臺灣博碩士論文加值系統

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。由於本實驗室己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 dexamethasone、beta-glycerophosphate、2-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：Trk 與 P75NTR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 NeuN 與 neurofilaments 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。

暫缺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 stromal 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.

目 錄
中文摘要……………………………………………………………1
英文摘要……………………………………………………………3
緒論…………………………………………………………………5
材料與方法…………………………………………………………14
結果…………………………………………………………………23
討論…………………………………………………………………30
圖表…………………………………………………………………37
參考文獻……………………………………………………………59
圖 表 目 錄
圖一：源於脂肪組織的基質幹細胞初代培養兩天後，細胞之型態與生長情形。
圖二：源於脂肪組織的基質幹細胞初代培養一週後，細胞之型態與生長情形。
圖三：源於脂肪組織的基質幹細胞第一次繼代培養兩天後，細胞之型態與生長情形。
圖四：源於脂肪組織的基質幹細胞第二次繼代培養兩天後，細胞之型態與生長情形。
圖五：源於脂肪組織的基質幹細胞以 osteogenic medium 培養兩週後，細胞之型態與生長情形。
圖六：源於脂肪組織的基質幹細胞以 osteogenic medium 培養兩週後，以 Alizarin red S 染色之結果。
圖七：源於脂肪組織的基質幹細胞以 adipogenic medium 培養兩週後，細胞之型態與生長情形。
圖八：以免疫化學細胞染色法偵測人類牙齦纖維母細胞上 P75NTR 的表現情況。
圖九：以免疫化學細胞染色法偵測人類牙齦纖維母細胞上 Trks 的表現情況。
圖十：以免疫化學細胞染色法偵測大鼠腦細胞上 P75NTR 的表現。
圖十一：以免疫化學細胞染色法偵測大鼠腦細胞上 Trks 的表現。
圖十二：以免疫化學細胞染色法偵測源於脂肪組織的基質幹細胞上 P75NTR 的表現。
圖十三：以免疫化學細胞染色法偵測源於脂肪組織的基質幹細胞上 Trks 的表現。
圖十四：源於脂肪組織的基質幹細胞不以任何抗體處理以作為免疫化學染色法的對照組。
圖十五：骨髓基質幹細胞以含有 NGF 200ng/mL 的培養液培養兩週後的型態與生長情形。
圖十六：源於脂肪組織的基質幹細胞以含有 NGF 200ng/mL 的培養液培養兩週後的型態與生長情形。
圖十七：以免疫化學細胞染色法偵測骨髓基質幹細胞以不含有 NGF 的培養液培養兩週後 nestin 的表現。
圖十八：以免疫化學細胞染色法偵測骨髓基質幹細胞以含有 NGF 50ng/mL 的培養液培養兩週後 nestin 的表現。
圖十九：以免疫化學細胞染色法偵測骨髓基質幹細胞以含有 NGF 200ng/mL 的培養液培養兩週後 nestin 的表現。
圖二十：以免疫化學細胞染色法偵測源於脂肪組織的基質幹細胞以不含有 NGF 的培養液培養兩週後 nestin 的表現。
圖二十一：以免疫化學細胞染色法偵測源於脂肪組織的基質幹細胞以含有 NGF 50ng/mL 的培養液培養兩週後 nestin 的表現。
圖二十二：以免疫化學細胞染色法偵測源於脂肪組織的基質幹細胞以含有 NGF 200ng/mL 的培養液培養兩週後 nestin 的表現。
圖二十三：以免疫化學細胞染色法偵測大鼠腦細胞上 GFAP 的表現。
圖二十四：以免疫化學細胞染色法偵測源於脂肪組織的基質幹細胞以不含有 NGF 的培養液培養兩週後 GFAP 的表現。
圖二十五：以免疫化學細胞染色法偵測源於脂肪組織的基質幹細胞以含有 NGF 200ng/mL 的培養液培養兩週後 GFAP 的表現。
圖二十六：以免疫化學細胞染色法偵測大鼠腦細胞上 Neurofilament 200KD 的表現。
圖二十七：以免疫化學細胞染色法偵測源於脂肪組織的基質幹細胞以不含有 NGF 的培養液培養兩週後 Neurofilament 200KD 的表現。
圖二十八：免疫化學細胞染色法偵測源於脂肪組織的基質幹細胞以含有 NGF 200ng/mL 的培養液培養兩週後 Neurofilament 200KD 的表現。
圖二十九：以免疫化學細胞染色法偵測大鼠腦細胞上 NeuN 的表現。
圖三十：以免疫化學細胞染色法偵測源於脂肪組織的基質幹細胞以含有 NGF 200ng/mL 的培養液培養兩週後 NeuN 的表現。
圖三十一：以免疫化學細胞染色法偵測大鼠腦細胞上 A2B5 的表現。
圖三十二：以免疫化學細胞染色法偵測源於脂肪組織的基質幹細胞以含有 NGF 200ng/mL 的培養液培養兩週後 A2B5 的表現。
表一：骨髓基質幹細胞與源於脂肪組織的基質幹細胞經過 NGF 處理之後，表現 nestin 的細胞數目佔全部細胞數目的比例的變化。
表二：源於脂肪組織的基質幹細胞經過 NGF 處理之後，表現 GFAP 的細胞數目佔全部細胞數目的比例的變化與表現 neurofilament 200KD 的細胞數目佔全部細胞數目比例的變化。

Altman,J. and Das,G.D. (1965) Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J.Comp Neurol., 124:319-335.
Andersson,S.G., Zomorodipour,A., Andersson,J.O., Sicheritz-Ponten,T., Alsmark,U.C., Podowski,R.M., Naslund,A.K., Eriksson,A.S., Winkler,H.H., and Kurland,C.G. (1998) The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature, 396:133-140.
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.
Benayahu,D., Kompier,R., Shamay,A., Kadouri,A., Zipori,D., and Wientroub,S. (1994) Mineralization of marrow-stromal osteoblasts MBA-15 on three-dimensional carriers. Calcif.Tissue Int., 55:120-127.
Bianco,P., Riminucci,M., Majolagbe,A., Kuznetsov,S.A., Collins,M.T., Mankani,M.H., Corsi,A., Bone,H.G., Wientroub,S., Spiegel,A.M., Fisher,L.W., and Robey,P.G. (2000) Mutations of the GNAS1 gene, stromal cell dysfunction, and osteomalacic changes in non-McCune-Albright fibrous dysplasia of bone. J.Bone Miner.Res., 15:120-128.
Bibel,M. and Barde,Y.A. (2000) Neurotrophins: key regulators of cell fate and cell shape in the vertebrate nervous system. Genes Dev., 14:2919-2937.
Brown,M., Keynes,R., and Lumsden,A. (2001) Neurotrophic Factors and their Receptors. In: The Developing Brain. Oxford University Press, Oxford, pp. 299-316.
Castro,R.F., Jackson,K.A., Goodell,M.A., Robertson,C.S., Liu,H., and Shine,H.D. (2002) Failure of bone marrow cells to transdifferentiate into neural cells in vivo. Science, 297:1299.
Eisenbarth,G.S., Walsh,F.S., and Nirenberg,M. (1979) Monoclonal antibody to a plasma membrane antigen of neurons. Proc.Natl.Acad.Sci.U.S.A, 76:4913-4917.
Gimble,J.M. (1990) The function of adipocytes in the bone marrow stroma. New Biol., 2:304-312.
Gimble,J.M., Robinson,C.E., Wu,X., and Kelly,K.A. (1996) The function of adipocytes in the bone marrow stroma: an update. Bone, 19:421-428.
Gronthos,S., Franklin,D.M., Leddy,H.A., Robey,P.G., Storms,R.W., and Gimble,J.M. (2001) Surface protein characterization of human adipose tissue-derived stromal cells. J.Cell Physiol, 189:54-63.
Halvorsen,Y.D., Bond,A., Sen,A., Franklin,D.M., Lea-Currie,Y.R., Sujkowski,D., Ellis,P.N., Wilkison,W.O., and Gimble,J.M. (2001) Thiazolidinediones and glucocorticoids synergistically induce differentiation of human adipose tissue stromal cells: biochemical, cellular, and molecular analysis. Metabolism, 50:407-413.
Hockfield,S. and McKay,R.D. (1985) Identification of major cell classes in the developing mammalian nervous system. J.Neurosci., 5:3310-3328.
Hung,S.C., Cheng,H., Pan,C.Y., Tsai,M.J., Kao,L.S., and Ma,H.L. (2002) In vitro differentiation of size-sieved stem cells into electrically active neural cells. Stem Cells, 20:522-529.
Johansson,C.B., Momma,S., Clarke,D.L., Risling,M., Lendahl,U., and Frisen,J. (1999) Identification of a neural stem cell in the adult mammalian central nervous system. Cell, 96:25-34.
Kaplan,F.S., Hahn,G.V., and Zasloff,M.A. (1994) Heterotopic Ossification: Two Rare Forms and What They Can Teach Us. J.Am.Acad.Orthop.Surg., 2:288-296.
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.
Liedtke,W., Edelmann,W., Bieri,P.L., Chiu,F.C., Cowan,N.J., Kucherlapati,R., and Raine,C.S. (1996) GFAP is necessary for the integrity of CNS white matter architecture and long-term maintenance of myelination. Neuron, 17:607-615.
Malaval,L., Modrowski,D., Gupta,A.K., and Aubin,J.E. (1994) Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures. J.Cell Physiol, 158:555-572.
Maniatopoulos,C., Sodek,J., and Melcher,A.H. (1988) Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res., 254:317-330.
Mezey,E., Chandross,K.J., Harta,G., Maki,R.A., and McKercher,S.R. (2000) Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science, 290:1779-1782.
Mullen,R.J., Buck,C.R., and Smith,A.M. (1992) NeuN, a neuronal specific nuclear protein in vertebrates. Development, 116:201-211.
Nuttall,M.E. and Gimble,J.M. (2000) Is there a therapeutic opportunity to either prevent or treat osteopenic disorders by inhibiting marrow adipogenesis? Bone, 27:177-184.
Otten,U., Ehrhard,P., and Peck,R. (1989) Nerve growth factor induces growth and differentiation of human B lymphocytes. Proc.Natl.Acad.Sci.U.S.A, 86:10059-10063.
Owen,M. (1988) Marrow stromal stem cells. J.Cell Sci.Suppl, 10:63-76.
Pietrangeli,C.E., Hayashi,S., and Kincade,P.W. (1988) Stromal cell lines which support lymphocyte growth: characterization, sensitivity to radiation and responsiveness to growth factors. Eur.J.Immunol., 18:863-872.
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.
Sanchez-Ramos,J., Song,S., Cardozo-Pelaez,F., Hazzi,C., Stedeford,T., Willing,A., Freeman,T.B., Saporta,S., Janssen,W., Patel,N., Cooper,D.R., and Sanberg,P.R. (2000) Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp.Neurol., 164:247-256.
Schnitzer,J. and Schachner,M. (1982) Cell type specificity of a neural cell surface antigen recognized by the monoclonal antibody A2B5. Cell Tissue Res., 224:625-636.
Scolding,N.J., Rayner,P.J., and Compston,D.A. (1999) Identification of A2B5-positive putative oligodendrocyte progenitor cells and A2B5-positive astrocytes in adult human white matter. Neuroscience, 89:1-4.
Terada,N., Hamazaki,T., Oka,M., Hoki,M., Mastalerz,D.M., Nakano,Y., Meyer,E.M., Morel,L., Petersen,B.E., and Scott,E.W. (2002) Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature, 416:542-545.
Wagers,A.J., Sherwood,R.I., Christensen,J.L., and Weissman,I.L. (2002) Little evidence for developmental plasticity of adult hematopoietic stem cells. Science, 297:2256-2259.
Wheeler,E.F. and Bothwell,M. (1992) Spatiotemporal patterns of expression of NGF and the low-affinity NGF receptor in rat embryos suggest functional roles in tissue morphogenesis and myogenesis. J.Neurosci., 12:930-945.
Wobus,A.M., Grosse,R., and Schoneich,J. (1988) Specific effects of nerve growth factor on the differentiation pattern of mouse embryonic stem cells in vitro. Biomed.Biochim.Acta, 47:965-973.
Wurmser,A.E. and Gage,F.H. (2002) Stem cells: cell fusion causes confusion. Nature, 416:485-487.
Ying,Q.L., Nichols,J., Evans,E.P., and Smith,A.G. (2002) Changing potency by spontaneous fusion. Nature, 416:545-548.
Zhu,Q., Couillard-Despres,S., and Julien,J.P. (1997) Delayed maturation of regenerating myelinated axons in mice lacking neurofilaments. Exp.Neurol., 148:299-316.
藍文謙, (1997) 生長因子對骨髓基質細胞的影響. 碩士論文
郭玟君, (2000) 神經生長因子誘導以酵素分離之牙乳突分化成類神經細胞. 碩士論文
陳庭鵬, (2002) 神經生長因子對肢芽組織基質細胞之影響. 碩士論文