upper fornix was observed under the light microscope with PAS staining. The number of conjunctival goblet cells in each high field of vision was calculated.
7. scanning electron microscope (SEM) was used to observe the morphology of corneal epithelium on scanning electron microscope (SEM) for forty-second days.
8. statistical method was used to compare the corneal fluorescein staining score of three groups of BALB/c mice at the same time point by Kruskal-Wallis H test. The corneal fluorescein staining score of the same group at different time points was compared with the Wilcoxon rank sum test. The corneal epithelium thickness of three groups at the same time point was compared with the variance analysis of the complete random design data, and the tear fluid was compared. The number of secretory and conjunctival goblet cells was measured by t test. The thickness of corneal epithelium, tear secretion and conjunctival goblet cells were compared at different time points in the same group.
Result
1. Schirmer I experiment zeroth days, the blank group, the control group, the experimental group of phenolic red cotton wetting length did not have statistical difference (P0.05). From the seventh day, the experimental group of phenolic red cotton wetting length and zeroth days was significantly reduced (P0.01). Seventh days of phenol red cotton wetting length decreased 28% in the zeroth world, forty-second days lower than the zeroth world down 66%.
2. the cornea fluorescein staining and scoring for zeroth days, the blank group, the experimental group and the control group had a little spot coloring of the cornea of BALB/c mice, and no statistical difference was found in the corneal score (P0.05). The experimental group BALB/c mice increased the central point coloring of the cornea, gradually appeared flaky coloring, and the lesions involved the cornea quadrant, with cornea From seventh days, the corneal fluorescein staining score of the experimental group was significantly higher than that of the zeroth day (P0.01). The corneal fluorescein staining score of the experimental group increased by 75%. on the seventh day of the experiment.
3. corneal tigers red staining for zeroth days, in the blank group, the experimental group and the control group, 3 BALB/c mice were stained with a small amount of spot coloring. The BALB/c mice in the experimental group started from seventh days, and the corneal dot coloring gradually increased, and the coloring of the cornea appeared, with the accumulative 4 quadrants of the cornea and the central and the nasal cornea as the weight.
4. tears fern experiment at each time point, take the blank group, the control group, the experimental group BALB/c mice tears, the tear liquid fern crystal image analysis under the optical microscope. The blank group, the control group can observe the formation of the well morphic ferns at each time point. The experiment group BALB/c mice tear crystallization ferns from seventh The days began to decrease and the connection between ferns was interrupted. Only twenty-eighth days later, only a few small ferns could be observed under microscope, or even disappeared completely.
5. corneal epithelium HE staining and corneal epithelial thickness measurement in blank group and control group BALB/c mouse cornea HE staining light microscope observation showed that epithelial layer was good, divided into 4~5 layers, basal cells arranged columnar, near the corneal surface and gradually become squamous epithelial cells. Experimental group BALB/c mice with the experimental progress of basal layer cells part In the zeroth and seventh days, the corneal epithelium thickness of BALB/c mice was not significantly different (P0.05). The thickness of corneal epithelium in the experimental group decreased significantly (P 0.05) compared with the control group and the blank group from the fourteenth day.
6. conjunctival epithelium PAS staining light microscopy and goblet cell count blank group and control group BALB/c mice conjunctival epithelial cells arranged neatly, goblet cell morphology complete, distributed in the epithelial cells, mainly distributed in the dome. The experimental group BALB/c mice with the experimental progress, epithelial cells disorder, even appearance of loss, goblet cell morphology On the forty-second day, the goblet cells in the fornix of partial BALB/c mice were completely absent.
7. scanning electron microscope was observed for forty-second days. The corneal epithelial cells of BALB/c mice were observed in the blank group, the control group and the experimental group. The epithelial cells of the blank group and the control group were polygonal, the cells were closely connected and the surface of the cells were covered with microvilli. The corneal surface microvilli lost and the corneal surface of the experimental group BALB/c mice The epithelial cells of the layer were destroyed.
conclusion
1. the use of hypertonic saline for BALB/c mice can induce similar signs and histopathological changes in cornea and conjunctiva of mice.
2. with hypertonic saline to BALB/c mice is a simple, reliable, reproducible model that can be applied to the pathogenesis of dry eye disease and the study of drug treatment.
3. high osmotic pressure of tears may play an important role in the pathogenesis of dry eye.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R777.2;R-332

【引证文献】