論文名稱:

生長因子對骨髓基質細胞的影響

 

Impact of growth factors on bone marrow stromal cells

研究生:

藍文謙  Wen-Chien Lan

 

(以作者名查詢陽明大學館藏系統)

 

(以作者名查詢全國圖書書目資訊網)

指導教授:

董醒任  Shing-Zeng Dung

        學位類別:

碩士

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

          學年度:

85

          語文別:

中文

          出版年:

86

關鍵字:

牙醫  DENTIST

 

牙醫學  DENTISTRY

 

口腔  ORAL-HEALTH

 

生長因子 

 

骨髓 

摘要:

臨床醫學研究發現,在治療嚴重骨缺損的患者時常遇到骨增生的量不足以及骨癒合之時間太長的問題。由於骨髓基質細胞是一種多潛力的細胞,它擁有分化形成各種細胞的能力;且骨生長因子有調控細胞分裂和分化的能力。本研究希望藉由瞭解骨髓基質細胞的特性,並以生長因子調控骨髓基質的生長與細胞分化,而加速骨癒合。因此以細胞培養的方式將骨髓細胞加以分離;在細胞近飽和時以血小板源生長因子 (Platelet-derived growth factorPDGF)、類胰島素生長因子(Insulin-like growth factor IGF-I)、和PDGF合併IGF-I處理,然後固定,並用光學顯微鏡與掃描式電子顯微鏡觀察細胞形態,而且使用細胞顆粒計數器分析細胞數量與大小分佈,以及免疫化學染色觀察integrin的表現。此外也以不同技術:MTT、鹼性磷酸酉每(alkaline phosphatase)、以及von Kossaalizarin red鈣化染色法,分析骨髓基質細胞生長及鈣化等功能之變化。實驗發現骨髓基質細胞是由許多不同形態、大小不一的細胞所組成;培養一週後,細胞大小是以10-20μm為主;以及有鹼性磷酸酉每的正反應與integrin α3αv的表現。PDGF能增加細胞的密度與細胞突觸的形成,以及提高40%細胞分裂增殖的能力且可促進integrin αv的表現,但卻抑制integrin α3的表現;IGF-I則會減少細胞分裂初期的細胞密度,增加細胞結節的形成,且同樣能促進integrin α3αv在細胞結節的表現,但對於細胞分裂增殖的能力,並無明顯的影響。在細胞鈣化的模型上,只有轉化生長因子-β(transforming growth factorTGF-β)在第一週即可增加細胞鈣化的速度及面積:PDGFIGF-I雖可增加細胞結節的數目,但在細胞鈣化的量及速度上似乎並無增加;而纖維原細胞生長因子 (fibroblast growth factorFGF)似有抑制鈣化之情形。由這些結果我們認為:骨髓基質細胞的分裂、分化、鈣化似乎可由不同生長因子來調控。骨再生是目前骨科及牙科研究的主流及治療的最終目標。本研究發現 不同骨生長因子可調控骨髓基質細胞而促進骨細胞的量及鈣化的速度。因此利用生長因子對於骨髓基質細胞的調控,並配合臨床之細胞與分子生物技術,應可為骨組織再生的領域帶來新的突破。

   
 

Growth factors play important roles in bone cell function. During bone healing, these factors may directly or indirectly regulate cells of the bone marrow stroma and re-establish the osteoblast phenotype and induce new bone formation. The purpose of the study was to explore the effects of bone gowth factors on cell morphology, cell proliferation, α3 and αv integrin expression, and in vitro mineralization. Bone marrow was flushed from rat femurs, the adherent cells were separated from the non-adherent cells, and be maintained in α-MEM containing 10 % FBS and ascorbic acid. Change of cell size and distribution after 0.5,3,6, or 24h treatment of PDGF-AB ( 20 ng/ml ), IGF-I ( 100 ng/ml), or the combination of PDGF-AB and IGF-I in serum-free α-MEM were evaluated by a particle size and distribution counter. Morphologic changes of primary bone marrow stromal cells after 0.5 or 3 h of PDGF-AB or IGF-I treatment were evaluated by light and electron microscopies. After 6 or 24 h treatment of bone marrow stromal cells with each of IGF-I, PDGF-AB or IGF-I+PDGF-AB, cell proliferation was determined by the MTT assay. The α3 integrin and fibronectin expression was evaluated by indirect immunoperoxidase localization using specific antibodies under light and laser confocal scanning microscopy. In an in vitro mineralization model, 10-8M dexamethasone, 10mM β-glycerophosphate, and 5 % FBS were used. Impact ofIGF-I, PDGF-AB, PDGF-AB combinate with IGF-I, bFGF  ( 10 ng/ml ), and TGF-β ( 10 ng/ml ) on the formation of mineralized nodule was evaluated by von Kossa and alizarin red stains. After 6 or 24 h of PDGF-AB or IGF-I treatment, the α3 and αv integrin expression was evaluated by indirect immunoperoxidase localization using specific antibodies. Results indicated that cells of 5-10μm decreased with time for all treatment groups and that 6 h PDGF-AB treatment showed two peaks in cells with size distribution ranged from 10 to 20μm. PDGF-AB increased cell density while IGF-I formed more small colonies. Scanning electron microscopic examinations also demonstrated that PDGF-AB increased cell

 

process in 30 min while it took 3 h for IGF-I to show a similar response. Cell proliferation assay showed that while PDGF-AB or PDGF-AB combinate with IGF-I increased 40 % of cell numbers, IGF-I had no effects on cell proliferation as compared to the controls. PDGF-AB or IGF-I appeared to enhance αv integrin expression. While α3 integrin expression was downregulated by PDGF-AB, αv integrin was upregulated by IGF-I.  Compared to the control, only TGF-P accelerated the rate and amount of mineralization in one week. PDGF-AB and IGF-I appeared to increase the number of mineralization nodules while the rate and amount of mineralization was relatively slower and smaller. In conclusion , bone growth factors appeared to have differential effects on the biological activities of bone marrow stromal cells and the molecular mechanisms involved in the modulation of these activities required further investigations.