地塞米松诱导兔眼高眼压的相关研究

发布时间：2018-05-26 22:12

本文选题：11β-羟类固醇脱氢酶1 + 生理功能　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:激素性青光眼又称糖皮质激素诱发性青光眼(glucocorticoid induced glaucoma,GIG),是一种因长时间全身或局部应用糖皮质激素引起的以眼压升高可伴视野损害为主要表现的眼部疾病。糖皮质激素因其强大的抗炎、抗过敏、免疫抑制作用而被广泛应用于临床治疗各种疾病,以致GIG的发病率较以往增加。其发生机理尚不完全清楚,目前公认的说法是糖皮质激素影响了小梁网组织正常的生长、代谢、结构及功能,从而导致房水排出受阻引起眼压升高。11β-羟类固醇脱氢酶1(11 beta-hydroxysteroid dehydrogenase type 1,11β-HSD1)被誉为“糖皮质激素效应的扩大器”,是糖皮质激素在组织水平上的受体前调节剂,可使局部组织无活性的可的松被激活,从而转化成为具有活性的皮质醇,增加局部组织活性皮质醇的浓度,促进糖皮质激素的作用。本研究通过对新西兰大耳白兔用0.5%地塞米松磷酸钠注射液结膜下注射联合0.1%地塞米松滴眼液眼表面点滴的方法,建立激素性高眼压(ocular hypertension,OHT)动物模型,观察并探讨11β-HSD1在兔OHT模型睫状体及小梁网中的表达和意义。方法:选15只雄性,兔龄在12-16周,体重为(2.5-3kg),经眼部经裂隙灯检查排除眼部疾病的健康新西兰大耳白兔(河北医科大学动物实验中心)纳入本实验。于清洁动物室内行适应性喂养7天,使其与实验人员接触,适应环境,同时训练白兔接受局部滴眼药(生理盐水),并适应在表面麻醉下进行眼压测量。饲养室温控制在21-24℃,相对湿度55%-65%,通风干燥,安静,12小时日光交替照射,每天定时饮食,自由饮水。采用随机数字表法将15只白兔随机分为正常对照组5只,实验组10只,以每只兔左眼为实验对象。丙美卡因滴眼液表面麻醉下,用schiotz眼压计测量眼压。每只眼测量2次,取平均值,测量完毕后,局部点妥布霉素滴眼液预防感染。于给药前3天的每日8:00、10:00、12:00测量每只白兔的双眼眼压,最后求取平均值以获得基础眼压值。第四天起,实验组选择固定时间(8:00)及固定地点每隔1天在兔左眼上方球结膜下注射0.5%地塞米松磷酸钠注射液5mg(1ml),每次注射后给予妥布霉素滴眼液点眼,晚上涂妥布霉素眼膏,用来避免感染,次日用0.1%地塞米松滴眼液(妥布霉地塞米松滴眼液)点白兔左眼4次,共持续7周;对照组按同样方法注射或眼表面点滴等容积无菌生理盐水。每3天监测眼压,以眼压升高达24mm Hg以上并能持续1周者为造模成功。给药7周后观察1周,以5ml利多卡因注入兔耳缘静脉过量致死,立即剥离眼球周围软组织,取下眼球。使用剪刀及锋利的手术刀片沿眼球赤道部将其剖开,弃去后半部分,小心取出晶状体。将剩下的包含角膜、虹膜、睫状体、房角及前部巩膜的眼前段组织沿纵轴于12点位对半剖开,一半固定于10%中性缓冲福尔马林液中48h后制作成石蜡标本用于HE染色和免疫组织化学染色;另一半标本将除小梁网及睫状体组织外的其余组织剔除后置于小液氮瓶中,于-80℃冰箱中保存,以备逆转录-PCR(reverse transcription-PCR,RT-PCR)检测。用冻存的睫状体组织匀浆提取总RNA,紫外分光光度计检测,逆转录合成c DNA,取每一个标本的扩增产物适量(6ul)于1%的含GV核酸染料的琼脂糖凝胶中进行电泳,以DNA Marker作为标准片段来标记,电泳后用紫外透射仪观察,并用数码相机照相,然后输入微机应用Quantity One凝胶图象分析软件,对目的电泳条带进行分析,而相应的内参电泳条带作为参照,结果用两者的积分吸光度比值来表示。统计学方法采用SPSS21统计学软件(美国IBM公司)进行统计分析。实验测试指标的数据资料经Shapiro-Wilk检验呈正太分布,以??S表示。采用完全随机分组两水平实验设计,实验组和对照组造模前后不同时间眼压值的差异比较采用重复测量两因素方差分析。两个组间llβ-HSD1基因在兔眼睫状体中相对表达量的差异比较采用独立样本t检验。P㩳0.05为差异有统计学意义。结果:15只参与实验的新西兰白兔的双眼平均基础眼压为18.97±2.92mm Hg。实验组和对照组的基础眼压分别为19.42±2.10mm Hg和18.53±2.99mm Hg(P㧐0.05)。实验组兔眼自用药第3周眼压开始升高26.07±2.17 mm Hg,到第4周时达到峰值26.77±4.37 mm Hg。从第3周开始,实验组眼压升高值与对照组及基础眼压比较,有显著统计学差异。在给药的7周时间内,对照组各时间点之间的眼压变化没有显著的统计学差异(P㧐0.05)。实验组眼压升高率为90%。实验组中,用光镜观察经HE染色后的兔眼小梁网组织,发现其较正常对照组细胞数量减少,核染色较深,细胞边界模糊,小梁细胞外基质增多,小梁网结构紊乱,小梁束之间的空隙缩小。睫状体组织在2组兔眼标本中未发现有明显的形态学改变。llβ-HSD1蛋白在实验组及对照组兔眼睫状体组织中均有表达,分布于睫状体组织无色素上皮细胞层,色素上皮层及基质层均未见表达,且实验组表达明显强于对照组。RT-PCR检测显示实验组兔眼模型睫状体组织中llβ-HSD1m RNA的表达水平较对照组升高。Quantity One软件定量分析显示,实验组兔眼llβ-HSD1表达量均值为0.86±0.07,对照组表达量平均值为0.35±0.06,2个组间的差异有统计学意义(P㩳0.05)。结论:1结膜下注射联合眼表面点滴地塞米松可以成功诱导新西兰大耳白兔眼压升高。2光镜下经HE染色的兔眼小梁网组织较正常对照组细胞减少,核染色较深,边界模糊,细胞外基质增多,小梁网结构紊乱,小梁束之间的空隙缩小。3 llβ-HSD1在实验组与对照组睫状体无色素上皮层中均有表达,实验组较对照组明显增多,提示局部外源性糖皮质激素增加可刺激llβ-HSD1表达,激活更多内源性糖皮质激素,促进眼压升高。
[Abstract]:Objective: hormone glaucoma, also known as glucocorticoid induced glaucoma (glucocorticoid induced glaucoma, GIG), is an ocular disease caused by prolonged systemic or local use of glucocorticoids with elevated intraocular pressure and visual field damage. It is widely used in the clinical treatment of various diseases, so that the incidence of GIG is more than before. Its mechanism is not completely clear. It is generally accepted that glucocorticoid affects the normal growth, metabolism, structure and function of trabecular meshwork, resulting in the increase of.11 beta hydrosteroid dehydrogenase 1 (11) caused by the obstruction of aqueous humor. Beta-hydroxysteroid dehydrogenase type 1,11 beta -HSD1, known as the "enlarger of glucocorticoid effect", is a receptor precursor of glucocorticoids at the tissue level, which can activate the inactive cortisone in local tissues, thus transforming into active Corticosterol, increasing the concentration of local tissue active cortisol, and promoting the concentration of the local tissue activity cortisol. The role of glucocorticoid in this study was to establish an animal model of ocular hypertension (OHT) by injection of 0.5% Dexamethasone Sodium Phosphate Injection conjunctiva and 0.1% dexamethasone eye drops in New Zealand white rabbits, and to observe and explore the 11 beta -HSD1 in rabbit OHT ciliary body and small Liang Wangzhong in rabbit model. Methods: 15 males were selected for 12-16 weeks of age and (2.5-3kg). The healthy New Zealand white rabbit (Hebei Medical University animal experiment center), which was checked out of the eye diseases by the slit lamp, was included in this experiment. For 7 days in the clean animal room, the rabbits were fed to the laboratory for 7 days to adapt to the environment and adapt to the environment. The white rabbits were trained to receive local eye drops (physiological saline) and adapt to the measurement of intraocular pressure under surface anaesthesia. Room temperature control at 21-24 C, relative humidity 55%-65%, ventilation, quiet, 12 hour daylight alternate irradiation, daily diet and free drinking water. 15 rabbits were randomly divided into 5 rats in a normal control group by random digital table method. Group 10, taking the left eye of each rabbit as the experimental object. Under the surface anaesthesia of the eye drops, the eye pressure was measured by the Schiotz tonometer. The average value was measured 2 times per eye. After the measurement, the local point Tobramycin Eye Drops was prevented from infection. The intraocular pressure of each white rabbit was measured by 8:00,10:00,12:00 daily 3 days before the administration, and the average of each white rabbit was measured at the end. Value for basic intraocular pressure (IOP). From fourth days, the experimental group selected the fixed time (8:00) and the fixed place to injecting 0.5% Dexamethasone Sodium Phosphate Injection 5mg (1ml) under the bulbar conjunctiva above the left eye of the rabbit every 1 days. After each injection, the eye was given to Tobramycin Eye Drops, and the Tobramycin Eye Ointment was painted at night to avoid infection, and 0.1% dexamethasone was used the next day. Eye drops (dexamethasone eye drops) for 4 times in the left eye of white rabbits for a total of 7 weeks. The control group was injected with the same volume of aseptic saline by the same method or the eye drops. The intraocular pressure was monitored every 3 days, the intraocular pressure was up to 24mm Hg above and the patients who were able to continue for 1 weeks were successful. The drug was injected with 5ml lidocaine into the rabbit ear margin for 1 weeks after 7 weeks. Dissection the soft tissue around the eyeball immediately and remove the eyeball. Use the scissors and sharp surgical blades to open it along the equator, discard the posterior part, carefully remove the lens. The left anterior segments of the cornea, iris, ciliary body, angle and anterior sclera are half opened along the longitudinal axis at 12 points, half fixed. Paraffin specimens were made after 48h in 10% neutral buffered formalin solution for HE staining and immunohistochemical staining; the other half of the specimens were removed from the small beam net and ciliary body tissues and stored in a small liquid nitrogen bottle and stored in the refrigerator at -80 C to prepare the reverse transcriptase -PCR (reverse transcription-PCR, RT-PCR) detection. The total RNA was extracted from the ciliary body homogenate, and the C DNA was synthesized by the ultraviolet spectrophotometer. The amplification products of each specimen (6ul) were obtained in 1% of the agarose gel containing GV nucleic acid dye, and were marked with DNA Marker as the standard fragment. After the electrophoresis, it was observed with the ultraviolet transmission instrument, and was photographed with a digital camera, and then input and input. Quantity One gel image analysis software is used to analyze the purpose of the electrophoresis strip, and the corresponding electrophoresis strip is used as the reference. The results are expressed by the ratio of the absorbance absorbance of the two. The statistical method is analyzed by the SPSS21 statistics software (American IBM company). The data of the experimental test index by Shapiro-Wi The LK test was a positive distribution, with the???? S. The two level experiment was designed with complete random grouping. The difference of intraocular pressure between the experimental group and the control group was compared with the two factor analysis of variance. The difference of the relative expression of ll beta -HSD1 gene in the ciliary body of the two groups was compared with the independent sample t test.P? 0 Results: the basic intraocular pressure of the 15 New Zealand white rabbits in the experimental group was 19.42 + 2.10mm Hg and 18.53 + 2.99mm Hg (P? 0.05), respectively. The intraocular pressure of the rabbits in the experimental group began to rise 26.07 + 2.17 mm Hg in the experimental group, and reached the peak to fourth weeks at the fourth week. The value of intraocular pressure in the experimental group was 26.77 + 4.37 mm Hg. from third weeks. There was significant difference between the experimental group and the control group and the basic intraocular pressure. In the 7 week period of the administration, there was no significant difference in the intraocular pressure between the control groups (P? 0.05). The increase rate of intraocular pressure in the experimental group was in the 90%. experimental group, and the HE staining was observed by light microscopy. After the trabecular meshwork, the number of cells in the trabecular meshwork in the rabbit eyes was reduced, the nucleus staining was deep, the cell boundary was blurred, the extracellular matrix of trabeculae increased, the structure of trabecular meshwork was disorganized and the gap between the trabeculae narrowed. The ciliary body tissues had not found obvious morphological changes of.Ll beta -HSD1 protein in the experimental group and the pair in the 2 groups of rabbit eyes. The expression of the ciliary body tissue in the group of rabbit eyes was expressed in the ciliary body tissue, the pigment epithelium and the matrix layer were not expressed, and the expression of the experimental group was significantly stronger than that of the control group.RT-PCR. The expression of ll beta -HSD1m RNA in the rabbit eye model of the experimental group was higher than that in the control group by.Quantity One software. Quantitative analysis showed that the mean value of ll beta -HSD1 expression in rabbit eyes was 0.86 + 0.07, and the average value of the control group was 0.35 + 0.06,2 (P? 0.05). Conclusion: 1 subconjunctival subconjunctival injection combined with dexamethasone can successfully induce the increase of intraocular pressure in New Zealand white rabbits by.2 light microscopy with HE stained rabbit eyes. The tissue of trabecular meshwork was less than that in the normal control group. The nucleus staining was deeper, the boundary was blurred, the extracellular matrix was increased, the structure of the trabecular meshwork was disorganized, the gap between the trabecular beams and the.3 ll beta -HSD1 was expressed in the experimental group and the control group, and the experimental group was significantly increased, suggesting local exogenous glucocorticoids. The increase can stimulate the expression of ll beta -HSD1, activate more endogenous glucocorticoids, and promote the increase of intraocular pressure.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R775

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