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研究生:蔡岳霖
研究生(外文):Yue-Lin Tsai
論文名稱:探討HOX-B4轉錄因子與Dkk-1分泌蛋白之異位表現對人類間質幹細胞生長的影響
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:蘇瑀蘇瑀引用關係
指導教授(外文):Yeu Su
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:57
中文關鍵詞:間質幹細胞
外文關鍵詞:MSCHOXB4Dkk-1
相關次數:
  • 被引用被引用:1
  • 點閱點閱:291
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  • 下載下載:16
  • 收藏至我的研究室書目清單書目收藏:0
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.
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