臺灣博碩士論文加值系統

軟骨的復元是極為緩慢的，對關節軟骨受傷的病患，目前多以軟骨移植的手段幫助關節恢復功能，但人體自身可取用的軟骨極為有限，這成為修復關節軟骨的一大障礙。間葉幹細胞具有分化為表皮、肌肉、脂肪、軟骨及骨母等細胞的能力，已經成功的用於關節軟骨的修補，但對於生長環境對間葉幹細胞分化路徑的影響，目前的了解還是有限。
關節軟骨的組成以水分最多，其次是第二型膠原蛋白(Collagen II)和胺多醣 (Glycosaminoglycans，GAG)。軟骨細胞可份泌膠原蛋白與胺多醣，軟骨便是由於collagen II和胺多醣結合，規則而緊密的排列而形成，文獻中顯示以第一型膠原蛋白(collagen I)為基質會誘導間葉幹細胞朝骨細胞分化，而在第一型膠原蛋白加入胺多醣，則會使間葉幹細胞朝軟骨細胞分化。另外有研究顯示，以瓊脂(agarose)包覆細胞進行培養，則間葉幹細胞也會朝軟骨細胞分化。
由此，我們懷疑培養間葉幹細胞基質的表面性質，將影響其分化方向，特別是表面的黏著性與細胞的貼附性。本研究擬以貼附性極佳的第一型膠原蛋白為基質，控制組是接上chondroitin sulfate做為適合軟骨細胞生長的控制組，實驗組是以 iodopropionic acid 進行collagen I的表面改質，形成降低蛋白貼附性的zwitterionic結構，用以降低細胞在基質表面的貼附性，試圖以改變間葉幹細胞的貼附性來探討貼附性對其分化的影響。我們以細胞內aggrecan，collagen II，及SOX9 mRNA的表現來偵測間葉幹細胞也會朝軟骨細胞分化的程度，結果發現增加chondrotin sulfate在collagen I的接枝量，的確會增加aggrecan，collagen II，及SOX9的表現量，而隨著增加1 wt%、3wt%、5 wt%、7 wt% Iodopropionic acid的接枝量，同樣會使aggrecan，collagen II，及SOX9的表現量增加，直到改質9wt% Iodopropionic acid，其aggrecan，collagen II，及SOX9的表現量呈現減少的狀態。
關鍵詞: 表面沾粘性、軟骨、間葉幹細胞

Cartilages are poorly vascularized tissues with weak capacity for self-repair. Therre are many causes of cartilage degeneration. The degenerated cartilages will never heal. Autologous transplants has been developed to repair articular cartilage and shown some success. Brittberg et al. has seeded autologous chondrocytes in wounded sites by extracting chondrocytes from a low weight-bearing region of the body and expanded in vitro. But we usually lacks the source of autologous chondrocytes. Mesenchymal stem cells (MSCs) have the capacity to differentiate toward epithelials, adiposes, myoblasts, osteoblasts, or chondrocytes, and have been successfully adopted in cartilage repair. However, the process of chondrogenesis of MSC has not been fully understood.
The major component of cartilage is water, and the rest are type II collagen and glycosaminoglycans. The type II collagen and glycosaminoglycans are actually secreted by chondrocytes. Studies has shown that type I collagen induced MSCs to differentiate toward osteoblasts. But the addition of GAGs shifted the differentiation toward chondrocytes. Other studies showed that MSCs cultivated on agarose differentiated toward chondrocytes.
This study aims at finding out the effect of surface adhesivity on mesenchymal stem cell differentiation. We tried to cultivate MSCs on the surface of collagen I. The surface adhesivity of collagen I was altered by grafting various amount of non-adhesive polymer, Iodopropionic acid. The expression of aggrecan, collagen II, and SOX9 were measured as the indication of chondrogenesis. It was found that the expression of aggrecan, collagen II，and SOX9 increased as the grafting density of chondroitin sulfate increased. Similarly, the chondrogenesis indication also increased with the increase in Iodopropionic acid content.
Key words: Surface adhesivity,Chondrocyes,Mesencymal stem cell

目錄
中文摘要………………………………………………………I
Abstract……………………………………………………………III
致謝………………………………………………………………..IV
目錄……………………………………………………………...…V
圖目錄…………………………………………………………...VII
表目錄……………………………………………………………...X
第一章 緒論…………………………………………………1
第一節 軟骨傷害及治療方法……………..………………..1
第二節 軟骨結構………………………………….………….3
第三節 細胞外間質分子對間葉幹細胞分化為軟骨細胞之影響……5
第四節 以果膠培養間葉幹細胞分化為軟骨細胞……..….7
第五節 研究目的..........................................................12
第二章　實驗材料及方法……………………………………13
第一節 實驗設備…………………………………………13
第二節 實驗藥品…………………………………………15
第三節 藥品製備…………………………………………17
第四節 細胞培養…………………………………………20
第五節 實驗設計…………………………………………21
第六節 實驗操作…………………………………………23
第三章 結果與討論…………………………………………31
第一節 Chondroitin sulfate (CS)改質Collagen I薄膜的目的與性質測定....................................................................31
第二節 Iodopropinoic acid(IPA) 改質Collagen I薄膜的目的與性質的測定.................................................................35
第三節 表面改質過後對大鼠骨髓間葉幹細胞貼附之影響
.....................................................................................40
第四節 表面改質過後對大鼠骨髓間葉幹細胞成長之影響
…………………………………………………………………43
第五節 表面改質過後對大鼠骨髓間葉幹細胞分化之影響
………………………………………………………………....47
第四章 結論......................................................................56
第五章 未來規劃………………………………………………..57
參考文獻..........................................................................58
附錄(電泳全圖)………………………………………………62

圖目錄
圖1-1 軟骨組織結構圖…………………………………………4
圖3-1 Collagen I 薄膜 X射線光電子全波段光譜...............33
圖3-2 Collagen I+10 wt% CS X射線光電子全波段光譜
.........................................................................................33
圖3-3 Collagen I+50wt% CS X射線光電子全波段光譜
..........................................................................................34
圖3-4 Collagen I 薄膜 X射線光電子分峰.........................36
圖3-5 Collagen I+1 wt% IPA X射線光電子分峰............36
圖3-6 Collagen I+3 wt% IPA X射線光電子分峰............37
圖3-7 Collagen I+5 wt% IPA X射線光電子分峰.............37
圖3-8 Collagen I+7 wt% IPA X射線光電子分峰.............38
圖3-9 Collagen I+9 wt% IPA X射線光電子分峰.............38

圖3-10表面改質六小時後大鼠骨髓間葉幹細胞改質後的貼附
.........................................................................................41
圖3-11表面改質六小時後，大鼠骨髓間葉幹細胞改質後的貼附率..................................................................................42
圖3-12表面改質後大鼠骨髓間葉幹細胞一個月生長情形
..........................................................................................44
圖3-13表面改質後大鼠骨髓間葉幹細胞第一天生長情況
..........................................................................................44
圖3-14表面改質後大鼠骨髓間葉幹細胞第七天生長情況
..........................................................................................45
圖3-15表面改質後大鼠骨髓間葉幹細胞第十四天生長情況
.........................................................................................45
圖3-16表面改質後大鼠骨髓間葉幹細胞第二十一天生長情況
..........................................................................................46

圖3-17表面改質後大鼠骨髓間葉幹細胞第二十八天生長情況
..........................................................................................46
圖3-18 collagen II、aggrecan、sox9 mRNA改質第一天的表現
..........................................................................................48
圖3-19 collagen II、aggrecan、sox9 mRNA在表面改質過後第七天的表現量
..........................................................................................49
圖3-20 collagen II、aggrecan、sox9 mRNA在表面改質過後第十四天的表現量
..........................................................................................50
圖3-21 collagen II、aggrecan、sox9 mRNA在表面改質過後第二十一天的表現量
.........................................................................................51
圖3-22 collagen II、aggrecan、sox9 mRNA在表面改質過後第二十八天的表現量
.........................................................................................52
圖3-23 collagen II mRNA表面改質過後第一天到第二十八天的表現量
..........................................................................................54
圖3-24 aggrecan mRNA表面改質過後第一天到第二十八天的表現量
.........................................................................................55
圖3-25 sox9 mRNA表面改質過後第一天到第二十八天的表現量
.........................................................................................55


表目錄
表3-1 表面改質後的硫碳比
…………………………………………………………………….34
表3-2表面改質後-COO/-CH含量
…………………………………………………………………….39
附錄(電泳全圖)………………………………………………62

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