作者:李雪,徐锦堂,崔浩,胡琦
作者单位:1(150001)中国黑龙江省哈尔滨市,哈尔滨医科大学第一附属医院眼科;2(510632)中国广东省广州市,暨南大学医学院第一附属医院眼科
【摘要】目的:通过检测角膜烧伤后前房水中TGFβ2含量的变化,探讨在眼部内环境下TGFβ2与角膜烧伤之间的关系,为角膜烧伤治疗提供依据。
方法:制备Wistar大鼠角膜碱、酸、热中度烧伤动物模型,分别于伤后4,8h;1,3,5,7,14,28,49d处死。采集房水:用20g/L BSA包被的毛细管于角膜缘处刺入角膜,吸取房水至20g/L BSA包被的离心管中,70℃保存。标准品和样品进行酸激活处理。使用ELISA方法检测角膜烧伤后前房水中TGFβ2的水平。
结果:大鼠角膜碱烧伤后4h TGFβ2的水平呈现一过性升高,然后下降,3d时TGFβ2含量再次升高,7d时达高峰,持续至7wk时降至正常。大鼠角膜酸烧伤后4h TGFβ2的水平呈现一过性升高,然后下降,低于正常对照组,两者差异显著。7d 时房水中TGFβ2升至正常,然后一直保持低平状态。49d时我们检测房水中TGFβ2水平仍低于正常。大鼠角膜热烧伤后4h TGFβ2的水平呈现一过性升高,1d时降至最低,3d时开始增长,直到7d时我们检测仍高于正常,然后缓慢下降。
结论:角膜烧伤后前房水中TGFβ2的含量发生变化,致伤原因不同,其变化方式不同。角膜碱烧伤和热烧伤后于7d时出现TGFβ2高峰,说明此时角膜处于免疫功能抑制状态,这对于维持角膜前房的免疫赦免状态非常重要,证明此时机体发挥了自我保护功能,避免产生过度的病理反应。角膜酸烧伤后TGFβ2水平始终低平,其原因尚有待于进一步研究。
【关键词】 转化生长因子β2;角膜;碱烧伤;酸烧伤;热烧伤
Expression of TGFβ2 in rat aqueous humor following corneal alkali burns, acid burns and thermal burns
Xue Li, JinTang Xu, Hao Cui , Qi Hu
Foundation item: Natural Science Foundation of Science and Technology Office of Heilongjiang Province (No. QC05C42)
1Department of Ophthalmology, the First Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China; 2 Department of Ophthalmology, the First Affiliated Hospital of Medical Institute Jinan University, Guangzhou 510632, Guangdong Province, China
Correspondence to: JinTang Xu. Department of Ophthalmology, the First Affiliated Hospital of Medical Institute Jinan University, Guangzhou 510632, Guangdong Province, China. [email protected]
AbstractAIM: To study the relationship between TGFβ2 and ocular burns by detecting the levels of TGFβ2 in rat aqueous humor following corneal alkali burns, acid burns and thermal burns and to provide experimental clues. METHODS: The models of alkali burns, acid burns and thermal burns in Wistar rats were established and the animals were sacrificed at 4, 8 hours, 1, 3 day, 1, 2, 4 and 7 weeks after burn. Aqueous humor was obtained by the anterior chamber paracentesis and frozen at 70℃. 1mol/L HCl was added to the samples to acid activation. The levels of TGFβ2 were assessed with a specificcapture ELISA kit.
RESULTS: The levels of TGFβ2 in rat aqueous humor following corneal alkali burns increased at 4 hours after injury and decreased quickly. The second increase was appeared at 3 days, reached to the peak at 1 week and came back to the normal at 7 weeks. The levels of TGFβ2 in rat aqueous humor following corneal acid burns increased at 4 hours after injury and decreased quickly. There was significant difference compared to the control. The levels of TGFβ2 increased to the normal at 1 week and lasted low expression as far as 7 weeks. The levels of TGFβ2 in rat aqueous humor following corneal thermal burns increased at 4 hours after injury and decreased quickly to the lowest expression at 1 day. We observed the second increase at 3 days and it lasted higher expression for long time.
CONCLUSION: The levels of TGFβ2 in rat aqueous humor changes following ocular burns. There is difference among different burns. The levels of TGFβ2s peak appears respectively at 1 week following alkali burns and thermal burns show the cornea be in immunosuppressive state this time and it is very important to maintain the immune privileged status of the anterior chamber. It is proved that the selfsafeguard of the body avoids excessive pathological reaction. The reason of low levels of TGFβ2 following acid burns needs more study. KEYWORDS: TGFβ2; cornea; alkali burn; acid burn; thermal burn
0引言
眼前房是典型的免疫赦免部位,保护精细的眼部组织避免免疫的损害,对外来抗原的刺激产生独特的免疫反应,这主要是因为:(1)血房水屏障的存在,限制血液中的细胞和分子进入眼内,从而减少特异性和非特异性的免疫反应发生;(2)角膜无血管和淋巴管;(3)房水中有许多细胞因子,如TGFβ,TNFα,IL1,IFN等,在完成房水的生理功能方面发挥着重要的调控作用[1]。目前认为,维持眼的免疫抑制环境,保持前房的免疫赦免特性,与房水中的抑制性免疫调节因子TGFβ,特别是TGFβ2密切相关[2,3]。Cousins等[4]用点杂交法证明房水中有TGFβ存在,其中80%~90%是TGFβ2,它在房水中的浓度高于血清,这对于维持前房的免疫赦免状态非常重要。许多研究业已证实,角膜碱烧伤后无论是在体液免疫方面还是在细胞免疫方面都表现为过度的免疫应答[57]。为了研究角膜碱、酸、热烧伤后TGFβ2在眼内环境中的作用,我们利用ELISA方法检测三种烧伤后房水中TGFβ2含量的变化。
1材料和方法
1.1材料
Wistar大鼠125只,体质量250g~260g,购自哈尔滨兽医研究所实验动物中心。大鼠TGFβ2 ELISA试剂盒(上海森雄公司),洗涤液(重蒸水1∶20稀释),20g/L牛血清白蛋白(BSA)(Promega),1mol/L HCl,1mol/L NaOH等。
1.2方法
1.2.1实验分组
按照随机数字表法分为正常对照组、碱烧伤组、酸烧伤组和热烧伤组,其中正常对照组5只,碱、酸、热烧伤组各40只。角膜烧伤组再根据烧伤后不同时间段4,8h;1,3,7,14,28,49d分为8组,每组5只。
1.2.2大鼠角膜中度烧伤动物模型制备
角膜碱烧伤模型:大鼠乙醚麻醉后,取直径为5mm的单层滤纸,浸入到1mol/L NaOH中10s,将浸碱的滤纸贴在大鼠双眼角膜中央部60s,除去滤纸,用生理盐水冲洗5min。双眼结膜囊内涂5g/L红霉素眼膏和10g/L阿托品眼膏。角膜酸烧伤模型:大鼠乙醚麻醉后,取直径为5mm的单层滤纸,浸入到0.5mol/L H2SO4中10s,将浸酸的滤纸贴在大鼠双眼角膜中央部60s,除去滤纸,用生理盐水冲洗5min。双眼结膜囊内涂5g/L红霉素眼膏和10g/L阿托品眼膏。角膜热烧伤模型:大鼠乙醚麻醉后,取直径为5mm的铁片,厚度为1mm,酒精灯加热30s,置于大鼠角膜中央约30s。双眼结膜囊内涂5g/L红霉素眼膏和10g/L阿托品眼膏。
1.2.3大鼠前房水标本采集
分别于角膜酸、碱、热烧伤后各时间段将大鼠麻醉,于角膜缘内偏下方将20g/L BSA包被的毛细管刺入角膜,吸取前房房水,用吸球吹入20g/L BSA包被的Eppendof管内,70℃保存。
1.2.4样品和标准品酸激活处理
将450 μL样品稀释液加入到一支1.5mL聚丙烯管中,再加10μL房水。加20 μL 1mol/L HCL,盖紧,上下混匀。4℃放置60min。加20μL 1mol/L NaOH,盖紧,上下混匀。
1.2.5 ELISA方法检测前房水中TGFβ2的含量
建立标准曲线:设标准孔8孔,每孔中各加入样品稀释液100μL,第1孔加标准品100μL,混匀后用加样器吸出100μL,移至第2孔。如此反复作对倍稀释至第7孔,最后从第7孔吸出100μL弃去,使之体积均为100μL。第8孔为空白对照。检测:待测孔中每孔加入已激活待测房水100μL,将反应板置37℃ 2h,用洗涤液将反应板充分洗涤4~6次,在滤纸上印干。每孔中加入第一抗体工作液50μL,将反应板充分混匀后置37℃ 1h,用洗涤液将反应板充分洗涤4~6次,在滤纸上印干。每孔加酶标抗体工作液100μL,将反应板置37℃ 1h。洗板。每孔加底物工作液100μL,置37℃暗处反应5~10min。每孔加入1滴终止液混匀,在492nm处测吸光值。结果计算:以标准品2000,1000,500,250,125,62.5,32,0ng/L的A值作图,画出标准曲线,根据样品的A值在该曲线图上查出该样品相应的TGFβ2的含量。
统计学处理:所得数据应用SPSS 10.0统计分析软件进行处理,统计学方法采用方差分析(F检验)。
2结果
大鼠角膜烧伤后前房水中TGFβ2的含量变化:(1)碱烧伤:大鼠角膜碱烧伤后4h TGFβ2的水平呈现一过性升高,然后下降,3d时TGFβ2含量再次升高,7d时达高峰,持续至49d时降至正常(图1)。(2)酸烧伤:角膜酸烧伤后4h TGFβ2的水平呈现一过性升高,然后下降,低于正常对照,两者差异显著。7d时房水中TGFβ2升至正常,然后一直保持低平状态。第49d时我们检测房水中TGFβ2水平仍低于正常(图2)。(3)热烧伤:角膜热烧伤后4h TGFβ2的水平有一过性升高,1d时降至最低,3d时开始增长,直到7d时我们检测仍高于正常,然后缓慢下降(图3)。而不同时间房水中TGFβ2含量变化(图4)。
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