原子力显微镜观测甲状腺相关眼病患者外周血T细胞

目的 应用原子力显微镜(AFM)对甲状腺相关眼病(TAO)患者外周血T细胞的膜表面进行观测,比较与正常T细胞的异同.方法 采用病例对照研究方法,选择42例TAO患者作为TAO组,根据病情将其分为活动期组(18例)和静止期组(24例),另外选择92例正常人作为正常组.经免疫磁珠法分离得到外周血T细胞,细胞培养进一步将T细胞纯化.流式细胞分析法对T细胞进行鉴定并检测其纯度,AFM原位扫描细胞样品.3组AFM观测数据采用单因素方差分析和Dunnett-t检验.结果 3组T细胞在形态上均呈现较为规则的圆形;正常组T细胞直径约在7～8μm,高度在600～850 nm之间;而TAO急性期T细胞直径偏大,约在9～11 μm之间,表面的凹凸不平较明显,可见颗粒状突起及凹陷,边缘还可观察到有细微的树枝状突起分布;静止期T细胞直径略小,约在8～10μm之间,而膜表而凹凸不平的程度介于其他两组之间.平均粗糙度、峰数、平均峰值高度、平均低谷高度、表面积差值指标在3组中有所不同(F=28.809,58.213,169.789,35.933,121.325;P<0.05),而峰数、表面积差值则存在两两差异(P=0.047,0.002).结论 应用AFM可观测到TAO患者外周血T细胞与正常T细胞膜表而的超微结构有所不同,而且TAO活动期和静止期的T细胞形态上也有差别.

Study on peripheral blood T lymphocyte cell membrane in thyroid associated ophthalmopathy by using atomic force microscopy

Objective To study the cell membrane of T-lymphocytes in thyroid associated ophthalmopathy (TAO) by using atomic force microscopy (AFM). Methods Case-control study. Forty-two patients were selected as the subjects, and divided into two groups: acute stage (18) and stationary stage(24) ones on the basis of symptom. Ninety-two healthy persons were in the normal groups. Dynal immunomagnetic beads isolation method was used to separate the T cells. CD3+ was chosen as a surface molecule marker of T cells to determine the purity of cells isolated. Cultivated T cells were observed by AFM. Amplitude and height images were obtained in the tapping mode with a scan rate of 2 Hz and an integral gain of 0.3 to 0.5. Statistical analysis was performed using single-factor analysis of variance and the P value was calculated. Results The topographies of these three groups of T cells showed significant difference(F=28.809,58.213,169.789,35.933,121.325;P<0.05). Average diameter and roughness of T cells in acute stage of TAO were significantly greater than those of the other two groups. One way analysis of variance showed that significant differences in various parameters (Ra, Peak count, Rpm, Rvm, Surface area diff) were found between these three groups (P=0.047,0.002). Conclusions The morphology and ultrastructure of lymphocytes in TAO are different from the normal subjects by observation with AFM. Lymphocytes in acute stage of TAO are also different from those in stationary stage.