^99Tc-亚甲基二磷酸盐对细胞因子刺激后人球后成纤维细胞增生的影响

Effect of ^99Tc-methylenediphosphonate on cytonike-induced activation of retroocular fibroblasts with Graves ophthalmopathy

背景Graves眼病是一种器官特异性自身免疫性疾病。研究表明，^99Tc-亚甲基二磷酸盐（^99Tc-MDP）在治疗Graves眼病方面有一定的疗效，但其机制尚不明了。目的观察^99Tc-MDP对细胞因子刺激后Graves眼病患者球后成纤维细胞（RFs）增生、合成透明质酸和表达人类白细胞DR抗原（HLA—DR）、细胞间黏附分子1（ICAM-1）的影响。方法球后结缔组织取自严重Graves眼病行球后减压术的女性患者2例，用组织块培养法在含质量分数20％胎牛血清（FBS）的DMEM培养液中培养RFs并进行传代，取2～7代细胞用于实验。分别将干扰素γ（IFN-γ）、白细胞介素1（IL-1）、肿瘤坏死因子α（TNF—α）以及不同质量浓度的^99Tc—MDP加入不同组的细胞培养液中孵育后，利用。H—TdR掺人法检测^99Tc—MDP对RFs增生的影响，采用放射免疫法检测培养细胞合成透明质酸的情况，用流式细胞仪检测HLA—DR、ICAM-1的表达。结果培养的细胞呈成纤维原细胞型，波形蛋白染色阳性，细胞角蛋白染色呈阴性。TNF—α、IFN-γ和IL-1作用后，培养的RFs增生值、透明质酸值、RFs中HLA—DR表达率及ICAM-1表达率分别为（4918．33±297．91）cpm、（505．83±41．29）mg／L、（56．88±14．67）％和（63．57±14．11）％，均明显高于正常对照组，差异均有统计学意义（P〈0．01），培养基中分别加入〉100mg／L ^99Tc—MDP后，RFs增生值、透明质酸值、RFs中HLA-DR表达率及ICMA-1表达率均明显低于TNF—α、IFN-γ、IL-1单独作用组，差异均有统计学意义（P〈0．01）。结论^99Tc—MDP能够以剂量依赖的方式抑制体外培养的人RFs的增生。

Background Graves ophthalmopathy is generally considered to be an organ-specific autoimmune disease. It was reported that ^99Tc-methylenediphosphonate （^99Tc-MDP） has therapeutic effect on Graves ophthalmopathy,though the mechanism has not been completely delineated. Objective This study was to explore the effects of ^99Tc-MDP on the proliferation of retroocular fibroblasts （RFs） ,hyaluronic acid （HA） synthesis,and the expression rate of human leucocyte antigen-DR （HLA-DR） and intercellular adhesion molecule-1 （ ICAM-1 ） in cultured RFs. Methods Retrooeular connective tissue was obtained from 2 eyes with Graves ophthalmopathy during the orbital decompression surgery. RFs were cultured with explant culture method in DMEM medium with 20% fetal bovine serum. The cells of 2-7 generations were used in the study. Interferon-γ, （IFN-γ） ,interlenkin-1 （IL-1） , tumor necrosis factor-α （TNF-α） was added in medium for 72 hours to stimulate the growth of the RFs,and then 10, 100 and 1000 mg/L ^99Tc-MDP was respectively used to co-culture the cells with IFN-γ, IL-1 or TNF-α. ^3 H-TdR ineorporation was used to detect the proliferation of RFs,and synthesis of HA,expression rate of HLA-DR and ICAM-1 in RFs were assayed by radio-immunoassay and flow cytometry, respectively. Results Cultured cells showed the fibrous shape under the inverted microscope with the positive response for vimentin and absent response for eytokeratine. After addition of TNF-α,IFN-γ and IL-1 ,the proliferation value of RFs, HA level,the expression rate of HLA-DR and ICMA-1 in RFs was （4918.33±297.91） counts/rain, （505.83±41.29）mg/L, （56.88±14.67） % and （ 63.57±14. 11 ） % respectively, showing significant difference in comparison with normal control group （ P〈0.01 ）. However, after co-culture of 〉 100 mg/L ^99Tc-MDP with TNF-α, IFN-γ and IL-1 , the proliferation value of RFs, HA level, the expressing rates of HLA-DR and ICMA-1 were significantly lower than those in the TNF-α, IFN-γ, and IL-1 only culture groups（P〈0.01 ） ,but were still higher than those in the normal group. Conclusions ^99Tc-MDP can suppress cytonikeinduced activation and growth of RFs derived from Graves ophthalmopathy in vitro at a dose-dependent manner.