本文選題：11β-羥類固醇脫氫酶1 + 生理功能�。� 參考：《河北醫(yī)科大學》2017年碩士論文

【摘要】：目的:激素性青光眼又稱糖皮質(zhì)激素誘發(fā)性青光眼(glucocorticoid induced glaucoma,GIG),是一種因長時間全身或局部應用糖皮質(zhì)激素引起的以眼壓升高可伴視野損害為主要表現(xiàn)的眼部疾病。糖皮質(zhì)激素因其強大的抗炎、抗過敏、免疫抑制作用而被廣泛應用于臨床治療各種疾病,以致GIG的發(fā)病率較以往增加。其發(fā)生機理尚不完全清楚,目前公認的說法是糖皮質(zhì)激素影響了小梁網(wǎng)組織正常的生長、代謝、結(jié)構及功能,從而導致房水排出受阻引起眼壓升高。11β-羥類固醇脫氫酶1(11 beta-hydroxysteroid dehydrogenase type 1,11β-HSD1)被譽為“糖皮質(zhì)激素效應的擴大器”,是糖皮質(zhì)激素在組織水平上的受體前調(diào)節(jié)劑,可使局部組織無活性的可的松被激活,從而轉(zhuǎn)化成為具有活性的皮質(zhì)醇,增加局部組織活性皮質(zhì)醇的濃度,促進糖皮質(zhì)激素的作用。本研究通過對新西蘭大耳白兔用0.5%地塞米松磷酸鈉注射液結(jié)膜下注射聯(lián)合0.1%地塞米松滴眼液眼表面點滴的方法,建立激素性高眼壓(ocular hypertension,OHT)動物模型,觀察并探討11β-HSD1在兔OHT模型睫狀體及小梁網(wǎng)中的表達和意義。方法:選15只雄性,兔齡在12-16周,體重為(2.5-3kg),經(jīng)眼部經(jīng)裂隙燈檢查排除眼部疾病的健康新西蘭大耳白兔(河北醫(yī)科大學動物實驗中心)納入本實驗。于清潔動物室內(nèi)行適應性喂養(yǎng)7天,使其與實驗人員接觸,適應環(huán)境,同時訓練白兔接受局部滴眼藥(生理鹽水),并適應在表面麻醉下進行眼壓測量。飼養(yǎng)室溫控制在21-24℃,相對濕度55%-65%,通風干燥,安靜,12小時日光交替照射,每天定時飲食,自由飲水。采用隨機數(shù)字表法將15只白兔隨機分為正常對照組5只,實驗組10只,以每只兔左眼為實驗對象。丙美卡因滴眼液表面麻醉下,用schiotz眼壓計測量眼壓。每只眼測量2次,取平均值,測量完畢后,局部點妥布霉素滴眼液預防感染。于給藥前3天的每日8:00、10:00、12:00測量每只白兔的雙眼眼壓,最后求取平均值以獲得基礎眼壓值。第四天起,實驗組選擇固定時間(8:00)及固定地點每隔1天在兔左眼上方球結(jié)膜下注射0.5%地塞米松磷酸鈉注射液5mg(1ml),每次注射后給予妥布霉素滴眼液點眼,晚上涂妥布霉素眼膏,用來避免感染,次日用0.1%地塞米松滴眼液(妥布霉地塞米松滴眼液)點白兔左眼4次,共持續(xù)7周;對照組按同樣方法注射或眼表面點滴等容積無菌生理鹽水。每3天監(jiān)測眼壓,以眼壓升高達24mm Hg以上并能持續(xù)1周者為造模成功。給藥7周后觀察1周,以5ml利多卡因注入兔耳緣靜脈過量致死,立即剝離眼球周圍軟組織,取下眼球。使用剪刀及鋒利的手術刀片沿眼球赤道部將其剖開,棄去后半部分,小心取出晶狀體。將剩下的包含角膜、虹膜、睫狀體、房角及前部鞏膜的眼前段組織沿縱軸于12點位對半剖開,一半固定于10%中性緩沖福爾馬林液中48h后制作成石蠟標本用于HE染色和免疫組織化學染色;另一半標本將除小梁網(wǎng)及睫狀體組織外的其余組織剔除后置于小液氮瓶中,于-80℃冰箱中保存,以備逆轉(zhuǎn)錄-PCR(reverse transcription-PCR,RT-PCR)檢測。用凍存的睫狀體組織勻漿提取總RNA,紫外分光光度計檢測,逆轉(zhuǎn)錄合成c DNA,取每一個標本的擴增產(chǎn)物適量(6ul)于1%的含GV核酸染料的瓊脂糖凝膠中進行電泳,以DNA Marker作為標準片段來標記,電泳后用紫外透射儀觀察,并用數(shù)碼相機照相,然后輸入微機應用Quantity One凝膠圖象分析軟件,對目的電泳條帶進行分析,而相應的內(nèi)參電泳條帶作為參照,結(jié)果用兩者的積分吸光度比值來表示。統(tǒng)計學方法采用SPSS21統(tǒng)計學軟件(美國IBM公司)進行統(tǒng)計分析。實驗測試指標的數(shù)據(jù)資料經(jīng)Shapiro-Wilk檢驗呈正太分布,以??S表示。采用完全隨機分組兩水平實驗設計,實驗組和對照組造模前后不同時間眼壓值的差異比較采用重復測量兩因素方差分析。兩個組間llβ-HSD1基因在兔眼睫狀體中相對表達量的差異比較采用獨立樣本t檢驗。P㩳0.05為差異有統(tǒng)計學意義。結(jié)果:15只參與實驗的新西蘭白兔的雙眼平均基礎眼壓為18.97±2.92mm Hg。實驗組和對照組的基礎眼壓分別為19.42±2.10mm Hg和18.53±2.99mm Hg(P㧐0.05)。實驗組兔眼自用藥第3周眼壓開始升高26.07±2.17 mm Hg,到第4周時達到峰值26.77±4.37 mm Hg。從第3周開始,實驗組眼壓升高值與對照組及基礎眼壓比較,有顯著統(tǒng)計學差異。在給藥的7周時間內(nèi),對照組各時間點之間的眼壓變化沒有顯著的統(tǒng)計學差異(P㧐0.05)。實驗組眼壓升高率為90%。實驗組中,用光鏡觀察經(jīng)HE染色后的兔眼小梁網(wǎng)組織,發(fā)現(xiàn)其較正常對照組細胞數(shù)量減少,核染色較深,細胞邊界模糊,小梁細胞外基質(zhì)增多,小梁網(wǎng)結(jié)構紊亂,小梁束之間的空隙縮小。睫狀體組織在2組兔眼標本中未發(fā)現(xiàn)有明顯的形態(tài)學改變。llβ-HSD1蛋白在實驗組及對照組兔眼睫狀體組織中均有表達,分布于睫狀體組織無色素上皮細胞層,色素上皮層及基質(zhì)層均未見表達,且實驗組表達明顯強于對照組。RT-PCR檢測顯示實驗組兔眼模型睫狀體組織中l(wèi)lβ-HSD1m RNA的表達水平較對照組升高。Quantity One軟件定量分析顯示,實驗組兔眼llβ-HSD1表達量均值為0.86±0.07,對照組表達量平均值為0.35±0.06,2個組間的差異有統(tǒng)計學意義(P㩳0.05)。結(jié)論:1結(jié)膜下注射聯(lián)合眼表面點滴地塞米松可以成功誘導新西蘭大耳白兔眼壓升高。2光鏡下經(jīng)HE染色的兔眼小梁網(wǎng)組織較正常對照組細胞減少,核染色較深,邊界模糊,細胞外基質(zhì)增多,小梁網(wǎng)結(jié)構紊亂,小梁束之間的空隙縮小。3 llβ-HSD1在實驗組與對照組睫狀體無色素上皮層中均有表達,實驗組較對照組明顯增多,提示局部外源性糖皮質(zhì)激素增加可刺激llβ-HSD1表達,激活更多內(nèi)源性糖皮質(zhì)激素,促進眼壓升高。
[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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R775

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