論文名稱:

成體中源於脂肪組織的基質幹細胞在體外分化為類似神經性細胞

 

Adult Adipose Tissue-Derived Stromal Stem Cells Differentiate into Neural-like Cells in Vitro

研究生:

陸柏年  Po-Nien Lu

 

(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:

楊世芳  Shih-Fang Yang

        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

            學號:

39017007

          學年度:

91

          語文別:

中文

          出版年:

92

關鍵字:

幹細胞  Stem Cell

 

基質細胞  stromal cell

 

肪組織  adipose tissue

 

神經性  neural

全文說明:        論文頁數:

61

摘要:

    幹細胞是一群具有自我增殖能力的未分化細胞的總稱。藉由誘導幹細胞分化為各種不同的細胞種類,可以應用於修復各種組織損傷。源於脂肪組織的基質幹細胞(Adipose Tissue Derived Stromal Stem Cell)和骨髓基質幹細胞具有的共同的來源,也和骨髓基質幹細胞一樣具有分化為各種中胚層組織的能力,但取得比骨髓基質幹細胞更容易,因此可能可以利用源於脂肪組織的基質幹細胞替代骨髓基質幹細胞應用於修補受損組織的技術上。由於本實驗室已能有效誘導牙乳突細胞分化成神經性細胞,若能誘導源於脂肪組織的基質幹細胞分化成神經細胞,其實用性可能遠大於骨髓基質幹細胞。因此本研究的主要目標即為測試是否能夠根據誘導骨髓基質幹細胞或牙乳突細胞分化成神經細胞的方法來誘導源於脂肪組織的基質幹細胞分化成神經細胞。

 

    在本研究中,以酵素分解S.D.大白鼠腹腔的脂肪組織,以離心的方法將密度低的脂肪細胞除去,分離出源於脂肪組織的基質幹細胞,並培養於Dulbecco's modified eagle media與胎牛血清組成的培養液中。以倒立式像位差光學顯微鏡觀察並記錄這些脂肪基質幹細胞的形態,同時觀察到當培養液中加入dexamethasonebeta-glycerophosphate2-phosphate ascorbate時,這些細胞可以分化為具堆積鈣質能力的細胞。以免疫化學染色法證實在實驗室中培養的源於脂肪組織的基質幹細胞中有部份細胞具有神經營養因子(neurotrophin)之受體:TrkP75NTR的表現。除此之外,以神經生長因子(nerve growth factor)加入源於脂肪組織的基質幹細胞的培養基,結果發現其細胞形態雖沒有產生明顯的改變,但以免疫化學染色法可觀察到幹細胞特有的標記nestin的表現下降,神經細胞的標記NeuNneurofilaments 200KD的表現則上升,星狀膠細胞(astrocyte)的標記GFAP的表現量上升,寡突膠細胞(oligodendrocyte)標記A2B5的表現量則無顯著改變。故可推測經神經生長因子處理的源於脂肪組織的基質幹細胞其分化可能傾向神經細胞或星狀細胞而非寡突細胞,且處於未完全分化的狀態。本研究結果顯示源於脂肪組織的基質幹細胞中確實有一些細胞能在神經生長因子的誘導下進行神經性分化。未來可繼續探討純化這些有神經分化能力的細胞的方法,以及如何有效的誘導源於脂肪組織的基質幹細胞分化成為具有功能性的神經元細胞。以期將來能夠運用這些細胞有效的治療神經損傷的病人。

 

    The term "stem cell" means a class of cells, which can self-renew and differentiate into different kinds of cells. The techniques of inducing stem cells to differentiate into all kinds of cells can be applied to repair damaged tissues. Adipose tissue derived stromal stem cell shares the same origin with bone marrow stromal cell, and also has the multi-potential of differentiation like bone marrow stromal cell. However, to acquire adipose tissue derived stromal stem cell is much easier, which makes it become a great replacement of bone stromal stem cell in the application of damaged tissue repairing. Our laboratory has already developed the technique of inducing dental papilla cell to differentiate into neuronal cells previously. On this base we believe that the adipose tissue derived tromal stem cell can be a better source than bone marrow stromal stem cells if the technique can be applied to adipose tissue derived stromal stem cell. Thus the main goal in this research is to apply this technique to study adipose tissue derived stromal stem cell.

 

    In this research, adipose tissues from adult male S. D. rat abdomens are digested by enzyme, and then the low-density adipocytes are eliminated by centrifugal. Isolated adipose tissue derived stromal stem cells are cultured in Dulbecco's modified eagle media and FBS. The morphology of these adipose tissue derived stromal stem cells is recorded by reversed phase contract microscope. When dexamethasone, beta-glycerophosphate, and 2-phosphate ascorbate are added to culture media, those cells differentiate into calcifying cells.

 

    Immunohistochemistry results show that cultured adipose tissue derived stromal stem cells express neurotrophin receptor Trk and P75NTR. Moreover, when nerve growth factor is added to the culture media of adipose tissue derived stromal stem cells, although no obvious morphology change is observed, the decrease of neuronal cell marker nestin, the increase of neuron maker NeuN, neurofilaments 200KD, and the increase of astrocyte maker GFAP are observed from the results of immunohistochemistry. There is no obvious change in the expression of oligodendrocyte maker A2B5. Infer from these results we think that the differentiation fate of nerve growth factor treated adipose tissue derived stromal cell has the inclination for neuron of astrocyte but not oligodendrocyte, and these cells are not terminally differentiated.

 

    In summary, the results of this research showed that adipose tissue derived stromal cell has the potential to differentiate into neural cells. The method of purification those multi-potential stem cells and the way to induce neuronal differentiation can direct the focuses of future studies.