【作者】 宋静；

【导师】 肖利华；

【作者基本信息】 河北医科大学 ， 眼科学， 2008， 硕士

【摘要】 目的:甲状腺相关眼病（ thyroid associated ophthalmopathy, TAO）属器官特异性自身免疫性疾病,眼球突出是其主要的临床体征,发病率居成年组眼眶病之首,约占20%。该病通常存在自限性,只有不足10%的患者会出现视力模糊、视神经障碍、角膜溃疡、全眼球炎甚至失明等严重后果。国内外一致认为自身免疫性疾病是机体免疫系统针对自身抗原产生免疫应答,以致破坏自身正常组织结构并引起相应的临床症状。TAO在环境、遗传等背景存在下,自身耐受遭到破坏,机体对自身抗原识别,产生了自身反应性T细胞,在多种细胞因子作用下,T细胞活化、迁移至眶后组织,引起特定组织损伤,导致疾病发生。临床实践中我们对TAO患者的T细胞活化状态的准确评估是获得较好疗效的前提。对于活动期TAO患者多采用皮质类固醇和免疫抑制剂药物联合应用,辅以局部放疗,小剂量化疗;而对于静止期的患者,药物疗效并不明显,在压迫性视神经病变引起视力急剧下降的情况下可采取提上睑肌延长术及眶减压术等手术治疗方案。T细胞膜表面存在IL-2受体α链（CD25）,该CD分子的表达为T细胞激活的第一事件,在维持自身免疫耐受中发挥重要作用。本实验应用原子力显微镜（Atomic Force Microscopy, AFM）对TAO患者外周血T细胞的膜表面进行观测,第一部分比较正常组与TAO患者T细胞之间的差异,探索可供研究的参数指标;第二部分进一步观测静脉注射激素治疗TAO患者用药前后,T细胞超微结构是否存在变化,这些改变是否能够通过AFM所提供的参数客观的表现出来;第三部分初步探索T细胞表面的超微结构改变与CD25分子含量的相关性,探讨AFM所观测的指标是否可以作为反映细胞功能活化状态的客观依据,从而能够反映TAO免疫活动程度。方法:将研究对象分为三组:正常对照组（n=12例）、TAO活动期组（n=18例）和TAO静止期组（n=22例）。1、外周血单个核细胞的分离:取晨起空腹肝素抗凝外周血3ml,用PBS缓冲液稀释到2倍体积,充分混匀后沿管壁缓慢地加入事先已装有3ml聚蔗糖-泛影葡胺淋巴细胞分离液的14ml一次性离心管内,水平式转头500xg离心20min,取悬浮于血浆与分层液的界面之间灰白层,移入另一离心管,用5倍以上PBS缓冲液充分洗涤,200xg离心10 min,2次。2、Dynal免疫磁珠分离纯化T细胞:将分离得到的单个核细胞沉淀放置于4℃下,加入一定量Dynabeads（72μl beads/ml PBMC）再加入1 ml左右的PBS（2 % FCS）重悬细胞;把装有此混合液的试管置于DynalMPC上2～3 min;弃去悬浮液,T细胞黏附在试管与DynalMPC接触的一侧;上述细胞悬浮液每100μl加入10μlDETACHaBEAD,放置室温孵育45～60min,不断温和混匀;再置于DynalMPC上2min;吸取悬浮液至新的试管中。3、将获得的T细胞一部分用于活性检测和鉴定:台盼蓝排斥实验检测细胞活性;HE、免疫细胞化学染色鉴定T细胞,流式细胞分析技术检测CD3⁺T细胞的纯度。4、细胞培养:将试管中其余所有T细胞置于5%CO2的孵箱中,于37℃培养48h。5、AFM样品的制备和观测:将培养细胞离心,用2.5%戊二醛溶液中固定30min,在三蒸水中浸泡过夜,载玻片置于超净台空间下自然晾干。AFM选择Tapping模式,对每个细胞进行整体扫描,然后选择最亮区域改变扫描范围为1um,扫描速率为2Hz进行局部扫描。一个血样制成一个样本,每个样本扫描10个细胞,每个细胞扫描20个区域,求平均数代表一个样本的测量数,图像经仪器平滑处理。数据采集和分析。6、应用上述方法对TAO活动期患者大剂量激素冲击治疗前后T细胞进行对比观测。7、应用流式细胞仪测得TAO外周血T细胞CD25分子表达百分含量,与AFM所测得的参数进行相关性分析。结果:1、经常规分离、培养的正常组和TAO组两个分期的T细胞在形状上均呈现较为规则的圆形;正常组T细胞直径约在5-8um,高度在600-850nm之间,表面总体较光滑但其超微结构也并非完全均一,而TAO急性期T细胞直径偏大,约在9-11um之间,表面的凹凸不平较明显,出现明显的颗粒状突起及凹陷,边缘还可观察到有细微的树枝状突起分布;静止期的T细胞直径略小,约在8-10um之间,而膜表面的粗糙程度介于正常组和TAO急性期组之间。2、应用单因素方差分析将三组T细胞的观测参数进行统计分析得出结果:Ra、Peak count、Rpm、Rvm、Surface area diff在α=0.05水平上,P<0.00,均存在统计学意义,有显著差异。3、应用多个样本间均数比较,Peak count, Surface area diff在三组间均存在差异,即正常对照组、TAO活动期组和静止期组两两之间存在差异。4、经过大剂量冲击治疗后,T细胞的直径变小,约为9-11um,表面的凹凸不平的程度也较治疗前有所下降。对AFM测量的指标,进行配对t检验,Peak count、Rpm、Rvm、Surface area diff存在显著性差异,表明活动期TAO患者激素治疗前后外周血T细胞膜表面存在超微结构的改变。5、应用多元线性回归以及曲线拟合方法,分析膜表面参数中Peak count、Rpm、Rvm、Surface area diff与T细胞膜表面CD25分子的相关性。CD25与AFM所测参数Rvm、Surface area diff、Peak count均存在相关性,运用逐步回归分析Rvm（x₂）、peakcount（x₁）,建立回归方程:Y=0.188x₁+0.56 x₂-0.219,本实验认为Rvm、peakcount可以作为衡量CD25表达含量多少的参考指标。结论:1、正常对照组与TAO活动期和静止期的T细胞在大小、膜表面超微结构均存在不同。经过多个样本间均数比较,有一些指标在三组之间存在两两差异,而一些指标在TAO静止期组和正常对照组之间并无差异,提示我们虽然静止期TAO的T细胞活性不如活动期表现明显,但是免疫系统仍处在某种不平衡的异常状态,可能只是细胞免疫应答的功能有所减退。2、使用激素前后,T细胞膜表面发生了超微结构的改变,抑制了T细胞的生物活性,从另一个角度推测糖皮质激素对于TAO患者的治疗可能是通过直接作用T细胞表面引起信号传导引起胞内变化而抑制T细胞自身免疫活性来发挥作用的。3、本实验认为TAO中活化T细胞表面表达的CD25分子含量与Rvm、peakcount具有相关性,可以通过测得Rvm、peakcount而间接估测CD25表达含量,以此了解T细胞的免疫活化程度。更多还原

【Abstract】 Objective: We wonder whether we can evaluate activation of T cell and the degree of inflammatory activity in TAO by AFM（Atomic Force Microscopy, AFM）.Thyroid associated ophthalmopathy （TAO） is an autoimmune disease, of which incidence is the highest in adult orbital disease, about 20%. It’s characterized clinically by exophthalmoses; eye muscle dysfunction and eyelid lag and retraction. Majorities of patients can relieve automatically, only less than 10% of whom will have incidences of blurred vision, keratohelcosis, even panophthalmitis. It is now generally accepted that TAO is an autoimmune disorder involving cell-mediated reactions against orbital antigens and humoral-mediated immunity. Clinically, it is prerequisite to evaluate the state of T cell’s activation in TAO for predominant therapy effect. There is no breakthrough in therapy, clinical doctors decide therapeutic regimen according to its severity and progressive inflammation of the orbital tissues. Administration of steroids or/and immunosuppressive drugs will be advised for a patient with progressive inflammation, followed by radiotherapy or chemical therapy. Operation will be a wisdom choice for one if the patient has optic nerve injury in the virtue of compression because there is no significant effect by drugs. Therapeutic effect has been not satisfying in the account of poor understanding of relationship between TAO and activation of T cell. Scholars are always eager for researching on etiology of TAO. CD25 is expressed in T cell membrane, which is the onset thing for activation of T cell, and plays a vital role in maintenance of immune tolerance. The objective of this research is to observe T cell membrane of TAO compared with normal group by AFM, to discover the differences in T cell membrane compared after administration of steroids drugs and clarify the correlation between CD25 levels of T cell membrane and parameters of AFM.Methods: Research objects are divided into three groups: normal group, acute stage and stationary stage of TAO ones.1 Extraction T cell from peripheral blood mononuclear cells: Whole blood was layered atop the upper gradient and the solution was centrifuged at 700xg for 30 min, which was isolated the mononuclear cells layer between plasma and Ficoll-Hypaque layers. All layers except mononuclear cells layer were discarded, and mononuclear cells were collected. The cells collected were then suspended in sterile distilled water to lyse any red blood cells contaminanted within the solution. Cells were centrifugated and suspended twice in RPMI to eliminate red blood cell ghosts. 2 Dynal immunomagnetic beads isolation method was used to separate T cell and CD3⁺ was chosen as a surface molecule marker of T cell to determine the purity of cells by FCM.3 Cultivate T cell in RPMI-1640 containing 10% fetal bovine serum and maintained in a 5% CO2 humidified atmosphere at 37℃for 48 hours.4 Fix T cells by 2.5% glutaraldehyde and soak them in ter-distilled water, and dried in air of super-clean bench.5 Before imaging, fixed cells were dehydrated by washing the fluid cell with 30, 50, and 70% solutions of ethanol for 7 min each. Treatment with ethanol removes the lipids from the membranes of cells but leaves behind skeletal proteins of the membranes when fixation is done with 2.5% glutaraldehyde alone. There is no evidence that the mild fixation with 2.5% glutaraldehyde used here appreciably disturbs the structures of the cells surfaces, at least to the resolution of these AFM studies. Amplitude and height images by AFM were obtained in the tapping mode with a scan rate of 2 Hz and an integral gain of 0.3 to 0.5. Statistical comparison was performed by using single-factor analysis of variance, and the results were presented with the P value.6 Compare with topographies of T cell after administration of steroid drugs by above methods.7 CD25 proliferations of T lymphocytes were determined with flow cytometry, analyse the correlation between CD25 levels and parameters of AFM for TAO. Results: 1 The topographies of three groups of T cell are different. The diameters and average roughnesses of T cells in acute stage of TAO are much more dominant than those of the other two kinds.2 Oneway- analysis of variance: The obvious differences are found between three groups in these parameters （Ra,Peak count,Rpm,Rvm,Surface area diff）. （P<0.05）3 Multiple comparisons: the mean differences（Peak count、Surface area diff） between two each groups are significant at the 0.05 level.4 One-sample T Test for analysis of T cells before and after administration of steroid drugs: the mean differences are significant at the 0.05 level for all statistic parameters.5 CD25 as dependent variables, P<0.05, there is a positive correlation between CD25 and three parameters (Rvm,Surface area diff,Peak count in AFM, analyze the correlation between CD25 and Rvm（x₂）、Peak count（x₁） by Stepwise method: Y=0.188x₁+0.56x₂-0.219.Conclusions: AFM is a powerful tool to study the morphology and ultrastructure of lymphocytes. There is significant difference in T cell membrane between before and after administration of steroids drugs, so we can hypothesis that the activation of T cell is directly repressed by steroids drugs. Elevated expression in situ of CD25 in peripheral blood may play an important role in the ongoing immune process. Moreover, its levels is positively correlated to the clinical activity score of TAO suggesting that maybe we can evaluate the activation of T cell in TAO by the parameter（Rvm、Peak count） of AFM.更多还原