https://scholars.lib.ntu.edu.tw/handle/123456789/145790
標題: | Hepsin蛋白對人類癌細胞的影響 The Effect of Hepsin Expression in Human Cancer Cell Lines |
作者: | 王忠琳 Wang, Chung-Lin |
關鍵字: | 第二型穿膜絲胺酸蛋白酶;裸鼠;人類癌細胞;細胞活體標定;hepsin;serine protease;flag tagging;tetracystei | 公開日期: | 2004 | 摘要: | Hepsin屬第二型穿膜絲胺酸蛋白酶,普遍表現於各組織細胞表面,其中又以肝臟表現量最高。近幾年研究發現,hepsin在前列腺癌細胞株有過量表現,與抑制癌細胞的生長及轉移相關;並能在缺乏組織因子狀態下活化血液第七凝血因子,啟動外源性凝血反應。然而,hepsin基因剔除小鼠不僅胚胎發育正常,成鼠也未出現凝血反應異常的現象,是以目前詳細弁鄐峓@用機轉仍不清楚。 為了解hepsin在癌細胞中扮演的角色,本論文以基因遺傳工程方法建構N端區域具偵測標記Flag及Teteracysteine (TC)之四種人類hepsin融合蛋白;包括野生型WT、蛋白活化區突變型R162A、酵素弁鈰洉蟔靮昤353Y及雙點突變型R162AS353Y融合蛋白。Hepsin融合蛋白可利用抗flag單株抗體之免疫螢光染色及FlAsH-EDT2染色偵測。接下來經反轉錄聚合酶鏈反應確認HeLa及Sk-Hep1兩種人類癌細胞株不表現hepsin後,將上述野生型及突變型hepsin基因轉入HeLa及Sk-Hep1,經過G418篩選後,並利用西方墨點及免疫沉澱法確定hepsin融合蛋白的表現。結果發現,相較於突變型hepsin融合蛋白穩定表現,野生型hepsin在兩種癌細胞株,均只能偵測到mRNA生合成,而無hepsin蛋白。然而,短暫表現轉染實驗証實HeLa細胞株確實能表現重組野生型hepsin,唯表現出的重組hepsin仍無法活化凝血第七因子FVII。在體外細胞生長實驗及活體裸鼠腫瘤形成實驗中亦觀察到,相較於野生型HeLa細胞,表現hepsin之細胞生長速率降低,並且在裸鼠形成之腫瘤大小、速度亦有明顯差異。此結果顯示hepsin可能具有未知活化機制影響表現,並可能在癌細胞的生長上扮演重要的角色。 Hepsin is a type II transmembrane serine protease, which is present in most tissues, with the highest level expressed in liver. Previous studies suggest that hepsin is involved in cell growth and migration. It is also shown to interact with coagulation Factor VII, and convert zymogen factor VII to factor VIIa. However, embryonic developmental defects and hemorrhage abnormality were not shown in hepsin knockout mice. Hence the biological role of hepsin remains unclear. To dissect the biological function of hepsin, we tried to express hepsin in cancer cell lines. After screening 4 cell lines with RT-PCR, we found that HeLa and Sk-Hep1 cells do not express hepsin. These two cell lines were transfected with Flag-Tetracysteine (TC) tagged-Hepsin expression vector. Besides wild type hepsin, we prepared 3 mutated hepsin expression vectors: activation domain mutation in residue R162A、S353Y、R162AS353Y ; Residue R162 is in activation domain and S353 is in enzymatic functional domain. Flag-TC tagged hepsin fusion protein can be detected with anti-Flag antibody with immunofluorescence staining and FlAsH-EDT2 staining. After G418 selection, we can only get cell lines with stably expressed mutated hepsin in both HeLa and Sk-Hep1 cells but not those with wild type hepsin fusion proteins. During transient expression, wild type hepsin fusion protein can be detected, but it was not able to cleave FVII to FVIIa in vitro. To prove the hepsin is involved in tumor cell growth in vivo ,we transplanted the wild type and mutant type hepsin-expressed HeLa cells via subcutaneous injection to nude mice. The growth rate and tumor size of hepsin-expressed HeLa cells in nude mice are significantly different. It suggests that hepsin may be involved in the regulation of cancer cell growth. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/62818 | 其他識別: | zh-TW |
顯示於: | 醫學檢驗暨生物技術學系 |
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