- 學位論文
血管周邊細胞增生分化為肌肉纖維母細胞導致纖維化腎臟的紅血球生成素產生不足
Transdifferentiation of Pericytes into Myofibroblasts is Responsible for the Inadequate EPO Production in Fibrotic Kidneys
摘要
幾乎所有的慢性腎臟病患都會產生貧血的現象。當健康的成人處於貧血的情況，血清中的紅血球生成素濃度會增加100到1000多倍以刺激造血；然而慢性腎衰竭病人血中的紅血球生成素卻不足以矯正貧血現象。我們在先前的研究證實，在腎臟纖維化的過程中，肌肉纖維母細胞主要的前驅細胞是血管周邊細胞。正常腎臟中的血管周邊細胞與血管內皮細胞共同擁有一基底膜，並具有合成基底膜、維持血管內皮細胞完整，與穩定血管的功能。利用Col1a1-GFPTg報導小鼠，我們發現血管周邊細胞與前人研究中所描述在腎臟中製造紅血球生成素的細胞具有類似的特徵，並表現許多與之相同的標幟蛋白。而在給予放血或缺氧刺激之後，血管周邊細胞能表現紅血球生成素。同時，我們也發現將血管周邊細胞的Hif2a剔除，會導致腎臟中的紅血球生成素表現降低。利用單側輸尿管結紮的動物疾病模式，我們發現在放血刺激前與刺激後，纖維化的腎臟比起對側控制組腎臟，其紅血球生成素表現有下降的趨勢。除此之外，當纖維化腎臟中的肌纖維母細胞受到放血刺激時，其製造的紅血球生成素，比起對側控制組腎臟當中的血管周邊細胞所製造的紅血球生成素，明顯較低。為了釐清紅血球生成素在血管周邊細胞轉分化為肌肉纖維母細胞時，其紅血球生成素表現降低的機制，我們分析了紅血球生成素基因的去氧核醣核酸甲基化修飾情形。我們確認了肌肉纖維母細胞的紅血球生成素基因，其啟動子與五端非轉譯區發生去氧核醣核酸過度甲基化的現象。我們也發現當給予5’-Azactiding以去除甲基化修飾後，腎臟中紅血球生成素的基因表現提升。因此我們的實驗結果支持：血管周邊細胞是腎臟臟中製造紅血球生成素的細胞；以及當血管周邊細胞轉分化為肌肉纖維母細胞，其紅血球生成素基因的啟動子與五端非轉譯區的過度甲基化，會導致在慢性腎臟病時，腎臟的紅血球生成素製造不足。
並列摘要
Anemia is present in almost all of the chronic kidney disease (CKD) patients. Serum erythropoietin (EPO) concentrations in healthy adults increase 100- to 1000-fold under anemia conditions to stimulate erythropoiesis, whereas EPO in CKD patients fail to response adequately to correct the anemia. Our previous studies shown that pericytes are the major progenitors of myofibroblasts during renal fibrosis. Using Col1a1-GFPTg reporter mice, we found that pericytes displayed similar characteristics and markers to renal EPO-producing cells (REPCs) described in literature, and produced EPO after phlebotomy or hypoxic stimulation. Also, we found that knockout of hypoxia-inducible factor 2 in pericytes led to decreased Epo expression in kidney. In animal model of unilateral ureteral obstruction (UUO), Epo expression was decreased in fibrotic UUO kidneys before and after phlebotomy when compared with contra-lateral kidneys (CLK). Besides, EPO induction in response to phlebotomy was inferior in UUO kidney myofibroblasts than that in CLK pericytes. To get an insight into the mechanisms responsible for the decreased Epo expression during pericyte-myofibroblast transition, we analyzed the DNA methylation modification of Epo gene. Hypermethylation of Epo promoter and 5’-UTR was confirmed in myofibroblasts. Demethylation by administration of 5’-azacytidine led to increased Epo expression in fibrotic kidneys. Hence our data support pericytes are the REPC and hypermethylation of Epo promoter and 5’-UTR during pericyte-myofibroblast transition is responsible for the inadequate EPO production in CKD.
參考文獻
延伸閱讀
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