鐵循環相關分子基因變異分析與其相關調控之探討

Abstract

鐵能幫助細胞電子傳遞、呼吸作用、氧氣輸送等正常生理功能，故鐵質對維持生物存活是必須物質。但因為鐵在體內沒有代謝管道，使得生物體對鐵的調控必須非常嚴密，防止鐵質含量不平衡所引起組織細胞損傷與疾病。近年來，研究人員陸續找到了與鐵相關的新蛋白群，這使得鐵在生物體內循環與調控更加清楚，也能以不同的角度看待鐵相關的機制與疾病。 Hepcidin是包含25個胺基酸的短鏈荷爾蒙，主要由肝臟製造，目前被認為是體內鐵質平衡的主要調控者。為了控制腸道細胞對於鐵質的吸收，以及巨噬細胞對於鐵質的釋放，hepcidin會降解細胞膜上的輸鐵蛋白ferroportin。目前已知有許多基因牽涉hepcidin表現調控，例如transferrin receptor 1 (TfR1)、transferrin receptor 2(TfR2)、hemojuvelin(HJV)、hemochromatosis (HFE)等。研究人員又發現難治性缺鐵性貧血患者與TMPRSS6變異相關，而找到TMPRSS6最重要的生理功能是藉由切割m-HJV，影響BMP/SMAD訊息傳遞來調節hepcidin的表現。 相較於國外對於HAMP(hepcidin gene)、HJV、FPN1(ferroportin gene)等基因變異有不少研究，台灣地區對於鐵相關的報告卻很少。鐵相關的疾病在於各種族之間發生的頻率與類型皆有不同，本研究針對台灣地區鐵質失衡患者，如鐵質缺乏的病患，以直接定序法直接定序分析TMPRSS6與其它相關基因的變異狀況。以47位缺鐵性貧血病患進行分析，在TMPRSS6重要的serine protease區域中，除了基因多型性變異外，並沒有發現有意義突變。此外，生物體含氧量與含鐵量為調控體內鐵平衡的兩大要素，本研究也針對TMPRSS6表現是否受到氧分壓改變所影響進行研究，TMPRSS6的啟動子區域上，確實包含數個HBS (Hypoxia-inducing factor binding sequence)；將Hep G2與Huh 7細胞培養於氧氣21 %(正常氧)與3 %(低氧)環境下，相較之下，發現當氧濃度降低，且經時越長，TMPRSS6的mRNA表現量越高。另外，也發現HAMP的mRNA在低氧狀況下，表現量有明顯上升。在Hep G2細胞的低氧實驗中，發現ceruloplasmin隨低氧處理的時間越長，轉錄明顯增多；TfR1的轉錄則是先下降後上升。

Iron is essential for living organisms to survive, e.g. it is involved in electron transport and energy metabolism. There is no excretory pathway for iron, the regulation of iron homeostasis should be tightly controlled. To prevent unbalanced iron concentration causing cells and tissues damage, the systemic iron metabolism is regulated by iron absorption, utilization, and recycling. Recently, several novel studies of iron-related moleculars and genes make the association between iron homeostasis and diseases more clear. Hepcidin, a 25-amino acid peptide hormone, is mainly produced in the liver and is considered to be the principal regulator of systemic iron concentration. To limit iron absorption in enterocytes and control iron release from macrophages, hepcidin would bind to the iron-exporter protein ferroportin and trigger entrocytosis to degrade ferroportin. The hepcidin expression is regulated through erythropoietic activity pathway, iron store pathway, and inflammation pathway. Additionally, severl moleculars are also involved in, such as transferring receptor 1 (TFR1), transferring receptor 2 (TFR2), hemochromatosis (HFE), hemojuvelin (HJV), bone morphogenetic protein (BMP), etc. Recently, the TMPRSS6 mutation is revealed to be related to iron-refractory iron deficiency anemia, subsequent reporters showed that the main function of TMPRSS6 is to inhibit hepcidin activation by cleaving m-HJV. The mutation of these genes were reported somewhere, however, there are few reporters to discover the mutations of HAMP, HJV, FPN1 and TMPRSS6 in Taiwan. The aim of the present research is to study iron-related genes in 47 patients with iron deficiency, especially the TMPRSS6. We didn’t find any meaningful mutations in the functional serine protease domain, besides some SNPs. Further, the second aim is to know the effect of differ oxygenation concentrations. We measured several genes mRNA of Hep G2 and Huh7 cell lines which are incubated in normoxia or hypoxia conditions. Compared with normoxia, hypoxia can induce the expression of TMPRSS6 and HAMP mRNA in both cell lines. As expected, we found ceruloplasmin mRNA increased under hypoxia in Hep G2 cells. Furthermore, hypoxia decreased TfR1 mRNA at first and then increased its expression.