醫學院: 醫學檢驗暨生物技術學研究所指導教授: 林淑華楊士鋒Yang, Shi-FengShi-FengYang2017-03-062018-07-062017-03-062018-07-062016http://ntur.lib.ntu.edu.tw//handle/246246/277426Hepsin 為第二型穿膜絲胺酸蛋白酶，主要表現於肝臟。體外實驗已證實Hepsin 可活化人類第七凝血因子及肝細胞生長因子前驅物等，推測 Hepsin 可能參與血液凝固及肝細胞生長，但 Hepsin 基因剔除 (KO) 小鼠在血液凝固功能、肝臟重量及肝臟再生能力並無任何異常，故 Hepsin 實際生理功能目前仍不清楚。先前本實驗室研究發現 Hepsin 基因剔除小鼠橫膈膜面積、直徑及肝臟末緣至肋骨最後一節距離，皆顯著小於野生型 (WT) 小鼠。橫膈膜病理切片也發現中央肌腱 (central tendon) 區域較野生型小鼠薄。此外， Hepsin 基因剔除小鼠肺功能在乙醯甲膽鹼 (Methacholine) 刺激下其氣道阻力 (resistance) 與肺順應性 (compliance) 皆劣於野生型小鼠，推論 Hepsin 缺失可能造成小鼠橫膈膜發育異常，造成類似橫膈上提 (diaphragmatic eventration) 的病理現像。本篇論文利用表現人類 Hepsin 基因轉殖小鼠 (Tg-hHPN) 動物模式探討 Hepsin 是否參與橫膈膜發育及相關機制。實驗首先建立 Hepsin 基因轉殖小鼠，分別表達野生型 Hepsin (WT) 和蛋白酶無法活化之突變型 Hepsin (RS) ，取得親代小鼠 (Founder G0) 後再與 Hepsin 基因剔除鼠進行配種，並利用西方點墨法分析子代肝臟 Hepsin 表現量，挑選表現最高並建立穩定表現之基因轉殖小鼠。其中於Tg-hHPNWT品系建立 line-68與 line-5小鼠而在Tg-hHPNRS建立 line-39 與 line-54 小鼠。本論文以 G2 代基因小鼠為分析對象。結果顯示，表現人類 hepsin 的基因剔除小鼠 (Tg+-hHPN；HPN-/-) 其橫膈膜直徑、面積與肝臟末緣至肋骨最後一節距離，皆顯著大於 Hepsin 基因剔除小鼠。顯示重新表現人類 Hepsin 能援救橫膈膜於基因剔除小鼠的發育缺陷。為瞭解 Hepsin 蛋白在橫膈膜發育中所扮演的角色，本論文亦分析胚胎 (E13.5) 橫膈膜前驅結構pleuroperitoneal fold (PPF) 面積、肌肉前驅細胞 (muscle progenitors) 與纖維母細胞 (Fibroblast) 數量，結果顯示 hepsin 基因剔除鼠中 PPF 面積與纖維母細胞數目皆無差異，而肌肉前驅細胞數量較野生型少(但無顯著差異，待累積n值)。綜合上述結果， Hepsin 可能透過影響肌肉前驅細胞，進而影響橫膈膜中央肌腱的發育，造成類似橫膈上提的病理現象。Hepsin, a type II transmembrane serine protease, is mainly expressed in the liver. It was investigated that in vitro Hepsin can activate the human factor VIIa and hepatocyte growth factor precursor (pro-HGF). It been proposed that Hepsin may be involved in blood coagulation and hepatocyte growth. Whereas Hepsin knockout mice have normal blood clotting function, liver weight and liver regeneration ability. The physiological function of hepsin remains unclear. In our previous studies showed that Hepsin knockout mice diaphragm area, diameter and the distance between liver edge to the last ribs are significantly less than the wild-type (WT) mice. Moreover, Diaphragm central tendon part of knockout mice was thinner than those in the wild-type mice. Under Methacholine stimulation, the airway resistance and lung compliance of Hepsin knockout mice was inferior to wild-type mice. It was proposed that the deficiency of Hepsin may induce the eventration of the diaphragm. In this studies, the human Hepsin transgene mice (Tg-hHPN) were used to investigate whether Hepsin participate in diaphragm development. Firstly, Tg-hHPN mice were established to express wild-type human Hepsin or the mutant form human Hepsin, HepsinRS. After obtaining the founders (G0), those mice crossed with Hepsin knockout mice. To obtain the highest and stable human Hepsin expressing transgene mice, the Hepsin expression level was detected in the liver of each founder offspring by Western blots. Wherein establishing the Tg-hHPNWT mice of line-68 and the line-5, strain and established the Tg-hHPNRS mice of line-39 and line-54.The G2 generation of the transgenic mice was used for the analisus in this study. The results showed that the diaphragm area, diameter and the distance between liver edge to the last ribs human Hepsin expressing with mice Hepsin-deficiency transgenic mice (Tg-hHPN; HPN - / -) of the significantly greater than Hepsin knockout mice. It showed that expression of human Hepsin can rescue developmental defects in the diaphragm in knockout mice. To understand the role of Hepsin in diaphragm development, we also measured diaphragm precursor structure the pleuroperitoneal folds (PPF) area and the number of muscle progenitors cell and Fibroblast in the transgene mive embryo (E13.5). The results showed that Hepsin knockout mouse had no difference in the PPF area and the number of fibroblasts, while the number of muscle precursor cells less than the wild-type (but no significant difference) continue to accrue n value). Based on the results, Hepsin may affect the muscle precursor cells, which resulted in the development of the central tendon in the diaphragm and caused diaphragm eventration.9799510 bytesapplication/pdf論文公開時間: 2021/8/26論文使用權限: 同意有償授權(權利金給回饋學校)Hepsin基因剔除小鼠基因轉殖小鼠橫膈膜發育先天性橫膈膜疝氣橫膈上提pleuroperitoneal foldHepsin knockout micetransgenic micediaphragm developmentcongenital diaphragmatic herniadiaphragmatic eventrationpleuroperitoneal foldsthesis10.6342/NTU201602085http://ntur.lib.ntu.edu.tw/bitstream/246246/277426/1/ntu-105-R03424015-1.pdf