| 摘要: | 胸腺素原(Prothymosin alpha, Ptma) 是一種約110 個胺基酸，12.5 kDa，等電點3.5的高度酸性小型核蛋白。在細胞培養的實驗中，胸腺素原被認為與細胞增生、抗細胞凋亡，以及細胞附著相關，而在許多癌症的診斷上也能作為一個有價值的標記。但既使如此，胸腺素原的確切功能仍然不甚清楚。本實驗以斑馬魚為模式，探討胸腺素原在胚胎發育時期的表現與生物體內的功能。在比較了數種已知的胸腺素原胺基酸序列後，發現它在各物種間的相似度相當高，斑馬魚的胸腺素原只有105個胺基酸，比起其他物種要少，但與牛、豬等哺乳類有五成以上的相似度，和人類是六成，而與兩棲類的相似度更高達七成。而在反轉錄聚合酶連鎖反應與全胚胎原位雜交實驗中，發現胸腺素原為母系遺傳基因，在發育最初的單一細胞期就有表現，並且持續到發育後期；表現的位置隨著發育階段的改變，從最初未分化的囊胚細胞團，到發育初期神經管、腦部、晶體、三叉神經前驅、原腎管、皮膚、胸鰭芽胞、鰓弧、後部側線神經前驅細胞、血管，到發育後期的心臟、胸腺、消化道、泳鰾，除了在骨骼、肌肉和體節上沒有發現，基本上其表現與器官組織發育的進程大致相同，顯示胸腺素原在細胞增生與器官與組織的發育上扮演重要角色。將胸腺素原與紅螢光蛋白RFP結合，並以keratin 18 驅動過度表現於斑馬魚的表皮，建立轉殖品系Tg(k18:ptma:rfp)，簡稱KPR，發現螢光表現明顯集中於細胞核。BrdU的標記與紅螢光的位置重疊顯示出過度表現胸腺素原的細胞正在增生中，而轉殖品系KPR表現出較多的細胞增生反應，並產生較厚的皮膚。而在紫外線UV-B照射的實驗中，發現過量表現胸腺素原的轉殖品系在較低劑量的紫外線照射下，與野生種相比會產生略微嚴重的細胞凋亡現象，但是在較高劑量的照射後轉殖品系KPR產生的凋亡現象反而比野生種輕微許多，表示胸腺素原在細胞凋亡的途徑中同時具有促進與抑制的作用。由以上結果，我認為胸腺素原可能促進細胞增生，並且控制細胞凋亡。
Prothymosin alpha (ptma) is a small (110 a.a., 12.5 kDa) and highly acidic (pI 3.5) nuclear protein. In cell culture experiments, ptma shows to be involved with proliferation, anti-apoptosis, and cell attachment. It also could be a useful marker in cancer diagnosis. However, its biological functions in vivo are still unclear. Here, I used zebrafish as a model to investigate the developmental expression and biological function of Ptma. Zebrafish ptma cDNA was encoded a 105-amino-acid-polypeptide. This sequence identified more than 50% with reported Ptma of mammalians, 60% with human, and up to 70% with amphibian. In RT-PCR and whole-mount in situ hybridization showed that ptma was a maternally inherited gene expressed at 1-cell stage, and continued to 7 dpf. At early blustrula stage, it expressed in all blastomeres. At segmentation stages, the ptma transcripts were restricted in the future neural tube, retina, otic placode, trigeminal nerve soma, pronephric ducts and brains, but were not found in somites, notochord or muscles. During pharyngeal period, ptma signals were observed in pectoral fin buds, skin, dorsal aorta and primordial of the posterior lateral lines. At early larval period (3-7 dpf), the expression was observed in aortic arches, heart, liver, thymus, swim bladder and guts. Ptma expression was basically conformed to the process of tissues and organs development except skeletons and muscles. These data suggested that Ptma played a role in cell proliferation that might be important for organogenesis. The germ line Tg(k18:ptma:rfp) (KPR) was established to over-expressed Ptma by promoter keratin 18 restricted in zebrafish epidermis labeled with red fluorescence. This transgenic line did not have exterior defects. The RFP signal was limited in nucleus and co-localized with BrdU signal. It suggested that Ptma over-expressed-cell was proliferating. KPR had more BrdU signals in the epidermis and thicker skin than WT did (KPR 0.0179 mm; WT 0.0125 mm), that meant over-expressed Ptma would activate cell proliferation. After low-doseage of UV-B irradiation, KPR showed a little more apoptotic signals compared to WT embryos. However, WT had much more serious damages than KPR after high-doesage UB-B exposure. On the basis of these observations, I propose that Ptma might promote proliferation and regulate apoptosis. |
