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研究生：	徐維彬 Hsu, Wei-Pin
論文名稱：	新穎肝細胞生長因子相似基因的生物功能研究 Biological function study of the novel hepatocyte growth factor-like gene
指導教授：	張明熙 Chang, Ming-shi
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2005
畢業學年度：	93
語文別：	中文
論文頁數：	87
中文關鍵詞：	肝細胞生長因子
外文關鍵詞：	HGF-like gene
相關次數：	點閱：56 下載：1
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　　Kringle domain最初是在凝血因子prothrombin中被發現，其含有幾個重複的由三個雙硫鍵連結所形成的相似區域。接著含這種結構區域的蛋白在許多不同的分子中也都有發現，這些包含kringle domain的分子，依據結構上及生物學上的特性，已被分類的很清楚。本實驗中所研究的Hepatocyte Growth Factor-Like(HGFL)分子，即是一個含kringle domain的新穎蛋白質。利用人類基因體資料庫分析，發現此基因位於第22對染色體上，且有兩種形式，我們分別命名為hHGFL長形式和hHGFL短形式；此外，也發現在老鼠基因庫中具有相應的序列，分別命名為mHGFL長形式和mHGFL短形式。利用ELM伺服器軟體分析hHGFL長形式和hHGFL短形式，結果兩個形式在胺基酸169-191的位置都有被預測為transmembrane domain，因此，我們推測它們為穿膜蛋白質。接著我們將extracellular domain和kringle domain構築到pPICZA載體中並且在Pichia pastoris中表現蛋白質。之前已有文獻指出HGF（hepatocyte growth factor）的kringle-1重組蛋白可以隨著濃度增加而抑制由bFGF(basic fibroblast growth factor)所刺激的牛主動脈內皮細胞(bovine aortic endothelial)的生長。因此，我們觀察在同時處理extracellular domain重組蛋白與bFGF或者同時處理kringle domain重組蛋白與bFGF對於牛肺動脈內皮細胞（Calf pulmonary artery endothelial,CPAE）的影響。由MTT assay結果顯示extracelluar domain重組蛋白在毫微克濃度的刺激下會抑制CPAE細胞的生長，但是在微克濃度刺激對於CPAE細胞似乎是有毒害的。進一步地，我們想要尋找出HGFL的ligand。因此利用多株抗體去模擬它的ligand，活化此接受器以及觀察其所引起的訊息傳導。初步的研究顯示p-JNK分子可能涉及此接受器的訊息傳導。在免疫組織化學染結果發現，許多組織都有表現HGFL而這個結果與RT-PCR相符。

　Kringle domain was originally identified in the blood coagulation factor, prothrombin, as repeating homologous triple-disulfide-linked peptide regions. These domains were thereafter identified in a variety of molecules and these kringle-containing molecules can be classified into distinct groups, based on structural and biological characteristics. In this study, we cloned Hepatocyte Growth Factor-Like (HGFL) molecule, a novel member of kringle-containing proteins. The gene locates at chromosome 22 and has two variants, named as hHGFL long form and hHGFL short form, which are discovered by human genomic database screening. We also found homologous sequence in mouse database and named as mHGFL long form and mHGFL short form. The hHGFL long and short form proteins contain transmembrane domain positioned at amino acids 169-191 predicted by ELM server. Thus, we presume they are membrane proteins. We cloned the extracellular domain and kringle-domain into pPICZαA vector and expressed the protein in Pichia pastoris. Previously, it was demonstrated that recombinant kringle-1 of HGF inhibits proliferation of bovine aortic endothelial (BAE) cell stimulated by basic fibroblast growth factor in a dose-dependent manner. Therefore, we treated the CPAE (Calf pulmonary artery endothelial) cells with basic fibroblast growth factor together with extracellular domain protein or kringle-domain protein. The results of MTT assay showed that the recombinant extracelluar domain protein of hHGFL inhibits CPAE cell proliferation at naro-gram level but are toxic for CPAE cell at micro-gram level. Furthermore, we intended to search for its ligand. We used polyclonal antibody to mimic its ligand to activate the receptor and induced its signal transduction. Preliminary study showed that JNK molecule may be involved in the signal transduction of the receptor.Furthermore, IHC staining demonstrates HGFL expressed in various tissues.The result of IHC staining consistents with RT-PCR.

中文摘要..................................................	1
英文摘要..................................................	2
誌謝......................................................	4
目錄......................................................	5
圖目錄....................................................	7
附錄目錄..................................................	8
縮寫檢索表................................................	10
儀器......................................................	11
序論......................................................	13
	1-1肝細胞生長因(HGF)-----------------------------------	13
	1-2血管新生抑制物與Angiostatin ------------------------	14
	1-3 Kringle Structure ----------------------------------	15
	1-4 研究動機與目的-------------------------------------	16
實驗材料與方法............................................	17
實驗材料................................................	17
	2-1-1菌株--------------------------------------------	17
	2-1-2質體--------------------------------------------	17
	2-1-3培養液及培養基----------------------------------	18
	2-1-4 Electrophoresis buffer -------------------------	19
	2-1-5 Western Blot Buffer-----------------------------	20
	2-1-6 FPLC buffer-------------------------------------	20
	2-1-7 Endoglycosidase H buffer------------------------	21
	2-1-8酵素--------------------------------------------	21
	2-1-9 CNBr-activated Sepharose 4 Fast Flow buffer system	21
實驗方法................................................	22
	2-2-1聚合脢連鎖反應----------------------------------	22
	2-2-2利用QIAGEN kit製備insert及vector--------------	22
	2-2-3限制脢處理--------------------------------------	23
	2-2-4接合反應----------------------------------------	23
	2-2-5 E.coli Transformation---------------------------	24
	2-2-6 Colony PCR--------------------------------------	25
	2-2-7 利用QIAGEN kit抽取出質體-----------------------	26
	2-2-8 PCR產物純化法-----------------------------------	26
	2-2-9 製備酵母菌的competent cell----------------------	26
	2-2-10 Pichia electroporation-------------------------	27
	2-2-11 TA cloning-------------------------------------	27
	2-2-12 Phenol/Chloroform 處理-------------------------	28
	2-2-13西方墨點法-------------------------------------	29
	2-2-14利用重組蛋白 A 管柱純化抗體--------------------	29
	2-2-15利用Pichia系統表現rhEX,kringle,重組蛋白質-----	30
	2-2-16 Endoglycosidase H------------------------------	31
	2-2-17 利用E.coli系統表現重組蛋白質------------------	31
	2-2-18 MTT assay--------------------------------------	31
	2-2-19製備recombinant human extracellular重組蛋白 (rhEX)單株抗體---------------------------------	32
	2-2-20製備recombinant human extracellular重組蛋白 (rhEX)多株抗體---------------------------------	34
	2-2-21選殖 human HGF-like long form------------------	35
	  2-2-22細胞免疫染色法---------------------------------	36
	2-2-23免疫組織化學染色法(Immunohistochemistry staining)-------------------------------------	36
	2-2-24 製備CNBr-activated Sepharose管柱--------------	37
結果......................................................	38
討論......................................................	45
參考文獻..................................................	48
結果圖表..................................................	52
附錄......................................................	79
自述......................................................	87

附錄目錄

附錄1:  pCR 2.1-Topo Vector Map	79
附錄2:  pcDNA 3.1 Vector Map	80
附錄3:  pPICZA Vector Map	81
附錄4:  pMAL-C2X Vector Map	82
附錄5:  pMAL 重組蛋白 Fusion and Purification System	83
附錄6:  引子鹼基序列整理	84
附錄7:  人類HGFL的分佈	85
附錄8. 不同細胞中以HGFL專一primer RT-PCR的結果	86
                                    

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校外：2007-08-04公開

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