臺灣博碩士論文加值系統

　　肝細胞生長因子（Hepatocyte growth factor, HGF），是一種能夠在組織和器官受損後，藉由與c-Met tyrosine kinase receptor結合，顯著促進組織修復和器官再生的細胞素(cytokine)。顧名思義，HGF能夠促使肝臟細胞再生，除此之外，它也和細胞吸附、遷移、增生、分化、血管新生、腫瘤生成等作用有關。
　　本實驗室以功能性基因體(Functional Genomics)為目標，在基因資料庫中發現類似HGF的新穎基因(novel gene)，並命名為肝細胞生長類似因子(Hepatocyte Growth Factor-Like gene, HGFL)。在更進一步的電腦基因資料庫搜尋下，我們找到了位於第22號染色體的人類HGFL genomic clone；且以library screen得到小鼠 HGFL genomic clone，並發現因alternative splicing所產生的另一種較長的HGFL variant，此現象無論在人類和小鼠中皆存在著。因此我們將這些新穎基因細分命名為： a﹚Mouse HGFL short form、b﹚Mouse HGFL long form、c﹚Human HGFL short form、d﹚Human HGFL long form。此四種基因所轉譯出的蛋白質皆為分泌性蛋白質(secretory protein)，其分子量為23~28 kDa。藉由電腦將此四種蛋白質的胺基酸序列與小鼠和人類的HGF比較，彼此之間於kringle domain有著約40 % 的相似性。
　　經由5’RACE結果得到全長的人類HGFL cDNA clone (full-length clone)，將全長cDNA sequence和genomic clone sequence在電腦上作比對，人類的HGFL short form是由6個exon及5個intron所組成；long form是由7個exon及6個intron所組成 ; 而小鼠HGFL short form在coding region中有5個exon和4個intron， long form 在coding region中有6個exon 和5個intron。我們分別由人類和小鼠的cDNA library中，以PCR的方式將上述四種cDNA分離出來並將其構築於載體上，並發現小鼠HGFL short form gene 和long form gene 會在心臟、腦、腎臟、脾臟、肝和十五天胚胎中表現。而人類HGFL gene在卵巢、睪丸、腎臟、肺臟、淋巴結、脾臟、胎盤、胸線胎兒心臟、胎兒肺臟和白血球中都有short form 和long from的存在；而在心臟、肝臟、小腸、骨髓、腦和胎兒肝中只能找到long from gene的存在。
　　藉由重複多次的5’RACE結果,我們定義出人類的HGFL的TSS (transcription start site)所在，並由TSS處往5’端挑選四個不同長度(pA, pB, pC, pD. 600bp~2100bp)的片段分別構築在含有luciferase的載體上，藉由luciferase的活性分析，發現最短片段的pD (-600bp)有著最強的luciferase活性。
　　此外，我們將小鼠HGFL short form 蛋白質和小鼠HGFL long form蛋白質 及 小鼠HGFL long form 專一性蛋白質以E.Coli系統來表達與純化，並將這些蛋白質作為抗原，施打至紐西蘭白兔中來產生多株抗體。並藉由此抗體(anti-HGFL)來進行免疫螢光染色，發現在小鼠的肝細胞和胃上皮細胞中可以找到HGFL的存在。
　　我們同時以intramuscular electroporation的方式將構築於pCDNA3.1的mouse HGFL short和long form plasmid DNA送入小鼠中，來了解這些蛋白質在體內的作用。以不同的條件(voltage, duration)來獲得最好的表現量，使得這些蛋白質能夠在老鼠體內表現。在給予多次電擊後，經由組織切片的結果顯示， mHGFL long form蛋白質在小鼠體內會造成嚴重的心肌病變(cardiomyopathy) ，這個由mHGFL蛋白質所導致的病變，其詳細的機轉仍待日後研究。

　　Hepatocyte growth factor (HGF), originally described as a strong mitogen for hepatocytes, is a multifunctional cytokine with a domain structure and proteolytic mechanism of activation. HGF is a heterodimer with a 69 kDa α-chain and a 34 kDa β-chain, linked by a single disulfide bridge. The α-chain contains the N-terminal hairpin structure and four homologous kringle domains and the β-chain has serine protease-like motif. HGF has pleiotropic effects on target cells, through its receptor c-Met tyrosine kinase receptor, including stimulation of growth, motility, and morphogenesis. HGF displays remarkable ability to promote tissue repair and organ regeneration after injury.
　　A homology screening of computer database using the HGF cDNA sequences as a query, we found a novel gene similar to HGF, named Hepatocyte Growth Factor-like gene (HGFL). We used computer database searches and library screening to find the human HGFL genomic clone locating at chromosome 22 and the mouse HGFL genomic clone. We also found two kinds of alternative splicing transcripts due to the post-transcriptional processing. According to the different length, we designated the two transcripts HGFL short form and HGFL long form. Thus, the four novel genes were named as: a) mHGFL short form, b) mHGFL long form, c) hHGFL short form, d) hHGFL long form. The gene encoded two secretory proteins containing 213 or 264 amino acids, and has a predicted molecular mass of 23 or 28 kDa. We compared amino acid sequences between HGFL and HGF in both mouse and human species. There are 40% similarities in HGF kringle domains.
　　The cDNA sequence of HGFL was compared with the genomic sequences to locate the exon/intron boundaries. We used 5’RACE to obtain full-length transcripts of human gene. For human HGFL short form, the gene contains six exons and five introns, while for the long form; the gene contains seven exons and six introns. There are five exons and four introns in coding region of mHGFL short form gene, and six exons and five introns in coding region of mHGFL long form gene. Using PCR amplification, we examined specific tissue distribution of short form and long form. Both mouse short form and long form cDNA were expressed in the tissues of 15 days embryo, brain, heart, kidney, liver, and spleen. The human short form cDNA was expressed in the tissues of fetal heart, fetal lung, kidney, leucocyte, lung, lymph node, ovary, planceta, spleen, testis, and thymus, while human long form cDNA was expressed in the tissue of bone marrow, brain, fetal heart, fetal liver, fetal lung, heart, kidney, leucocyte, liver, lung, lymph node, ovary, planceta, small intestine, spleen, testis, and thymus. To understand human HGFL gene regulation, we analyze its promoter activity in human 293 and dog MDCK kidney cell lines. The pD (about 600bp upstream of transcription start site) showed the highest promoter activity, 5- (in 293 cells) and 18- (in MDCK cells) fold higher than the negative control of the promoterless enhancer vector.
　　We also have expressed the mHGFL short form recombinant protein and mHGFL long form specific recombinant protein in E.coli to generate their specific polyclonal antibody. To use the anti-HGFL pAb to perform immunohistochemistry of HGFL. We could find that HGFL expressed in mouse hepatocyte and gastric epithelial cells. To analyze the in vivo biological function of the gene, we used intramuscular electroporation to deliver the mouse HGFL short and long form cDNAs into mouse. After four time intramuscular electroporation, we found the mice which were treated with mHGFL long form gene have serious cardiomyopathy. The mechanism of cardiomypathy induced by HGFL awaits further study.

1. Introduction 1
2. Materials and methods 7
3. Results 15
4. Discussion 21
5. Figures 25
6. Appendix 61
7. Reference 86

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