本文選題：干眼病 + 淚液�。� 參考：《第三軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 背景與目的 干眼病(dry eye disease)又稱角結(jié)膜干燥癥(keratoconjunctivitis sicca)或者淚液功能不全綜合征(dysfunctional tear syndrome),是指任何原因引起的淚液質(zhì)和量的異�；騽恿W(xué)異常,導(dǎo)致淚膜穩(wěn)定性下降,并伴有眼部不適,導(dǎo)致眼表組織病變?yōu)樘卣鞯募膊】偡Q,是最常見的眼表疾病。干眼病嚴(yán)重影響患者的生活質(zhì)量。 干眼病發(fā)病機制復(fù)雜,多種因素均可以導(dǎo)致干眼病。美國眼科研究所干眼病研究小組將干眼病分為淚液生成不足型(包括Sj?gren’s綜合癥和非Sj?gren’s綜合癥淚液分泌不足)和淚液蒸發(fā)過強型。目前雖然有許多種干眼病模型,但是至今尚無一種動物模型可以準(zhǔn)確模仿出該病頻繁發(fā)生,逐漸惡化,機制復(fù)雜的特點。雖然干眼病誘發(fā)因素眾多,機制復(fù)雜,但是該病發(fā)生的共同最終通路是淚膜不穩(wěn)定,淚液中電解質(zhì)濃度升高導(dǎo)致淚液滲透壓升高。淚液的高滲透壓是干眼病發(fā)病機制中的關(guān)鍵因素。 研究表明,正常人淚液滲透壓是302±9.7mOsm/L,干眼病患者淚液的滲透壓是326.9±22.1mOsm/L。用316mOsm/L作為干眼病的診斷指標(biāo),其總體準(zhǔn)確度優(yōu)于其它任何一項干眼病診斷指標(biāo)。 本研究通過用高滲鹽水給小鼠點眼,提高淚液的滲透壓,制作淚液高滲透壓性干眼病模型,觀察是否可以引起和干眼病類似的體征和組織病理學(xué)損害,初步探討淚液高滲透壓在干眼病發(fā)病中的作用。 方法 1.動物的選擇與分組6～8周齡雌性BALB/c小鼠60只,用隨機數(shù)字表法分為空白組(20只)、對照組(20只)、實驗組(20只),雙眼取用。對照組采用308mOsm/L氯化鈉溶液點眼,每天5次,實驗組采用500 mOsm/L氯化鈉溶液點眼,每天5次,空白組不點眼。 2. SchirmerⅠ實驗在裂隙燈顯微鏡下,用顯微鑷夾住酚紅棉線,置于BALB/c小鼠眼睛的外眥部,60秒后取出,測量酚紅棉線濕潤的長度。 3.角膜熒光素染色和評分使用微量加液器在BALB/c小鼠下方結(jié)膜囊內(nèi)滴入1μl 5%熒光素鈉溶液,3分鐘后在裂隙燈顯微鏡下采用鈷藍光檢查并照相記錄和評分。 4.角膜虎紅染色使用生理鹽水浸濕的虎紅試紙,將浸濕的部分輕輕接觸BALB/c小鼠的角膜。在裂隙燈顯微鏡下照相和記錄。 5.淚液蕨類實驗用毛細玻璃管采集BALB/c小鼠下穹隆部淚液,涂在潔凈的玻片上,室溫干燥,48小時內(nèi)在光學(xué)顯微鏡下進行蕨類結(jié)晶圖像分析,并照相記錄。 6.角膜上皮和結(jié)膜上皮的組織學(xué)觀察采用HE染色在光鏡下觀察角膜上皮形態(tài),用圖像分析軟件測量中央角膜上皮厚度。取上方穹窿部結(jié)膜,采用PAS染色在光鏡下觀察上方穹窿部結(jié)膜上皮的形態(tài)。計算每個高倍視野下結(jié)膜杯狀細胞的數(shù)量。 7.掃描電子顯微鏡觀察在第42天,用掃描電子顯微鏡觀察角膜上皮的形態(tài)。 8.統(tǒng)計學(xué)方法采用Kruskal-Wallis H檢驗比較同一時間點三組BALB/c小鼠的角膜熒光素染色評分。采用Wilcoxon秩和檢驗比較同一組不同時間點角膜熒光素染色評分。采用完全隨機設(shè)計資料的方差分析比較同一時間點三組的角膜上皮厚度、淚液分泌量、結(jié)膜杯狀細胞數(shù)量。采用t檢驗比較同一組不同時間點的角膜上皮厚度,淚液分泌量,結(jié)膜杯狀細胞數(shù)量。 結(jié)果 1. SchirmerⅠ實驗第0天,空白組、對照組、實驗組的酚紅棉線浸濕長度未見統(tǒng)計學(xué)差異(P0.05)。從第7天開始,實驗組酚紅棉線浸濕長度和第0天比較明顯減少(P0.01)。第7天酚紅棉線浸濕長度較第0天下降了28%,第42天較第0天下降66%。 2.角膜熒光素染色和評分第0天,空白組、實驗組、對照組均有BALB/c小鼠角膜少許點狀著色,角膜評分各組未見統(tǒng)計學(xué)差異(P0.05)。實驗組BALB/c小鼠隨著實驗的進行,角膜中央點狀著色增加,逐漸出現(xiàn)片狀著色,病變累及角膜各個象限,以角膜中央為重。從第7天起,實驗組角膜熒光素染色評分和第0天相比明顯升高(P0.01)。實驗組第7天角膜熒光素染色評分較第0天升高了75%。 3.角膜虎紅染色第0天,空白組、實驗組、對照組均有3只BALB/c小鼠角膜虎紅染色可見少量點狀著色。實驗組BALB/c小鼠從第7天開始,角膜點狀著色逐漸增加,并且出現(xiàn)片狀著色,累計角膜4個象限,以中央和鼻側(cè)角膜為重。 4.淚液蕨類實驗在各個檢查時間點,取空白組、對照組、實驗組BALB/c小鼠淚液,在光學(xué)顯微鏡下進行淚液蕨類結(jié)晶圖像分析�？瞻捉M、對照組在各個檢查時間點均可觀察到形態(tài)良好的蕨類物形成。實驗組BALB/c小鼠淚液結(jié)晶中的蕨類物從第7天開始逐漸減少,蕨類物之間的連接中斷。第28天以后顯微鏡下僅能觀察到稀少的小蕨類物,甚至完全消失。 5.角膜上皮HE染色的光鏡觀察和角膜上皮厚度測量空白組和對照組BALB/c小鼠角膜HE染色光鏡觀察可見上皮分層良好,分4～5層,基底部細胞排列呈柱狀,靠近角膜表面逐漸變成鱗狀上皮細胞。實驗組BALB/c小鼠隨實驗進展基底層細胞部分缺失,上皮細胞分層紊亂,細胞層數(shù)減少,出現(xiàn)空泡。第0天和第7天,空白組、對照組、實驗組BALB/c小鼠的角膜上皮層厚度未見統(tǒng)計學(xué)差異(P0.05)。從第14天起,實驗組較對照組和空白組角膜上皮層厚度明顯減少(P 0.05)。 6.結(jié)膜上皮PAS染色光鏡觀察和杯狀細胞計數(shù)空白組和對照組BALB/c小鼠的結(jié)膜上皮細胞排列整齊,杯狀細胞形態(tài)完整,分布在上皮細胞間,主要分布在穹隆部。實驗組BALB/c小鼠隨實驗進展,上皮細胞排列紊亂,甚至出現(xiàn)缺失,杯狀細胞形態(tài)不一致,數(shù)量減少。在第42天部分BALB/c小鼠上方穹隆部杯狀細胞完全缺失。 7.掃描電子顯微鏡觀察在第42天,用掃描電子顯微鏡觀察空白組、對照組、實驗組BALB/c小鼠角膜上皮細胞�？梢娍瞻捉M和對照組上皮細胞形態(tài)呈多邊形,細胞間緊密連接,細胞表面滿布微絨毛。實驗組BALB/c小鼠角膜表面微絨毛丟失,角膜表層上皮細胞破壞。 結(jié)論 1.用高滲鹽水給BALB/c小鼠點眼可以在小鼠角膜和結(jié)膜引起與干眼病患者類似的體征和組織病理學(xué)變化。 2.用高滲鹽水給BALB/c小鼠點眼是一個簡單的,可靠,重復(fù)性好的模型,這個模型可以運用于干眼病發(fā)病機制和藥物治療的研究。 3.淚液高滲透壓可能在干眼病發(fā)病機制中起重要作用。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R777.2;R-332

【引證文獻】

相關(guān)碩士學(xué)位論文 前1條

1 張捷;干眼模型的制作與透明質(zhì)酸鈉/殼聚糖滴眼治療干眼癥的體內(nèi)研究[D];華南理工大學(xué);2012年

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本文編號：1777432