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研究生(外文):Yue-Lin Tsai
論文名稱(外文):Evaluation of the effects of an ectopically-expressed HOX-B4 and Dkk-1 on the growth of human mesenchymal stem cells
指導教授(外文):Yeu Su
  • 被引用被引用:1
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取自骨髓和臍帶血的間質幹細胞(mesenchymal stem cells, MSC)受到不同刺激可分化為中胚層所衍生的組織,如血管平滑肌肉細胞、骨細胞、脂肪細胞,也能轉分化成內胚層或外胚層所衍生的細胞系,如肝臟上皮細胞、肺臟上皮細胞、腸內皮細胞、星細胞等,為研究幹細胞分化與增殖之分子機制的最佳材料。但由於MSC在體外長期且大量培養不易,限制了它們的應用。近年的研究顯示HOXB4轉錄因子能促進造血幹細胞(HSC)之更新(self-renewal),且不影響其分化能力;而Dkk-1分泌蛋白則可藉由抑制Wnt訊息傳遞而促進MSC暫時性增殖。反之,活化Wnt訊息傳遞可促進HSC的增殖,因此我們想瞭解HOXB4與Dkk-1對人類MSC生長及分化的影響。本研究中我們嘗試利用蛋白質傳輸(protein transduction)與重組腺病毒的方式分別將HOXB4與Dkk-1的蛋白或其基因送入MSC內。我們發現, PTDTAT-GFP融合蛋白質可於1小時內傳輸至HaCaT細胞中,而Pep-1□肽/GFP複合物也能被傳輸至Rat-1細胞中。利用重組腺病毒感染MSC,發現當MOI等於三千即可達到80 %的感染率,但當MOI超過一萬時,會造成部分MSC死亡的現象。以重組腺病毒轉殖之LacZ報告基因,其在MSC內的表現至少維持21天。最有趣的是,我們發現經AdHOXB4與AdDkk-1感染之MSC,其增殖狀況優於受AdLacZ感染的細胞,目前我們正進一步研究這些經不同基因轉殖的MSC之分化能力,以評估此種促進間質幹細胞體外增殖的策略,未來在臨床應用的可行性。
Human bone marrow-derived mesenchymal stem cells (hMSCs) are multipotent, capable of differentiating into at least three lineages (osteogenic, chondrogenic, and adipogenic) when cultured under appropriate conditions. Because of these unique features, hMSCs, either on their own or in combination with therapeutic gene therapy, hold great promises for treating a variety of diseases. However, methods to promote the ex vivo expansion of hMSCs must be developed before the routine use of these cells in transplantation therapy can be realized. In this regard, Dickkopf-1(Dkk-1), a potent secreted Wnt antagonist that allows hMSCs to reenter the cell cycle, is a good candidate. Another one worthy of testing is HOXB4 since transduction of its gene and/or protein into hematopoietic stem cells (HSCs) enhances their ex vivo proliferation without impairing their function. To examine the feasibility of using large quantity of HOXB4 or Dkk-1 to facilitate the ex vivo expansion of hMSCs, strategies including direct protein transduction and gene transfer through adenovirus infection were explored in the present study. We showed that green fluorescence protein (GFP) could be transduced efficiently into various types of cells either as a chimeric protein fused with a protein transduction domain (PTD) derived from the Tat protein of HIV-1 or as a complex associated with Pep-1. However, we were unable to overproduce PTDtat-HOXB4 to further examine its effect on hMSCs growth. In the meantime, we detected high expression levels of LacZ gene transduced by recombinant adenoviruses in hMSCs when multiplicity of infection (MOI) of the virus was 3,000. Moreover, results from both MTS assay and direct cell counting demonstrated an increased proliferation of hMSCs ex vivo by enforced expression of virally transduced HOXB4 or Dkk-1 gene.
Alonso, L. and Fuchs, E. (2003). Stem cells in the skin: waste not, Wnt not. Genes Dev 17, 1189-1200.
Antonchuk, J., Sauvageau, G., and Humphries, R. K. (2002). HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 109, 39-45.
Azizi, S. A., Stokes, D., Augelli, B. J., DiGirolamo, C., and Prockop, D. J. (1998). Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats--similarities to astrocyte grafts. Proc Natl Acad Sci U S A 95, 3908-3913.
Benihoud, K., Yeh, P., and Perricaudet, M. (1999). Adenovirus vectors for gene delivery. Curr Opin Biotechnol 10, 440-447.
Berkner, K. L. (1988). Development of adenovirus vectors for the expression of heterologous genes. Biotechniques 6, 616-629.
Bjornsson, J. M., Larsson, N., Brun, A. C., Magnusson, M., Andersson, E., Lundstrom, P., Larsson, J., Repetowska, E., Ehinger, M., Humphries, R. K., and Karlsson, S. (2003). Reduced proliferative capacity of hematopoietic stem cells deficient in Hoxb3 and Hoxb4. Mol Cell Biol 23, 3872-3883.
Blatt, C., Lotem, J., and Sachs, L. (1992). Inhibition of specific pathways of myeloid cell differentiation by an activated Hox-2.4 homeobox gene. Cell Growth Differ 3, 671-676.
Brun, A. C., Fan, X., Bjornsson, J. M., Humphries, R. K., and Karlsson, S. (2003). Enforced adenoviral vector-mediated expression of HOXB4 in human umbilical cord blood CD34+ cells promotes myeloid differentiation but not proliferation. Mol Ther 8, 618-628.
Caron, N. J., Torrente, Y., Camirand, G., Bujold, M., Chapdelaine, P., Leriche, K., Bresolin, N., and Tremblay, J. P. (2001). Intracellular delivery of a Tat-eGFP fusion protein into muscle cells. Mol Ther 3, 310-318.
Chang, C. P., Brocchieri, L., Shen, W. F., Largman, C., and Cleary, M. L. (1996). Pbx modulation of Hox homeodomain amino-terminal arms establishes different DNA-binding specificities across the Hox locus. Mol Cell Biol 16, 1734-1745.
Deans, R. J. and Moseley, A. B. (2000). Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol 28, 875-884.
Dennis, J. E. and Charbord, P. (2002). Origin and differentiation of human and murine stroma. Stem Cells 20, 205-214.
Frankel, A. D. and Pabo, C. O. (1988). Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55, 1189-1193.
Friedenstein, A. J. (1976). Precursor cells of mechanocytes. Int Rev Cytol 47, 327-359.
Green, M. and Loewenstein, P. M. (1988). Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell 55, 1179-1188.
Gregory, C. A., Singh, H., Perry, A. S., and Prockop, D. J. (2003). The Wnt signaling inhibitor dickkopf-1 is required for reentry into the cell cycle of human adult stem cells from bone marrow. J Biol Chem 278, 28067-28078.
He, T. C., Zhou, S., da Costa, L. T., Yu, J., Kinzler, K. W., and Vogelstein, B. (1998). A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 95, 2509-2514.
Helgason, C. D., Sauvageau, G., Lawrence, H. J., Largman, C., and Humphries, R. K. (1996). Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro. Blood 87, 2740-2749.
Ho, A., Schwarze, S. R., Mermelstein, S. J., Waksman, G., and Dowdy, S. F. (2001). Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo. Cancer Res 61, 474-477.
Jiang, Y., Jahagirdar, B. N., Reinhardt, R. L., Schwartz, R. E., Keene, C. D., Ortiz-Gonzalez, X. R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., et al. (2002). Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418, 41-49.
Jin, H. K., Carter, J. E., Huntley, G. W., and Schuchman, E. H. (2002). Intracerebral transplantation of mesenchymal stem cells into acid sphingomyelinase-deficient mice delays the onset of neurological abnormalities and extends their life span. J Clin Invest 109, 1183-1191.
Kopen, G. C., Prockop, D. J., and Phinney, D. G. (1999). Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci U S A 96, 10711-10716.
Krosl, J., Baban, S., Krosl, G., Rozenfeld, S., Largman, C., and Sauvageau, G. (1998). Cellular proliferation and transformation induced by HOXB4 and HOXB3 proteins involves cooperation with PBX1. Oncogene 16, 3403-3412.
Krosl, J. and Sauvageau, G. (2000). AP-1 complex is effector of Hox-induced cellular proliferation and transformation. Oncogene 19, 5134-5141.
Kuppuswamy, M., Subramanian, T., Srinivasan, A., and Chinnadurai, G. (1989). Multiple functional domains of Tat, the trans-activator of HIV-1, defined by mutational analysis. Nucleic Acids Res 17, 3551-3561.
Kwon, H. Y., Eum, W. S., Jang, H. W., Kang, J. H., Ryu, J., Ryong Lee, B., Jin, L. H., Park, J., and Choi, S. Y. (2000). Transduction of Cu,Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into mammalian cells. FEBS Lett 485, 163-167.
Lawrence, H. J., Sauvageau, G., Ahmadi, N., Lopez, A. R., LeBeau, M. M., Link, M., Humphries, K., and Largman, C. (1995). Stage- and lineage-specific expression of the HOXA10 homeobox gene in normal and leukemic hematopoietic cells. Exp Hematol 23, 1160-1166.
Lee, O. K., Kuo, T. K., Chen, W. M., Lee, K. D., Hsieh, S. L., and Chen, T. H. (2004). Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 103, 1669-1675.
Li, Y., Chen, J., Chen, X. G., Wang, L., Gautam, S. C., Xu, Y. X., Katakowski, M., Zhang, L. J., Lu, M., Janakiraman, N., and Chopp, M. (2002). Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery. Neurology 59, 514-523.
Lill, M. C., Fuller, J. F., Herzig, R., Crooks, G. M., and Gasson, J. C. (1995). The role of the homeobox gene, HOX B7, in human myelomonocytic differentiation. Blood 85, 692-697.
Magli, M. C., Largman, C., and Lawrence, H. J. (1997). Effects of HOX homeobox genes in blood cell differentiation. J Cell Physiol 173, 168-177.
Mai, J. C., Shen, H., Watkins, S. C., Cheng, T., and Robbins, P. D. (2002). Efficiency of protein transduction is cell type-dependent and is enhanced by dextran sulfate. J Biol Chem 277, 30208-30218.
Morris, M. C., Depollier, J., Mery, J., Heitz, F., and Divita, G. (2001). A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat Biotechnol 19, 1173-1176.
Ortiz, L. A., Gambelli, F., McBride, C., Gaupp, D., Baddoo, M., Kaminski, N., and Phinney, D. G. (2003). Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A 100, 8407-8411.
Owens, B. M. and Hawley, R. G. (2002). HOX and non-HOX homeobox genes in leukemic hematopoiesis. Stem Cells 20, 364-379.
Pereira, R. F., O''Hara, M. D., Laptev, A. V., Halford, K. W., Pollard, M. D., Class, R., Simon, D., Livezey, K., and Prockop, D. J. (1998). Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta. Proc Natl Acad Sci U S A 95, 1142-1147.
Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S., and Marshak, D. R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science 284, 143-147.
Prockop, D. J., Gregory, C. A., and Spees, J. L. (2003). One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues. Proc Natl Acad Sci U S A 100 Suppl 1, 11917-11923.
Reya, T., Duncan, A. W., Ailles, L., Domen, J., Scherer, D. C., Willert, K., Hintz, L., Nusse, R., and Weissman, I. L. (2003). A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423, 409-414.
Satoh, Y., Matsumura, I., Tanaka, H., Ezoe, S., Sugahara, H., Mizuki, M., Shibayama, H., Ishiko, E., Ishiko, J., Nakajima, K., and Kanakura, Y. (2004). Roles for c-Myc in self-renewal of hematopoietic stem cells. J Biol Chem 279, 24986-24993.
Schiedlmeier, B., Klump, H., Will, E., Arman-Kalcek, G., Li, Z., Wang, Z., Rimek, A., Friel, J., Baum, C., and Ostertag, W. (2003). High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation. Blood 101, 1759-1768.
Schwarz, E. J., Alexander, G. M., Prockop, D. J., and Azizi, S. A. (1999). Multipotential marrow stromal cells transduced to produce L-DOPA: engraftment in a rat model of Parkinson disease. Hum Gene Ther 10, 2539-2549.
Schwarze, S. R., Hruska, K. A., and Dowdy, S. F. (2000). Protein transduction: unrestricted delivery into all cells? Trends Cell Biol 10, 290-295.
Semenov, M. V., Tamai, K., Brott, B. K., Kuhl, M., Sokol, S., and He, X. (2001). Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6. Curr Biol 11, 951-961.
Shake, J. G., Gruber, P. J., Baumgartner, W. A., Senechal, G., Meyers, J., Redmond, J. M., Pittenger, M. F., and Martin, B. J. (2002). Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg 73, 1919-1925; discussion 1926.
Tremain, N., Korkko, J., Ibberson, D., Kopen, G. C., DiGirolamo, C., and Phinney, D. G. (2001). MicroSAGE analysis of 2,353 expressed genes in a single cell-derived colony of undifferentiated human mesenchymal stem cells reveals mRNAs of multiple cell lineages. Stem Cells 19, 408-418.
Vieille-Grosjean, I. and Huber, P. (1995). Transcription factor GATA-1 regulates human HOXB2 gene expression in erythroid cells. J Biol Chem 270, 4544-4550.
Wadia, J. S. and Dowdy, S. F. (2002). Protein transduction technology. Curr Opin Biotechnol 13, 52-56.
Wu, S., Suzuki, Y., Ejiri, Y., Noda, T., Bai, H., Kitada, M., Kataoka, K., Ohta, M., Chou, H., and Ide, C. (2003). Bone marrow stromal cells enhance differentiation of cocultured neurosphere cells and promote regeneration of injured spinal cord. J Neurosci Res 72, 343-351.
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