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標題: | 犬YKL-40重組蛋白之表現及其在犬乳腺腫瘤細胞之作用 Recombinant Canine YKL-40 Expression and Its Effects on Canine Mammary Gland Tumor Cells |
作者: | Chun-Yu Lin 林均豫 |
指導教授: | 廖泰慶(Tai-Ching Liao) |
關鍵字: | YKL-40,CHI3L1,犬乳腺腫瘤, YKL-40,CHI3L1,canine mammary gland tumor, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | YKL-40 首次被發現於人類的骨瘤細胞株MG63中,為一分泌型之醣蛋白。其命名來自其蛋白胺基酸序列N端之前三個胺基酸,分別是酪胺酸(Y)、賴胺酸(K)以及白胺酸(L),取其縮寫並加上其分子重量約為40 kDa,因此命名為YKL-40。此外由於YKL-40具有類似幾丁酶可結合幾丁質之能力而有別名CHI3L1(chitinase-3-like-1),但其主要作用區域之胺基酸由白胺酸(leucine)所取代,因而失去幾丁酶之活性。過往關於YKL-40的研究皆以人類疾病及實驗動物為主,與犬隻相關的研究發表屈指可數。而在YKL-40與癌症相關的研究中,一般認為血清中增量之YKL-40與許多種類之癌症及預後不良有關。近期的研究也指出YKL-40具有調控血管新生的能力。然而YKL-40確切的功用與作用機制仍尚未明瞭,更遑論犬隻YKL-40的研究仍屬稀少。本研究將以BALB/3T3細胞進行轉染帶有犬隻YKL-40基因的載體,透過真核系統之細胞株穩定表現此分泌型醣蛋白。合成後的蛋白會被分泌至培養液中,收集後經濃縮後進行純化。之後利用犬隻乳腺癌細胞株CMT-1及MPG來進行YKL-40生物功能的測試與評估,包括:細胞增生試驗、細胞移行試驗以及細胞侵入能力試驗等。在最低100 ng/mL的濃度下,YKL-40對於促進細胞移行及侵入具有一定的效果,然而在細胞增生的能力並不顯著。而在細胞訊息傳遞路徑的探索上,發現YKL-40具有活化AKT的能力,推測其主要透過此一路徑進行訊息傳遞。 YKL-40 was first identified as a secretory glycoprotein from a human osteosarcoma cell line, MG63. This protein is named based on its molecular weight of 40 kDa and N-terminal amino acids tyrosine (Y), lysine (K), and leucine (L). It is also known as CHI3L1 (chitinase-3-like-1), which has the chitin-binding affinity but lacks the chitinase activity due to the substitution of an essential glutamic acid with leucine in the chitinase-3-like catalytic domain. During the past decade, most of YKL-40 studies were focused on human diseases and experimental animal models such as mice, and little information of canine research was presented so far. Elevated serum YKL-40 has been associated with worse prognosis in a variety of human cancers. Recent studies suggested that YKL-40 plays a role in regulation of tumor angiogenesis. However, the real function and molecular mechanism of YKL-40 still remained unknown, and the investigation on canine source YKL-40 protein was also rare. We have reported previously the molecular cloning of the canine YKL-40 gene and a predicted YKL-40 protein structure. In this study, a transfection was performed on BALB/3T3 cell to establish a stable cell line which can overexpress canine YKL-40 protein. Recombinant canine YKL-40 protein was then concentrated and purified from the cultured medium of the transfected cell line. The biological functions of this protein were evaluated on the canine mammary gland tumor (MGT) cells including proliferation, migration and invasion assays, etc.. YKL-40 at a concentration as low as 100 ng/mL seem to has effect in cell migration and invasion, yet little effect in cell proliferation. The alterations of molecular signaling induced by addition of YKL-40 protein will be also evaluated on canine MGT cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/5880 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 獸醫學系 |
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