纖維母細胞生長因子對甲狀腺眼病變病患眼內纖維母細胞的影響

Influences of fibroblast growth factor on orbital fibroblast in thyroid eye disease

施智偉(Chih-Wei Shih)

指導教授 ： 廖述朗

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201702502

摘要

甲狀腺眼病變是最常見的眼窩疾病，有多達半數以上的Graves’ disease 患者有眼病變。許多眼病變無法以現有治療方式治癒(例如眼藥水、全身性類固醇、眼窩放射性治療、眼窩減壓手術)。甲狀腺眼病變的症狀包括結膜充血、眼球突出、動眼肌病變、視神經病變等。這些症狀是由眼窩脂肪組織增生與動眼肌肥厚引起，而這些眼窩內組織的變化與纖維母細胞(fibroblast)有關。纖維母細胞表面受體受到特定細胞因子刺激後，可以製造琉璃醣碳基酸(hyaluronic acid)與前列腺素衍生物素(prostanoids)，也可以增生和分化成脂肪細胞與肌纖維母細胞。這些能夠刺激纖維母細胞的細胞因子包括thyroid-stimulating immunoglobulins，interleukin-6 (IL-6)， IL-8，和prostaglandin E2 (PGE2)等。纖維母細胞生長因子(fibroblast growth factor, FGF)也能影響纖維母細胞的表現，但它在甲狀腺眼疾的研究中較少被提及，角色也較不明確。本研究希望可以釐清纖維母細胞生長因子、纖維母細胞與甲狀腺眼病變之間的相關性，並測纖維母細胞生長因子受體(fibroblast growth factor receptor, FGFR) 抑制劑是否可能影響纖維母細胞而推測它是否可能用來治療甲狀腺眼病變。
我們的實驗方法是收集血液樣本，包括甲狀腺眼患者（31位）與Graves’ disease 而無眼病變患者（24位），測量血中纖維母細胞生長因子、甲狀腺功能、甲促素受體抗體等指數以比較照分析。我們也收集兩群眼窩纖維母細胞，分別來分別來自甲狀腺眼疾（31位）與非甲狀腺眼疾（34位）受試者的眼窩組織，從中分離出纖維母細胞。用western blot與reverse transcription quantitative PCR (RT-qPCR)來測試FGFR mRNA的表現量。我們使用Hs68上皮纖維母細胞株測試FGF1，與FGFR抑制劑 (AZD4547， BGJ398，和SUS5402) 對於Hs68細胞生長與分化的影響。
關於實驗結果，在血液樣本的部分，甲狀腺眼病變病患與Graves’ disease 而無眼病變患者相較，血液中的FGF1濃度較高（中位數19.18 pg/mL v.s. 7.06pg/mL, P=0.025）。用western blot探測FGFR可以發現甲狀腺眼疾與非甲狀腺眼疾兩組病患的眼窩纖維母細胞均表現出較多的FGFR1，與非常少量的FGFR2,3,4，但表現在兩組間並無明顯差異。用RT-qPCR則可發現甲狀腺眼疾與非甲狀腺眼疾兩組病患的眼窩纖維母細胞都表現出較多的FGFR1 mRNA，與少量的FGFR2,3,4 mRNA，其表現量在兩組間也沒有明顯的差異。在Hs68細胞株上，可以發現FGF1有促進Hs68細胞生長與分化為脂肪細胞的作用。另外，FGF1有抑制Hs68分化為肌纖維母細胞的作用。我們測試了FGFR抑制對細胞生長和分化的影響。其中AZD4547和BGJ398可以拮抗FGF1對於促進Hs68生長的作用。AZD4547和BGJ398可以拮抗FGF1抑制Hs68分化為肌纖維母細胞的影響。
我們的實驗對於FGF和眼窩纖維母細胞在甲狀腺眼疾的致病機轉提供了初步的資訊，FGF對纖維母細胞的的生長與分化有一定的調控角色。我們也發現FGFR抑制劑可以抑制眼窩纖維母細胞的增生，FGFR抑制劑可能是一個新的治療甲狀腺眼疾的方法。但仍然有許多主題需要進一步釐清，例如FGF對於Hs68細胞株生長分化的影響能否在受試者眼窩纖維母細胞上表現，FGFR抑制劑對眼窩纖維母細胞分化的影響也需要進一步研究來確定。藉由這些相關的研究，我們才能對甲狀腺眼疾的致病機轉與治療更加瞭解。

關鍵字

甲狀腺眼病變 ； 纖維母細胞生長因子 ； 纖維母細胞 ； 纖維母細胞受體 ； 葛雷夫氏症

並列摘要

Thyroid eye disease (TED) is the most common orbital disease. Oophthalmopathy is seen in up to 50% more than half of the patients with Graves’ disease. Many patients with TED can’t tolerate or be cured by current treatments including topical ophthalmic medications, systemic steroid, orbital radiotherapy and orbital decompression surgery. Clinical signs of TED include conjunctival injection, proptosis, eyelid retraction, restrictive extraocular myopathy, and compressive optic neuropathy. These signs are mainly caused by exraocular muscle hypertrophy and expanded orbital adipose tissue. Orbital fibroblasts play a major role in the pathogenesis of TED. When stimulated by thyroid-stimulating immunoglobulins and other cytokines, for example, interlukin-6, ilterlukin-8, and prostaglandin E2, orbital fibroblasts can produce hyaluronic acid and inflammatory prostanoids, they can also proliferate or differentiate into adipocytes. Fibroblast growth factors (FGFs), when binding to FGF receptors (FGFRs) on the fibroblasts, may also have some effect on cell proliferation and differentiation. However, the role of FGFs in TED is unclear. In this study, we investigate the relationship of FGF, fibroblast and TED. We also observe the influence of FGFR inhibitors on orbital fibroblasts.
Serum samples were collected from patients with TED (31 patients) and Graves’ disease without orbitopathy (24 patients). Levels of FGF, thyrotropin, thyroxin, thyrotropin binding inhibitory immunoglobulin (TBII) were measured and analyzed. Orbital fibroblasts were isolated from orbital tissue, which was obtained from orbital surgeries due to TED (31 patients) and other diseases (34 patients). FGFRs of orbital fibroblasts were observed by western blot and expression of FGFR mRNA were measured by RT-qPCR. We evaluate the effect of FGF1 and FGFR inhibitors (AZD4547, BGJ398, and SUS5402) on Hs68 fibroblast cell line. Cell proliferation, adipogenic differentiation and myofibroblastic differentiation were observed and measured.
Serum FGF1 was higher in cases of TED than in Graves’ disease without orbitopathy (19.18 pg/mL v.s. 7.06pg/mL, p=0.025). Abundant FGFR1 and very low level of FGFR2,3,4 of orbital fibroblasts were revealed by western blot in TED and non-TED patients. There was no significant difference of FGFRs expression between TED and non-TED patients. In the result of RT-qPCR, there was abundant expression of FGFR1 mRNA and low level of FGFR2,3,4 mRNA in TED and non-TED patients. There was no difference of FGFR mRNA expression between TED and non-TED groups. In Hs68 cells, FGF1 promoted cell proliferation and adipogenic differentiation. We also observed that FGF1 inhibited fibroblastic differentiation in Hs68 cells. AZD4547 and BGJ398 could antagonize the effect of FGF1 on fibroblasts proliferation and myofibroblastic differentiation.
Our study provides preliminary information about the role of FGF on orbital fibroblasts in TED. We also observed that FGFR inhibitors could inhibit orbital fibroblast proliferation. FGF-FGFR pathway could be a new treatment target for TED in the future. Further studies are needed, for example, the effect of FGF on orbital fibroblasts, the effect of FGFR inhibitors on orbital fibroblasts. We could understand more about the pathogenesis and treatment of TED through further studies on FGF.

並列關鍵字

Thyroid eye disease ； Fibroblast growth factor ； Fibroblast ； Fibroblast growth factor receptor ； Graves’ disease

參考文獻

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