硬皮症具臨床意義的血清標記與纖維母細胞轉型機轉研究

The study of clinical significance of serum markers and fibroblast transformation mechanism in systemic sclerosis

吳青穎(Ching-Ying Wu)

指導教授 ： 余幸司

高雄醫學大學/醫學院/醫學研究所博士班/博士(2019年)

摘要

全身性硬皮症是一個結合基因、免疫調控失衡而以異常纖維化為表現的系統性疾病。因為全身性硬皮症會侵犯的許多器官與系遍及全身，除了皮膚組織的漸進式硬化外，尤其以心血管、肺臟及腎臟受到影響的層面最為廣泛。倘若能藉由一些血液檢測即能及早預知病人後續可能會面臨到的臨床徵象，將可以及時給予醫療的介入甚至是預防。本研究的第一個部分，將透過找尋血清中的生物標記的方法，針對臨床上患者的病程與預後做一預測。在過去的研究中發現，乙酰肝素酶(heparanase)是一種內切-β-D-葡醣醛酸酶 (endo-β-D-glucuronidase)，它可裂解硫酸肝素蛋白多醣 (heparin sulfate proteoglycans)的硫酸乙酰肝素側鏈，通過細胞外基質(extracelllar matrix)的降解和重塑以及促血管生成因子(pro-angiogenic factor)的釋放，參與傷口癒合、發炎、血管新生和腫瘤進展的過程。由於 heparanase媒介的生物過程與一些自體免疫機轉有關，而全身性硬皮症一是個涉及免疫失衡、發炎及血管病變而導致廣泛性纖維化的自體免疫疾病，heparanase極有可能在全身性硬皮症的疾病進展中扮演某種角色，我們因此著手研究了全身性硬皮症患者血清heparanase濃度與其臨床疾病進展的相關性。研究結果發現血清heparanase濃度在SSc患者中與健康個體相較是明顯升高的，而瀰漫性硬皮症和侷限性硬皮症這兩個次分組之間則沒有差異。另一方面，患有肢端潰瘍傷口的SSc患者的血清heparanaes濃度明顯低於沒有遠端潰瘍傷口的患者，這些結果表明血清heparanase濃度會在SSc患者中升高，可能反映了heparanase所參與的的生理過程對SSc疾病進展具有貢獻。由於跟heparanase 互相結合的促血管生成因子如血管內皮生長因子的釋放增加，可以保護具有較高血清heparanase濃度的SSc患者免於肢端潰瘍傷口的發生。因此，具有較高的血清hepraranse濃度可以作為SSc患者中針對肢端潰瘍傷口的保護性標記。
另外一個我們有興趣的因子: Syndecan-1是跨膜(transmembrane)硫酸乙酰肝素蛋白多醣(heparan sulfate proteoglycan)家族的成員，其膜結合和可溶形式(form)參與傷口癒合、發炎和血管發生的過程。因為這些生理現象與全身性硬化症（SSc）的發病機理有關，我們研究了SSc中血清syndecan-1濃度與臨床病程的關聯性。不論在瀰漫性硬皮症dcSSc或限性硬皮症lcSSc這兩組當中，患者血清 syndecan-1 濃度都顯著高於健康人。其中，在晚期dcSSc患者（發病時程> 6年），血清syndecan-1濃度顯著高於正常對照組，而早期dcSSc患者（發病病程≤6年）則無明顯升高。在臨床意義方面，血清 syndecan-1濃度升高的SSc患者當中，毛細血管擴張症患病率較高、右心室收縮壓升高的比例較高、肺部瀰漫性肺炎彌散能力(DLCO)則明顯較差而且低於正常濃度。因此，可溶性syndecan-1可能與SSc患者中增生血管病變的進展有關。在臨床的應用方面可以考慮在syndecan-1有上升的患者，應加強心臟及間質性肺病的檢查，右心室收縮壓升高是心衰竭的前兆，而DLCO的減少是間質性肺病惡化的一個早期的指標，若能藉由這些生物標記及早預知後續可能合併的全身併發症，將可以率先以藥物(比如cyclophosphamide)控制避免疾病的迅速進展。
研究的第二個部分在於探討如何有效能遏制住全身性硬皮症當中持續不綴地惡化的纖維化過程。在過去，轉化生長因子-β（TGF-β1）被視為是治療全身性硬皮症（SSc）的重要靶標，因為它在細胞的纖維化過程中扮演著重要角色。然而，我們的研究顯示了SSc患者的組織檢體與纖維母細胞中能呈現出三種不同的 TGF-β1表現，闡明了單靠抑制TGF-β1並不足以治療SSc。過去的文獻所發表的阻斷TGF-β1的臨床試驗也同樣顯示出令人失望的結果。因此，我們的研究試圖尋找另外一種潛在的新方法，以期找尋出能調控纖維母細胞免於過度纖維化的方法。之前研究Ingenuity Pathway Analysis（IPA）顯示，SSc的途徑與Ca2+信號Store-operated Ca2+ entry (SOCE)的調節有密切相關，並且發現SOCE活性在 SSc患者的纖維母細胞中是增加的。如果使用SOCE抑制劑，如2APB和相關的鈣離子通道抑制劑SKF96365和indomethacin來進一步處理SSc患者的纖維母細胞，發現SOCE抑制劑可選擇性地降低這些細胞纖維化的程度並可以改變細胞形態。因此，SOCE抑制劑，尤其是2APB和indomethacin，通過細胞骨架重塑和改變其膠原蛋白的分泌可導致SSc患者纖維母細胞的去分化並恢復回較正常的細胞活動性。
我們進一步解釋了SSc發病機轉與SOCE如何影響纖維母細胞分化。從SSc患者纖維母細胞的培養皿中發現，倘若加入一些外源性因子，比如加入gelatin-1、FAM20A和白蛋白，對於調節具有較高濃度的SOCE和α-SMA的纖維母細胞的分化是重要的。結論是，為了治療SSc，我們提出可以阻斷SSc中增高的SOCE活性。如果SOCE的活性被阻斷，那麼可以藉此達到誘導SSc纖維母細胞的去分化並同時改變細胞外基質（ECM）結構的結果，這點對於限制SSc過度纖維化這個致病機轉是有幫助的，也為全身性硬皮症的纖維硬化治療帶來了一線曙光。
總結一下本研究結合了臨床預後與新治療標的的探討。第一部分，利用血清中的臨床標記，可以幫助辨別硬皮症病人的臨床預後與相關風險。而第二部分則闡明利用SOCE抑制劑可做為治療細胞過度纖維化的一個介入點。期待本研究可以為讓世人對全身性硬皮症的全貌能增進多一點的認識。利用SOCE抑制劑的研究目前僅有在細胞階段，下一步將進行動物研究，將小鼠施打bleomycin以模擬硬皮症的組織變化，並找出適合濃度的SOCE抑制劑以深入了解以抑制劑治療在動物身上的可行性。
因全身性硬皮症的病因涉及免疫、基因、血管病變與異常纖維化的綜合作用，而且患者的臨床表徵因涉及全身各系統，未來仍有很大可以深掘研究的層面諸如免疫、基因與血管病變等面向，都仍有許多課題等待深掘，以期解開全身性硬皮症持續進展纖維化之謎。

關鍵字

硬皮症 ； 血清標記 ； 纖維母細胞

並列摘要

Systemic sclerosis(SSc) is a systemic disease characterized by genetic predisposition, immune dysregulation and abrrent microvasculopathy leading to fibrosis. In SSc, multiple organs and systems are commonly affected all over the body. Aside from the remarkable and characteristic progressive hardening of skin tissue, the cardiovascular, lung, and kidney are particularly involved. If there were some blood tests that can be used to predict the clinical progession of the disease that patients may face in the future, medical treatment or even preventive intervention can be given in time to help the patients.
The first part of the study will provide a prediction of the clinical course and prognosis of patients by looking for suitable biomarkers in serum. In previous studies, it has been found that heparanase is an endo-β-D-glucuronidase that cleaves heparan sulfate proteoglycans. The side chain is involved in the process of wound healing, inflammation, angiogenesis, and tumor progression through degradation and remodeling of the extracelllar matrix and release of pro-angiogenic factors. Given that the biological process of heparanase is related to certain autoimmune mechanisms, and systemic sclerosis is an autoimmune disease involving immune dysfunction, inflammation and vascular anormalites, we believe that heparanase may be play certain role in the progression of SSc. We seek to study the correlation between serum heparanase concentration and clinical progression in patients with SSc.
Serum heparanase concentrations were significantly higher in SSc patients compared to healthy individuals, while there was no difference between the two subgroups of diffuse cutaneous SSc and and limited cutaneous SSc. On the other hand, serum heparanaes levels in patients with digital ulcers were significantly lower than those in patients without digital ulcer ulcers. The finding may reflect the physiological processes in which heparanase contributes somehow to the progression of SSc disease. Due to the increased release of angiogenesis factors such as vascular endothelial growth factors, which are associated with heparanase, SSc patients with higher serum heparanase levels can be protected from the occurrence of digital ulcers. Therefore, a higher serum hepraranse concentration can be used as a protective marker for predicting digital ulcers in SSc patients.
Another factor that we were intested in is syndecan-1. Syndecan-1 is a member of the heparan sulphate proteoglycan family, whose membrane binding and soluble forms are involved in wound healing, inflammation, and vascular processes. Because these physiological phenomena are related to the pathogenesis of SSc, we studied the correlation between serum syndecan-1 concentrations in SSc and its associated clinical courses. In both subgroups of SSc patients, dcSSc and lcSSc groups, their serum syndecan-1 concentrations were significantly higher than healthy controls. Among them, serum syndecan-1 concentration of those late dcSSc (disease duration more than 6 years) was significantly higher than those in the normal control group, while the early dcSSc patients (disease duration less than 6 years) did not increase. In terms of clinical significance, SSc patients with elevated serum syndecan-1 concentrations has higher prevalence of capillary vasculopathies, higher prevalence of the elevated right ventricular systolic pressure, and a higher prevalence in the deterioration of the diffuse capacity of carbon monoxide (DLCO). Given that the right ventricular systolic pressure is a precursor to heart failure, and the reduction of DLCO is an early indicator of the deterioration of interstitial pulmonary disease, SSc patients with elevated syndecan-1 should be encouraged to take heart examination and screen for interstitial pulmonary disease with prompt.
The second part of the study is to explore if there is an alternative way that can correct the fibrosis process in SSc, In the past, transforming growth factor-beta (TGF-β1) was regarded as an important target for the treatment of SSc because it plays an important role in the process of fibrosis. However, our study showed discrepancies in the levels of TGF-β1 expression in SSc patients, indicating that inhibiting TGF-β alone is not sufficient to treat SSc. A previous clinical trial also revealed disappointing results. Thus, our study attempted to search for a potential novel approach. Ingenuity Pathway Analysis (IPA) indicated that the SSc pathological pathways were closely associated with store-operated Ca2+ entry (SOCE)-regulated signals, and SOCE activity was found to be increased in SSc fibroblasts. Further treatment of SSc fibroblasts with SOCE inhibitors, 2APB, and associated calcium channel inhibitors SKF96365, and indomethacin, showed that the SOCE inhibitors selectively decreased the expression of fibrosis markers and altered the cell morphology. Consequently, SOCE inhibitors, especially 2APB and indomethacin, caused the dedifferentiation of SSc fibroblasts via cytoskeleton remodeling and altered collagen secretion and restored the cell mobility. We further explained SSc pathogenesis as fibroblast differentiation with SOCE. Treatment with exogenous factors, gelatin-1, FAM20A and human albumin, which were identified from the conditioned medium of SSc fibroblasts, were important for regulating the differentiation of fibroblasts with higher levels of SOCE and α-SMA. Conclusively, to treat SSc, blockage of the increased SOCE activity in SSc induces the dedifferentiation of SSc fibroblasts and simultaneously changes the extracellular matrix (ECM) structure to limit SSc pathogenesis.
To sum up, this study emcompasses the clinical correlation and prognosis prediction, and also provide a potential treatment target as using SOCE inhibitors. The first part,we advocate the application of a simple blood test to test hyparanase and sydecan-1 as biomarkers can help early identify the clinical prognosis and associated risks in patients with SSc. The second part we clarified that the treatment of SOCE inhibitors can be used as a theupeutic target for the correction of excessive fibrosis.
I hope this study can be a contribution to knowledge and to the understanding of SSc, even though the disease itself is complex and the mechanism remains elusive. Current research using SOCE inhibitors is currently confined in the cell level (in vitro). The next step is to conduct animal studies. The mice administered with bleomycin would be a choice to mimic the tissue changes of SSc, and administered with SOCE inhibitors in different levels could help to figure out the ideal dosage and effect in vivo. SSc involves a combination of genetic predisposition and immune dysfunction, which was initiated by abberrent vascular functioning and eventually ended up with fibrosis. Given that SSc is a disease of complex that is associated with various clinical manifestations and involvements contributed by the SSc etiology, there are still many aspects for researchers to explore in the future.

並列關鍵字

systemic sclerosis ； serum biomarker ； fibroblast

參考文獻

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