- 學位論文
結核桿菌誘導肺部纖維化的轉譯研究
Translational Studies of Mycobacterium Tuberculosis-Induced Lung Fibrosis
摘要
結核桿菌在肺部的感染造成肺纖維化，並導致不可逆的肺功能降低。結締組織生長因子(connective tissue growth factor, CTGF)的表現被確認在肺纖維化中扮演重要角色，然而其訊息傳遞路徑及在肺纖維母細胞中結核桿菌誘導結締組織生長因子表現的機制尚不明瞭。我們的研究發現在纖維母細胞中，結核桿菌誘導CTGF蛋白表現增加有時間及劑量關係。機轉探討中也發現結核桿菌誘導CTGF蛋白表現透過TLR2而非TLR4。藉由啟動子活性分析發現結核桿菌誘導CTGF活性增加主要是由啟動子上-747至-184片段調控，這個片段中主要有STAT3和AP1的結合位。進一步研究發現，curcumin會抑制結核桿菌誘導CTGF蛋白表現，結核桿菌亦可誘導AP-1轉錄因子活性增加，並增加c-Jun/c-Fos的結合至CTGF啟動子的活性。藉由c-Jun siRNA可抑制結核桿菌誘導的CTGF蛋白表現及AP-1的轉錄因子活性。JNK的抑制劑(SP6000125)和JNK的dominant-negative mutant (DN)可抑制結核桿菌誘導的CTGF蛋白表現。我們也發現結核桿菌可刺激JNK和c-Jun的磷酸化，且SP600125可抑制結核桿菌誘導的c-Jun磷酸化及AP-1的轉錄因子活性。結核桿菌誘導fibronectin的表現可被anti-CTGF抗體所抑制。綜上所述，這部分研究闡明結核桿菌藉由TLR2活化JNK並增加c-Jun/c-Fos的DNA結合能力，最終誘導CTGF蛋白表現及胞外基質的堆積。另一方面，纖維球的活化、增生及分化反應被認為是參與傷口修復和纖維化的重要的關鍵因子。然而纖維球和結核病之間的關係目前仍不清楚。我們的研究發現結核病患者的纖維球的比例、分化程度及CTGF的表現量皆高於正常人。經過培養後，結核病患者的纖維球的分化、增生容量及CTGF蛋白表現量皆高於正常人。且經過培養後，結核病患者的纖維球的分化、增生容量及CTGF的表現情形皆提高，但正常人的纖維球則沒有變化。而進一步研究發現，結核病患者纖維球在給予CXCL12抑制劑(AMD3100)可降低CTGF及α-SMA的蛋白表現並降低增生反應。CXCL12刺激正常人纖維球的分化、增生反應及CTGF蛋白表現可被AMD3100、TGF-β抑制劑(SB431524)及anti-CTGF抗體所抑制。綜上所述，我們的研究首次發現肺結核患者纖維球的α-SMA、CTGF的蛋白表現及細胞數量皆高於正常人，且CXCL12藉由活化CTGF和TGF-β調控結核病患者中的纖維球活化。以上研究或許可提供一個具潛力的治療結核病造成的肺纖維化的策略。
並列摘要
Mycobacterium tuberculosis (M.tb) infection in lung leads to pulmonary fibrosis, which causes the irreversible reduction of pulmonary function. Connective tissue growth factor (CTGF) expression has been confirmed to play an indispensable role in pulmonary fibrosis. However, the underlying signal pathway and effect of M.tb on CTGF expression in human lung fibroblasts are unclarify. Our results demonstrated that M.tb resulted time- and concentration-dependent increase in CTGF expression in human lung fibroblasts. A mechanistic investigation demonstrated that M.tb mediated CTGF expression through TLR2, but not TLR4. The promoter activity assay demonstrated that M.tb-mediated CTGF activity was mainly regulated by the promoter region at −747 to −184 bp, which involved AP-1 and STAT3 binding sites. Moreover, curcumin (an AP-1 inhibitor) restricted M.tb-mediated CTGF expression. M.tb also mediated increases in AP-1-luciferase activity and DNA binding activity of c-Jun/c-Fos on the CTGF promoter. Furthermore, the knockdown of c-Jun by siRNA restrained M.tb-mediated CTGF expression and AP-1-luciferase activity. A JNK inhibitor (SP600125) and a JNK dominant-negative mutant inhibited M.tb-induced CTGF expression. We also observed that M.tb could mediate the phosphorylations of JNK and c-Jun. Furthermore, SP600125 attenuated M.tb-mediated c-Jun phosphorylation and AP-1- luciferase activity. M.tb-mediated fibronectin expression was restricted by anti-CTGF antibody. In summary, these results demonstrate that M.tb is activated through TLR2 to mediate JNK activation, further increasing the DNA binding activity of c-Jun/c-Fos and finally promoting CTGF expression and ECM production. Moreover, fibrocyte activation, proliferation and differentiation are considered to be a key factor of wound healing and pulmonary fibrosis. However, the relationship between TB and fibrocytes are still unclear. Our studies found that the increased of the level of differentiation, proliferation, and CTGF expression in fibrocytes from TB patients were higher than in normal participates. After cultured, the differentiation, proliferation capacity and CTGF expression was increased in fibrocytes from patients with TB, but not from those of normal participants. Furthermore, treatment with the CXCL12 antagonist (AMD3100) reduced the expression of CTGF and α-SMA in fibrocytes and fibrocyte proliferation in patients with TB. Treatment with AMD3100, TGF-β antagonist (SB431524), or anti-CTGF antibody attenuated the expression of CTGF and α-SMA in fibrocytes and fibrocyte proliferation induced by CXCL12 in fibrocytes from normal participants. Our study for the first time demonstrated that the increase of α-SMA, CTGF expression and cell number in fibrocytes from patients with tuberculosis were higher than those in normal participates, and the role of the CXCL12 in mediating fibrocyte activation through CTGF and TGF-β in TB patients, which were indicated that these finding have pathological significance, and may play a potential therapeutic strategy for the development of fibrosis in patients with Tuberculosis.
參考文獻
延伸閱讀
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