臺灣博碩士論文加值系統

因生活習慣及飲食習慣改變，罹患糖尿病之患者年年趨增，其中以第二型糖尿病人數最多，占糖尿病患者總人數 95% 以上。目前能有效治療第二型糖尿病的藥物有限。源自低侵入性手術之脂肪抽取物的脂肪間質幹細胞除了具備良好的再生及分化能力，其免疫調節相關之細胞激素表現量亦頗高。本實驗探討人類脂肪間質幹細胞分化為類胰島 β 細胞之可行性，和以 STZ-NA 誘發糖尿病小鼠模式探討人類脂肪間質幹細胞移植於第二型糖尿病之效果及追蹤。研究中利用三種不同分化策略證實人類脂肪間質幹細胞具有分化為類胰島 β 細胞之潛力；而其中又以共培養與分化培養基複合式分化策略之分化效果最佳。由動物實驗得知，藉由螢光染劑（CM-Dil）標定人類脂肪間質幹細胞，可以追蹤移植細胞於化學誘導第二型糖尿病小鼠之去向。於小鼠尾靜脈注射植入人類脂肪間質幹細胞一個月後，由組織切片觀察得知植入之細胞多發現於胰臟及肝臟組織中，且細胞移植後可改善胰臟組織浸潤及胰島小球嚴重破壞情形。綜合以上結果，人類脂肪間質幹細胞除了具有分化為類胰島 β 細胞能力外，於治療第二型糖尿病亦有極大發展潛力。

Due to major changes in lifestyle and diet an increasing number of the population are becoming diabetic. Among the four types of diabetes, the type II diabetes is the most prevalent with 95% of all diabetes around the world; however, its effective treatment is still an unmet medical need.Adipose-derived stem cells can be easily obtained from donors using fat extraction by minimally invasive surgery. They are highly proliferative, multipotent and able to regulate immune and inflammatory responses.The objective of this study is to investigate the ability of human adipose-derived stem cells (hADSCs) to differentiate into pancreatic β-like cells and to examine the fate of hADSCs transplanted in STZ-NA-induced type II diabetic mice. In vitro experiments showed hADSCs could be induced to pancreatic β-like cells by three different strategies. Among them, the method combined with co-culture and differentiation medium was shown to be the most effective for the induction. In the mice model, CM-Dil dye was used to label the hADSCs for the purpose of tracking the cells. Cell transplantation was performed by tail vein injection of labeled hADSCs. By the microscopic observations on tissues sections from mice, it was found that transplanted hADSCs appeared mainly in pancreas and liver during the first month after transplantation. The labeled cells in the targeted organs were enumerated during the first two months after transplantation. The infiltration of immune cells to pancreatic tissue was decreased and the damage of the islet cells was ameliorated in the hADSCs transplanted mice compared to the sham treatment. Based on these results, it could be concluded that the hADSCs can differentiate into pancreatic β-like cells and have great potential in the application of type II diabetes treatment.

摘要
Abstract
表目錄
圖目錄
壹、研究動機與目的
貳、研究背景及重要性簡介
2.1 糖尿病
2.1.1 糖尿病之簡介
2.1.2 糖尿病的診斷
2.2 幹細胞
參、 材料與方法
3.1 人類脂肪間質幹細胞培養與繼代
3.1.1 間質幹細胞鑑定分析
3.2 人類脂肪間質幹細胞分化為類胰島 β 細胞
3.2.1 分化培養基誘導分化策略
3.2.2 人類脂肪間質幹細胞與小鼠胰臟組織共培養
3.2.3 複合式分化策略
3.2.4 萃取 total mRNA及製備 cDNA
3.2.5 聚合酶連鎖反應
3.2.6 即時定量聚合酶連鎖反應
3.2.7 Dithizone 染色
3.3 以 STZ-NA 誘發糖尿病小鼠模式探討人類脂肪間質幹細胞治療效果與追蹤
3.3.1非肥胖型第二型糖尿病小鼠模型
3.3.2 標定人類脂肪間質幹細胞與細胞移植
3.3.3 組織處理及包埋
3.3.4 常規組織染色
3.3.5 組織切片螢光拍攝前處理與免疫組織化學染色
3.4 統計分析
肆、實驗結果
4.1 幹細胞特性分析
4.1.1 幹細胞表面抗原分析
4.1.2 人類脂肪間質幹細胞分化能力
4.2 人類脂肪間質幹細胞分化為類胰島 β 細胞
4.2.1 誘導分化之細胞型態
4.2.2 誘導分化之細胞基因表現
4.2.3 誘導分化之細胞經 DTZ 染色分析
4.3 動物生理分析
4.3.1 CM-Dil 螢光染劑標定之人類脂肪間質幹細胞分化能力
4.3.2 STZ-NA 化學誘導第二型糖尿病小鼠建立及細胞移植效果
4.3.3 追蹤人類脂肪間質幹細胞
伍、討論
陸、結論與未來展望
柒、參考文獻

Ackermann, A.M., Gannon, M., 2007. Molecular regulation of pancreatic beta-cell mass development, maintenance, and expansion. Journal of molecular endocrinology 38, 193-206.
Blaber, S.P., Webster, R.A., Hill, C.J., Breen, E.J., Kuah, D., Vesey, G., Herbert, B.R., 2012. Analysis of in vitro secretion profiles from adipose-derived cell populations. Journal of translational medicine 10, 172.
Ebrahimie, M., Esmaeili, F., Cheraghi, S., Houshmand, F., Shabani, L., Ebrahimie, E., 2014. Efficient and simple production of insulin-producing cells from embryonal carcinoma stem cells using mouse neonate pancreas extract, as a natural inducer. PloS one 9, e90885.
Gabr, M.M., Zakaria, M.M., Refaie, A.F., Ismail, A.M., Abou-El-Mahasen, M.A., Ashamallah, S.A., Khater, S.M., El-Halawani, S.M., Ibrahim, R.Y., Uin, G.S., Kloc, M., Calne, R.Y., Ghoneim, M.A., 2013. Insulin-producing cells from adult human bone marrow mesenchymal stem cells control streptozotocin-induced diabetes in nude mice. Cell transplantation 22, 133-145.
Gatti, R.A., Meuwissen, H.J., Allen, H.D., Hong, R., Good, R.A., 1968. Immunological reconstitution of sex-linked lymphopenic immunological deficiency. Lancet 2, 1366-1369.
Gonzalez, P., Santos, T.M., Calil, A., Corradi Perini, C., Percegona, L.S., Silva, I.C., Kuligovski, C., Aguiar, A.M., Camara, N.O., Aita, C.A., 2010. Expression of pancreatic endocrine markers by prolactin-treated rat bone marrow mesenchymal stem cells. Transplantation proceedings 42, 566-569.
Hao, H., Liu, J., Shen, J., Zhao, Y., Liu, H., Hou, Q., Tong, C., Ti, D., Dong, L., Cheng, Y., Mu, Y., Liu, J., Fu, X., Han, W., 2013. Multiple intravenous infusions of bone marrow mesenchymal stem cells reverse hyperglycemia in experimental type 2 diabetes rats. Biochemical and biophysical research communications 436, 418-423.
Harn, H.J., Lin, S.Z., Hung, S.H., Subeq, Y.M., Li, Y.S., Syu, W.S., Ding, D.C., Lee, R.P., Hsieh, D.K., Lin, P.C., Chiou, T.W., 2012. Adipose-derived stem cells can abrogate chemical-induced liver fibrosis and facilitate recovery of liver function. Cell transplantation 21, 2753-2764.
Ho, J.H., Tseng, T.C., Ma, W.H., Ong, W.K., Chen, Y.F., Chen, M.H., Lin, M.W., Hong, C.Y., Lee, O.K., 2012. Multiple intravenous transplantations of mesenchymal stem cells effectively restore long-term blood glucose homeostasis by hepatic engraftment and beta-cell differentiation in streptozocin-induced diabetic mice. Cell transplantation 21, 997-1009.
Hung, H.Y., Qian, K., Morris-Natschke, S.L., Hsu, C.S., Lee, K.H., 2012. Recent discovery of plant-derived anti-diabetic natural products. Natural product reports 29, 580-606.
Karaoz, E., Genc, Z.S., Demircan, P.C., Aksoy, A., Duruksu, G., 2010. Protection of rat pancreatic islet function and viability by coculture with rat bone marrow-derived mesenchymal stem cells. Cell death & disease 1, e36.
Lee, J., Han, D.J., Kim, S.C., 2008. In vitro differentiation of human adipose tissue-derived stem cells into cells with pancreatic phenotype by regenerating pancreas extract. Biochemical and biophysical research communications 375, 547-551.
Li, Y.V., 2014. Zinc and insulin in pancreatic beta-cells. Endocrine 45, 178-189.
Lin, G., Wang, G., Liu, G., Yang, L.J., Chang, L.J., Lue, T.F., Lin, C.S., 2009. Treatment of type 1 diabetes with adipose tissue-derived stem cells expressing pancreatic duodenal homeobox 1. Stem cells and development 18, 1399-1406.
Matsuyama-Yokono, A., Tahara, A., Nakano, R., Someya, Y., Shiraki, K., Hayakawa, M., Shibasaki, M., 2009. Antidiabetic effects of dipeptidyl peptidase-IV inhibitors and sulfonylureas in streptozotocin-nicotinamide-induced mildly diabetic mice. Metabolism: clinical and experimental 58, 379-386.
Mohamad Buang, M.L., Seng, H.K., Chung, L.H., Saim, A.B., Idrus, R.B., 2012. In vitro generation of functional insulin-producing cells from lipoaspirated human adipose tissue-derived stem cells. Archives of medical research 43, 83-88.
Moriscot, C., de Fraipont, F., Richard, M.J., Marchand, M., Savatier, P., Bosco, D., Favrot, M., Benhamou, P.Y., 2005. Human bone marrow mesenchymal stem cells can express insulin and key transcription factors of the endocrine pancreas developmental pathway upon genetic and/or microenvironmental manipulation in vitro. Stem cells 23, 594-603.
Murtaugh, L.C., 2007. Pancreas and beta-cell development: from the actual to the possible. Development 134, 427-438.
Najar, M., Raicevic, G., Boufker, H.I., Fayyad-Kazan, H., De Bruyn, C., Meuleman, N., Bron, D., Toungouz, M., Lagneaux, L., 2010. Adipose-tissue-derived and Wharton's jelly-derived mesenchymal stromal cells suppress lymphocyte responses by secreting leukemia inhibitory factor. Tissue engineering. Part A 16, 3537-3546.
Nam, J.S., Kang, H.M., Kim, J., Park, S., Kim, H., Ahn, C.W., Park, J.O., Kim, K.R., 2014. Transplantation of insulin-secreting cells differentiated from human adipose tissue-derived stem cells into type 2 diabetes mice. Biochemical and biophysical research communications 443, 775-781.
Okura, H., Komoda, H., Fumimoto, Y., Lee, C.M., Nishida, T., Sawa, Y., Matsuyama, A., 2009. Transdifferentiation of human adipose tissue-derived stromal cells into insulin-producing clusters. Journal of artificial organs : the official journal of the Japanese Society for Artificial Organs 12, 123-130.
Ouyang, J., Huang, W., Yu, W., Xiong, W., Mula, R.V., Zou, H., Yu, Y., 2014. Generation of insulin-producing cells from rat mesenchymal stem cells using an aminopyrrole derivative XW4.4. Chemico-biological interactions 208, 1-7.
Storb, R., Thomas, E.D., Buckner, C.D., Clift, R.A., Fefer, A., Fernando, L.P., Giblett, E.R., Johnson, F.L., Neiman, P.E., 1976. Allogeneic marrow grafting for treatment of aplastic anemia: a follow-up on long-term survivors. Blood 48, 485-490.
Susman, S., Rus-Ciuca, D., Soritau, O., Tomuleasa, C., Buiga, R., Mihu, D., Pop, V.I., Mihu, C.M., 2011. Pancreatic exocrine adult cells and placental stem cells co-culture. Working together is always the best way to go. Romanian journal of morphology and embryology = Revue roumaine de morphologie et embryologie 52, 999-1004.
Thatava, T., Ma, B., Rohde, M., Mayer, H., 2006. Chromatin-remodeling factors allow differentiation of bone marrow cells into insulin-producing cells. Stem cells 24, 2858-2867.
Timper, K., Seboek, D., Eberhardt, M., Linscheid, P., Christ-Crain, M., Keller, U., Muller, B., Zulewski, H., 2006. Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells. Biochemical and biophysical research communications 341, 1135-1140.
Wilson, M.E., Scheel, D., German, M.S., 2003. Gene expression cascades in pancreatic development. Mechanisms of development 120, 65-80.
Yeung, T.Y., Seeberger, K.L., Kin, T., Adesida, A., Jomha, N., Shapiro, A.M., Korbutt, G.S., 2012. Human mesenchymal stem cells protect human islets from pro-inflammatory cytokines. PloS one 7, e38189.

網路資訊
衛生福利部，Ministry of health and welfare. http://www.mohw.gov.tw/
International Diabetes Federation, IDF. http://www.idf.org/