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標題: | 巨噬細胞在腹膜纖維化中的角色 Macrophage in peritoneal fibrosis |
作者: | Chi-Chun Lin 林紀均 |
指導教授: | 林水龍(Shuei-Liong Lin) |
關鍵字: | 巨噬細胞,肌纖維母細胞,腹膜纖維化,間皮下纖維母細胞,連體共生, macrophage,peritoneum fibrosis,myofibroblast,submesothelial fibroblast,parabiosis, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 末期腎病的起因大多來自於糖尿性腎病變、腎絲球腎炎和高血壓性腎病變所引起。末期腎病的病人需要進行透析或腎臟移植等治療以取代其喪失的腎臟功能並維持健康,腹膜透析是其中的一個選擇。比起血液透析,腹膜透析有許多優點,包含早期存活率較高、可自行在家操作、費用較低…等,因此吸引許多病人選擇這種治療。然而長時間的腹膜透析仍會造成許多問題的產生,其中最嚴重的問題為腹膜纖維化的形成,使得病人沒辦法繼續進行透析而必須採取其他治療方法。因此,預防及減緩腹膜纖維化成為臨床上必須解決的問題。在我們先前的研究中發現,在腹膜纖維化的過程中,間皮下纖維母細胞 (submesothelail fibroblast) 是肌纖維母細胞的主要來源,並非來自骨髓的纖維細胞或間皮細胞。然而在這過程中巨噬細胞所扮演的角色仍尚未被釐清,因此我們想探討不同來源的巨噬細胞在腹膜纖維化的過程中分別扮演什麼角色。
我們利用Csf1r-Cre/Esr1Tg;Rs26fstdTomato/+ 小鼠,其巨噬細胞給予Tamoxifen的誘導下可產生紅色螢光,可藉此觀察當腹膜纖維化時,巨噬細胞的數量及位置。在我們的實驗結果中發現,當給予次氯酸鈉的刺激之後,巨噬細胞的數量會顯著的增加並且累積在腹膜中。因為巨噬細胞顯著增加在受傷的腹膜上,我再利用基因技術讓小鼠巨噬細胞可以因為注射白喉毒素而將巨噬細胞去除,發現將巨噬細胞去除時,原本因纖維化而增厚的腹膜厚度不僅減少,連肌纖維母細胞的數量也減少了。此表示巨噬細胞對於間皮下纖維母細胞轉變為肌纖維母細胞的過程有特定的作用。除此之外,我們也發現這些巨噬細胞並不會表現肌纖維母細胞特定因子(α-smooth muscle actin ; α-SMA)及膠原蛋白 (collagen),這個結果顯示巨噬細胞在我們的實驗模式中並不會轉變為肌纖維母細胞。為了探討來自循環及組織駐留的巨噬細胞,我們利用連體共生(parabiosis)將CAG-EGFPTg 和 C57BL/6 小鼠縫合,經過四週後會產生微循環,彼此的周邊血液可進行交換,但腹腔中的細胞並不受影響。實驗結果顯示在次氯酸鈉刺激後,在腹腔內會有約40%的巨噬細胞來自於循環中,而剩下60%則是組織中的巨噬細胞經由刺激之後增生而來。利用連體共生與基因技術去除來自循環或組織駐留的巨噬細胞,並無法釐清究竟來自循環或組織駐留的巨噬細胞對於腹膜纖維化的影響何者較為重要。 總結來說,當腹膜纖維化時,巨噬細胞會堆積在受傷的腹膜中,但不會轉變成為肌纖維母細胞,因此其扮演的角色可能是藉由細胞間的交互作用影響間皮下纖維母細胞轉變為肌纖維母細胞。循環中及組織中的巨噬細胞在腹膜纖維化過程中,其數量的平衡及表型的變化需要進一步研究。 Diabetic kidney disease, glomerulonephritis, and hypertensive nephropathy are the leading causes of end-stage renal disease (ESRD). Peritoneal dialysis (PD) is one of therapies for ESRD. PD holds many therapeutic advantages including early survival benefit, the convenience of home therapy, and lower healthcare costs, which are particularly attractive to patients. However, patients will face some challenges especially peritoneal fibrosis after long-term dialysis. Prevention and attenuation of peritoneal fibrosis is an unmet medical need for patients with peritoneal dialysis. In our previous study, we proved that submesothelial fibroblasts are the major origin of myofibroblasts in peritoneal fibrosis. However, the roles of monocytes/macrophages during peritoneal fibrosis are not clear. We tried to figure out the roles of circulating monocyte- derived macrophages and tissue resident macrophages during peritoneal fibrosis in this study. We used Csf1r-Cre/Esr1Tg;Rs26fstdTomato/+ mice whose Csf1r-expressing cells including bone-marrow-derived macrophages and yolk sac macrophages expressed red fluorescence protein after tamoxifen-induced genetic recombination. Besides, parabiotic model made by two mice were used to create chimeric mice for study of circulating monocyte-derived macrophages. Sodium hypochlorite was used to induce peritoneal fibrosis. We first analyzed tissue-resident macrophages in peritoneal cavity and grouped them into two subsets, large peritoneal macrophage (LPM) and small peritoneal macrophage (SPM), as previous studies. After hypochlorite injury, we observed many macrophages accumulated in peritoneum. Selective macrophage ablation in Csf1r-Cre/Esr1Tg;Rs26fstdTomato/+;Rs26iDTR/+ mice was performed by administration of diphtheria toxin during peritoneal fibrosis. The thickness of injured peritoneum and the number of myofibroblasts were decreased markedly under macrophage depletion. Immunofluorescence staining showed that macrophages did not express collagen and a-SMA markers, suggesting macrophages did not transit into myofibroblasts during peritoneal fibrosis. In parabiotic model made between CAG-EGFPTg and wild type C57BL/6 mice, we demonstrated that about 40% of peritoneal macrophages were derived from circulating monocytes and the rest came from tissue-resident macrophages during peritoneal fibrosis. Selective ablation by diphtheria toxin in the parabiotic model made from Csf1r-Cre/Esr1Tg;Rs26iDTR/+ and Rs26iDTR/+ littermate could not clearly clarify the contribution of circulating monocytes-derived and resident macrophages to peritoneal fibrosis. In conclusion, our study implicated that macrophages have decisive effects on peritoneal fibrosis but not through macrophage-myofibroblast transition (MMT) pathway. Cross-talk between peritoneal macrophages and submesothelial fibroblasts should be one the underlying mechanisms for the differentiation and scar formation of peritoneal myofibroblasts after injury. The importance of circulating monocyte-derived macrophages and tissue-resident macrophages balance needs further study. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7821 |
DOI: | 10.6342/NTU201700152 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 生理學科所 |
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