Study On T Lymphocyte Cell Membrane Of Thyroid Associated Ophthalmopathy From Peripheral Blood By Atomic Force Microscopy

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.