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. Aihara, H., and J. Miyazaki. 1998. Gene transfer into muscle by electroporation in vivo.Nat Biotechnol 16:867.
2. Aoki, M., R. Morishita, Y. Taniyama, I. Kida, A. Moriguchi, K. Matsumoto, T. Nakamura, Y. Kaneda, J. Higaki, and T. Ogihara. 2000. Angiogenesis induced by hepatocyte growth factor in non-infarcted myocardium and infarcted myocardium: up-regulation of essential transcription factor for angiogenesis, ets.Gene Ther 7:417.
3. Boccaccio, C., M. Ando, L. Tamagnone, A. Bardelli, P. Michieli, C. Battistini, and P. M. Comoglio. 1998. Induction of epithelial tubules by growth factor HGF depends on the STAT pathway.Nature 391:285.
4. Bottaro, D. P., J. S. Rubin, D. L. Faletto, A. M. Chan, T. E. Kmiecik, G. F. Vande Woude, and S. A. Aaronson. 1991. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product.Science 251:802.
5. Bussolino, F., M. F. Di Renzo, M. Ziche, E. Bocchietto, M. Olivero, L. Naldini, G. Gaudino, L. Tamagnone, A. Coffer, and P. M. Comoglio. 1992. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth.J Cell Biol 119:629.
6. Bussolino, F., M. F. Di Renzo, M. Ziche, E. Bocchietto, M. Olivero, L. Naldini, G. Gaudino, L. Tamagnone, A. Coffer, and P. M. Comoglio. 1992. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth.J Cell Biol 119:629.
7. Cioce, V., K. G. Csaky, A. M. Chan, D. P. Bottaro, W. G. Taylor, R. Jensen, S. A. Aaronson, and J. S. Rubin. 1996. Hepatocyte growth factor (HGF)/NK1 is a naturally occurring HGF/scatter factor variant with partial agonist/antagonist activity.J Biol Chem 271:13110.
8. Dai, Y., M. Roman, R. K. Naviaux, and I. M. Verma. 1992. Gene therapy via primary myoblasts: long-term expression of factor IX protein following transplantation in vivo.Proc Natl Acad Sci U S A 89:10892.
9. Dupuis, M., K. Denis-Mize, C. Woo, C. Goldbeck, M. J. Selby, M. Chen, G. R. Otten, J. B. Ulmer, J. J. Donnelly, G. Ott, and D. M. McDonald. 2000. Distribution of DNA vaccines determines their immunogenicity after intramuscular injection in mice.J Immunol 165:2850.
10. Funakoshi, H., and T. Nakamura. 2001. Identification of HGF-like protein as a novel neurotrophic factor for avian dorsal root ganglion sensory neurons.Biochem Biophys Res Commun 283:606.
11. Heller, R., M. Jaroszeski, A. Atkin, D. Moradpour, R. Gilbert, J. Wands, and C. Nicolau. 1996. In vivo gene electroinjection and expression in rat liver.FEBS Lett 389:225.
12. Hoover, F., and J. Magne Kalhovde. 2000. A double-injection DNA electroporation protocol to enhance in vivo gene delivery in skeletal muscle.Anal Biochem 285:175.
13. Horton, H. M., D. Anderson, P. Hernandez, K. M. Barnhart, J. A. Norman, and S. E. Parker. 1999. A gene therapy for cancer using intramuscular injection of plasmid DNA encoding interferon alpha.Proc Natl Acad Sci U S A 96:1553.
14. Jakubczak, J. L., W. J. LaRochelle, and G. Merlino. 1998. NK1, a natural splice variant of hepatocyte growth factor/scatter factor, is a partial agonist in vivo.Mol Cell Biol 18:1275.
15. Jiang, W. G., M. B. Hallett, and M. C. Puntis. 1993. Hepatocyte growth factor/scatter factor, liver regeneration and cancer metastasis.Br J Surg 80:1368.
16. Kwon, M., C. S. Yoon, S. Fitzpatrick, G. Kassam, K. S. Graham, M. K. Young, and D. M. Waisman. 2001. p22 is a novel plasminogen fragment with antiangiogenic activity.Biochemistry 40:13246.
17. Lai, J. F., S. C. Kao, S. T. Jiang, M. J. Tang, P. C. Chan, and H. C. Chen. 2000. Involvement of focal adhesion kinase in hepatocyte growth factor- induced scatter of Madin-Darby canine kidney cells.J Biol Chem 275:7474.
18. Li, S., F. C. MacLaughlin, J. G. Fewell, Y. Li, V. Mehta, M. F. French, J. L. Nordstrom, M. Coleman, N. S. Belagali, R. J. Schwartz, and L. C. Smith. 1999. Increased level and duration of expression in muscle by co-expression of a transactivator using plasmid systems.Gene Ther 6:2005.
19. Li, S., X. Zhang, X. Xia, L. Zhou, R. Breau, J. Suen, and E. Hanna. 2001. Intramuscular electroporation delivery of IFN-alpha gene therapy for inhibition of tumor growth located at a distant site.Gene Ther 8:400.
20. Liu, Y., J. N. Centracchio, L. Lin, A. M. Sun, and L. D. Dworkin. 1998. Constitutive expression of HGF modulates renal epithelial cell phenotype and induces c-met and fibronectin expression.Exp Cell Res 242:174.
21. Liu, Y., J. N. Centracchio, L. Lin, A. M. Sun, and L. D. Dworkin. 1998. Constitutive expression of HGF modulates renal epithelial cell phenotype and induces c-met and fibronectin expression.Exp Cell Res 242:174.
22. Liu, Y., E. M. Tolbert, L. Lin, M. A. Thursby, A. M. Sun, T. Nakamura, and L. D. Dworkin. 1999. Up-regulation of hepatocyte growth factor receptor: an amplification and targeting mechanism for hepatocyte growth factor action in acute renal failure.Kidney Int 55:442.
23. Maruyama, H., K. Ataka, F. Gejyo, N. Higuchi, Y. Ito, H. Hirahara, I. Imazeki, M. Hirata, F. Ichikawa, T. Neichi, H. Kikuchi, M. Sugawa, and J. Miyazaki. 2001. Long-term production of erythropoietin after electroporation-mediated transfer of plasmid DNA into the muscles of normal and uremic rats.Gene Ther 8:461.
24. Mathiesen, I. 1999. Electropermeabilization of skeletal muscle enhances gene transfer in vivo.Gene Ther 6:508.
25. Matsumoto, K., and T. Nakamura. 1996. Emerging multipotent aspects of hepatocyte growth factor.J Biochem (Tokyo) 119:591.
26. Matsumoto, K., and T. Nakamura. 1997. Hepatocyte growth factor (HGF) as a tissue organizer for organogenesis and regeneration.Biochem Biophys Res Commun 239:639.
27. Mir, L. M., M. F. Bureau, J. Gehl, R. Rangara, D. Rouy, J. M. Caillaud, P. Delaere, D. Branellec, B. Schwartz, and D. Scherman. 1999. High-efficiency gene transfer into skeletal muscle mediated by electric pulses.Proc Natl Acad Sci U S A 96:4262.
28. Miyazawa, K., T. Shimomura, A. Kitamura, J. Kondo, Y. Morimoto, and N. Kitamura. 1993. Molecular cloning and sequence analysis of the cDNA for a human serine protease reponsible for activation of hepatocyte growth factor. Structural similarity of the protease precursor to blood coagulation factor XII.J Biol Chem 268:10024.
29. Miyazawa, K., H. Tsubouchi, D. Naka, K. Takahashi, M. Okigaki, N. Arakaki, H. Nakayama, S. Hirono, O. Sakiyama, and et al. 1989. Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor.Biochem Biophys Res Commun 163:967.
30. Mumper, R. J., J. Wang, S. L. Klakamp, H. Nitta, K. Anwer, F. Tagliaferri, and A. P. Rolland. 1998. Protective interactive noncondensing (PINC) polymers for enhanced plasmid distribution and expression in rat skeletal muscle.J Control Release 52:191.
31. Nakagami, H., R. Morishita, K. Yamamoto, Y. Taniyama, M. Aoki, K. Matsumoto, T. Nakamura, Y. Kaneda, M. Horiuchi, and T. Ogihara. 2001. Mitogenic and antiapoptotic actions of hepatocyte growth factor through ERK, STAT3, and AKT in endothelial cells.Hypertension 37:581.
32. Nakamura, T., T. Nishizawa, M. Hagiya, T. Seki, M. Shimonishi, A. Sugimura, K. Tashiro, and S. Shimizu. 1989. Molecular cloning and expression of human hepatocyte growth factor.Nature 342:440.
33. Nishi, T., K. Yoshizato, S. Yamashiro, H. Takeshima, K. Sato, K. Hamada, I. Kitamura, T. Yoshimura, H. Saya, J. Kuratsu, and Y. Ushio. 1996. High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation.Cancer Res 56:1050.
34. O'Malley, B. W., Jr., K. A. Cope, C. S. Johnson, and M. R. Schwartz. 1997. A new immunocompetent murine model for oral cancer.Arch Otolaryngol Head Neck Surg 123:20.
35. Pohl, J. F., H. Melin-Aldana, G. Sabla, J. L. Degen, and J. A. Bezerra. 2001. Plasminogen deficiency leads to impaired lobular reorganization and matrix accumulation after chronic liver injury.Am J Pathol 159:2179.
36. Rijken, D. C., and D. V. Sakharov. 2001. Basic principles in thrombolysis: regulatory role of plasminogen.Thromb Res 103 Suppl 1:S41.
37. Rizzuto, G., M. Cappelletti, D. Maione, R. Savino, D. Lazzaro, P. Costa, I. Mathiesen, R. Cortese, G. Ciliberto, R. Laufer, N. La Monica, and E. Fattori. 1999. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation.Proc Natl Acad Sci U S A 96:6417.
38. Rubin, J. S., D. P. Bottaro, and S. A. Aaronson. 1993. Hepatocyte growth factor/scatter factor and its receptor, the c-met proto-oncogene product.Biochim Biophys Acta 1155:357.
39. Rubin, J. S., A. M. Chan, D. P. Bottaro, W. H. Burgess, W. G. Taylor, A. C. Cech, D. W. Hirschfield, J. Wong, T. Miki, P. W. Finch, and et al. 1991. A broad-spectrum human lung fibroblast-derived mitogen is a variant of hepatocyte growth factor.Proc Natl Acad Sci U S A 88:415.
40. Shima, N., M. Nagao, F. Ogaki, E. Tsuda, A. Murakami, and K. Higashio. 1991. Tumor cytotoxic factor/hepatocyte growth factor from human fibroblasts: cloning of its cDNA, purification and characterization of recombinant protein.Biochem Biophys Res Commun 180:1151.
41. Shimomura, T., K. Denda, A. Kitamura, T. Kawaguchi, M. Kito, J. Kondo, S. Kagaya, L. Qin, H. Takata, K. Miyazawa, and N. Kitamura. 1997. Hepatocyte growth factor activator inhibitor, a novel Kunitz-type serine protease inhibitor.J Biol Chem 272:6370.
42. Skibinski, G., A. Skibinska, and K. James. 2001. The role of hepatocyte growth factor and its receptor c-met in interactions between lymphocytes and stromal cells in secondary human lymphoid organs.Immunology 102:506.
43. Thewke, D. P., and N. W. Seeds. 1996. Expression of hepatocyte growth factor/scatter factor, its receptor, c- met, and tissue-type plasminogen activator during development of the murine olfactory system.J Neurosci 16:6933.
44. van der Voort, R., T. E. Taher, P. W. Derksen, M. Spaargaren, R. van der Neut, and S. T. Pals. 2000. The hepatocyte growth factor/Met pathway in development, tumorigenesis, and B-cell differentiation.Adv Cancer Res 79:39.
45. van der Voort, R., T. E. Taher, R. M. Keehnen, L. Smit, M. Groenink, and S. T. Pals. 1997. Paracrine regulation of germinal center B cell adhesion through the c- met-hepatocyte growth factor/scatter factor pathway.J Exp Med 185:2121.
46. Vicat, J. M., S. Boisseau, P. Jourdes, M. Laine, D. Wion, R. Bouali-Benazzouz, A. L. Benabid, and F. Berger. 2000. Muscle transfection by electroporation with high-voltage and short- pulse currents provides high-level and long-lasting gene expression.Hum Gene Ther 11:909.
47. Weidner, K. M., N. Arakaki, G. Hartmann, J. Vandekerckhove, S. Weingart, H. Rieder, C. Fonatsch, H. Tsubouchi, T. Hishida, Y. Daikuhara, and et al. 1991. Evidence for the identity of human scatter factor and human hepatocyte growth factor.Proc Natl Acad Sci U S A 88:7001.
48. Wolff, J. A., R. W. Malone, P. Williams, W. Chong, G. Acsadi, A. Jani, and P. L. Felgner. 1990. Direct gene transfer into mouse muscle in vivo.Science 247:1465.
49. Yamashita, Y. I., M. Shimada, H. Hasegawa, R. Minagawa, T. Rikimaru, T. Hamatsu, S. Tanaka, K. Shirabe, J. I. Miyazaki, and K. Sugimachi. 2001. Electroporation-mediated interleukin-12 gene therapy for hepatocellular carcinoma in the mice model.Cancer Res 61:1005.
50. Yang, J., S. Chen, L. Huang, G. K. Michalopoulos, and Y. Liu. 2001. Sustained expression of naked plasmid DNA encoding hepatocyte growth factor in mice promotes liver and overall body growth.Hepatology 33:848.
51. Yin, D., and J. G. Tang. 2001. Gene therapy for streptozotocin-induced diabetic mice by electroporational transfer of naked human insulin precursor DNA into skeletal muscle in vivo.FEBS Lett 495:16.
52. Yu, C. Z., H. Hisha, Y. Li, Z. Lian, T. Nishino, J. Toki, Y. Adachi, M. Inaba, T. X. Fan, T. Jin, T. Iguchi, S. Sogo, N. Hosaka, T. H. Song, J. Xing, and S. Ikehara. 1998. Stimulatory effects of hepatocyte growth factor on hemopoiesis of SCF/c- kit system-deficient mice.Stem Cells 16:66.
53. Zarnegar, R., and G. K. Michalopoulos. 1995. The many faces of hepatocyte growth factor: from hepatopoiesis to hematopoiesis.J Cell Biol 129:1177.