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研究生:陳柔縈
研究生(外文):Rou-Ying Chen
論文名稱:胎盤絨毛膜蜕膜間葉幹細胞於臨床應用之研究
論文名稱(外文):Clinical Application Studies of Placenta Choriodecidual Membrane-derived Mesenchymal Stem Cells
指導教授:黃彥華黃彥華引用關係
指導教授(外文):Yen-Hua Huang
口試委員:符文美黃朝慶林泰元
口試委員(外文):Wen-Mei FuChao-Ching HuangThai-Yen Ling
口試日期:2015-07-13
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:62
中文關鍵詞:胎盤絨毛膜蜕膜間葉幹細胞臨床應用
外文關鍵詞:Placenta Choriodecidual Membrane-derived Mesenchymal Stem CellsClinical Application
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幹細胞具有自我更新以及分化的能力,幹細胞的來源可以分為:胚胎幹細胞、誘導性多功能幹細胞以及成體幹細胞。由胚胎幹細胞分離建立牽涉倫理道德及法律上的疑慮。此外,誘導性多功能幹細胞易導致細胞不正常的增生,而使得移植生物體中的細胞,較不易受控制而生成畸胎瘤。因此,在臨床上的應用就以成體幹細胞為多數。成體幹細胞可在體內許多組織取得,例如骨髓是最常見也是最早取得幹細胞的所在,另一豐富的幹細胞來源為脂肪組織,但這兩種取得幹細胞的方式都具有侵入性。因此,從醫療廢棄物為取得幹細胞的來源就顯得方便、簡單許多,例如胎盤、臍帶血和羊水等。胎盤是存在於母體內,但不會被母體的免疫系統攻擊排斥。因其同時具有免疫調控和免疫抑制的功能,而被認為在臨床的應用上具有較高的價值。本實驗室先前已成功的利用分離方法取得絨毛膜蜕膜間葉幹細胞,並申請到專利: I 419971字號以及M 462274。我們經由分析細胞的分化能力和細胞表面標誌後,證實所分離的為胎盤絨毛膜蜕膜間葉幹細胞。並想釐清胎盤絨毛膜蜕膜間葉幹細胞使用於臨床應用的可能性。首先證實了細胞的安全性:將pcMSCs打入小鼠體內經過180天的觀察後,不具致腫瘤能力。進一步測試pcMSCs是否抑制腫瘤生成能力,我們將pcMSCs 與肝癌細胞以及乳癌細胞混合一起注入重症聯合免疫缺陷小鼠皮下,觀測腫瘤的形成,發現胎盤絨毛膜蜕膜間葉幹細胞具有抑制腫瘤生成的能力。第三:我們進行pcMSCs對傷口癒合能力的測試,發現在加入胎盤絨毛膜蜕膜間葉幹細胞的傷口處有較佳的癒合情形,並對傷口組織進行分析,發現在加入胎盤絨毛膜蜕膜間葉幹細胞的傷口處,可以偵測到傷口癒合時期會高度表現的第一型以及第三型人類膠原蛋白的表現量,同時由FISH的實驗結果證實,而證實胎盤絨毛膜蜕膜間葉幹細胞具有促進表皮傷口癒合的能力。
Stem cells have self-renewal and differentiation capacity. Stem cells sources can be divided into embryonic stem cells, adult stem cells and induced pluripotent stem cells. The isolation of embryonic stem cells involves the ethical and legal issues. The induced pluripotent stem cells, which induced by Oct4、Sox2、c-Myc、Klf4, were generated by Shinya Yamanaka in 2006. However, c-Myc is substantially contributed to tumor formation. Therefore, the clinical application of adult stem cells is the majority. Adult stem cells can be obtained from the many tissues, such as bone marrow and adipose tissues. The bone marrow stem cells is the most common also the first to obtain stem cells in human. The adipose-derived mesenchymal stem cells can be isolated and don’t use the intrusive way. The recent studies have demonstrated the mesenchymal stem cells can be isolated from medical waste, like placenta, umbilical cord blood, and amniotic fluid. Immune tolerance and immunosuppressive function in pregnancy are the absence of a maternal immune response against the fetus and placenta, which thus may be viewed as unusually successful allografts. Therefore, the mesenchymal stem cells, which derived from placenta, will be a good resource for clinic cell therapy. We have been successfully setup the protocol of separation of decidual -chorionic mesenchymal stem cells (pcMSCs), and got the patents (I 419971 and M 462274). First, we confirmed the differentiation ability and cell surface marker expression of pcMSCs. Second, we confirmed the safety of pcMSCs by estabilishing that pcMSCs would not form tumor on their own, and in a ratio of 9:1 with either Hep 3B liver cancer cells, or MDA-MB-231 breast cancer cells, would suppress tumor growth. Third, functional assay for wound healing demonstrated that pcMSCs would accelerate wound closure via increased endogenous collagen formation.
目錄 I
中文摘要 1
英文摘要 2
一、前言 3
1.1 幹細胞的分類 3
1.2 間葉幹細胞 4
1.2.1 骨髓間葉幹細胞 6
1.2.2 脂肪間葉幹細胞 7
1.2.3 胎盤間葉幹細胞 8
1.3 細胞激素-6 10
1.4 白血病抑制因子 10
1.5 肝癌細胞 Hep 3B 11
1.6 乳癌細胞MDA-MB-231 11
1.7 傷口癒合 11
二、研究動機 13
三、材料與方法 14
3.1 細胞萃取與培養 14
3.2 細胞誘導分化實驗 14
3.2.1 骨分化實驗 14
3.2.2 脂分化實驗 15
3.2.3 軟骨分化實驗 16
3.3 細胞表面標記實驗 16
3.4 基因表現測定 16
3.4.1 RNA萃取 16
3.4.2 反轉錄聚合酶連鎖反應 17
3.4.3 即時偵測聚合酶連鎖反應 17
3.4.4 聚合酶連鎖反應 17
3.4.5 洋菜膠體電泳 18
3.5 細胞安全性實驗 18
3.5.1 胎盤絨毛膜蜕膜間葉幹細胞的安全性 18
3.5.2胎盤絨毛膜蜕膜間葉幹細胞促進癌細胞增生之能力檢測 19
3.6 傷口癒合實驗 19
3.7 統計與分析 19
四、實驗結果 20
4.1 胎盤絨毛膜蜕膜間葉幹細胞細胞形態 20
4.2 胎盤絨毛膜蜕膜間葉幹細胞體外誘導分化 20
4.3 胎盤絨毛膜蜕膜間葉幹細胞於無血清系統及血清中之細胞表面標記分析 20
4.4 胎盤絨毛膜蜕膜間葉幹細胞、骨髓間葉幹細胞以及脂肪間葉幹細胞細胞之表面標記分析 21
4.5 胎盤絨毛膜蜕膜間葉幹細胞具有較低之細胞激素-6表現 22
4.6胎盤絨毛膜蜕膜間葉幹細胞不同細胞代數之LIF表現量 22
4.7 胎盤絨毛膜蜕膜間葉幹細胞不具致腫瘤能力 22
4.8 胎盤絨毛膜蜕膜間葉幹細胞具有降低腫瘤生成之能力 23
4.9 胎盤絨毛膜蜕膜間葉幹細胞具有促進傷口癒合的能力 24
五、討論 26
5.1 間葉幹細胞與腫瘤之關係 26
5.2 間葉幹細胞與傷口癒合之關係 28
六、圖表 29
圖一、比較胎盤絨毛膜蜕膜間葉幹細胞、骨髓間葉幹細胞及脂肪間葉幹細胞之細胞型態 29
圖二、胎盤絨毛膜蜕膜間葉幹細胞之分化能力 30
圖三、胎盤絨毛膜蜕膜間葉幹細胞細胞表面標記分析 31
圖四、胎盤絨毛膜蜕膜間葉幹細胞 (pcMSCs)、骨髓間葉幹細胞 (BMSCs) 及脂肪間葉幹細胞 (ADMSCs) 細胞表面標記分析 32
圖五、比較骨髓間葉幹細胞 (BMSCs)、脂肪間葉幹細胞 (ADMSCs) 與胎盤絨毛膜蜕膜間葉幹細胞 (pcMSCs) 之IL-6表現量 33
圖六、胎盤絨毛膜蜕膜間葉幹細胞不同細胞代數之LIF表現量 34
圖七、肝癌細胞株 Hep3B 細胞與胎盤絨毛膜蜕膜間葉幹細胞 (pcMSCs) 於肝癌癌幹細胞表面標誌表現量之比較 35
圖八、肝癌細胞腫瘤和肝癌細胞混合胎盤絨毛膜蜕膜間葉幹細胞、肝癌細胞混合骨髓間葉幹細胞以及肝癌細胞混合脂肪間葉幹細胞所形成腫瘤大小、腫瘤生長曲線、腫瘤體積與腫瘤重量示意圖 36
圖九、 肝癌細胞 Hep 3B 腫瘤、肝癌細胞 Hep 3B 混合胎盤絨毛膜蜕膜間葉幹細胞 (pcMSCs) 腫瘤,以及肝癌細胞 Hep 3B 混合骨髓間葉幹細胞 (BMSCs) 腫瘤之 IL-6 mRNA 表現量 38
圖十、 肝癌細胞 Hep 3B 腫瘤、肝癌細胞 Hep 3B 混合胎盤絨毛膜蜕膜間葉幹細胞 (pcMSCs) 腫瘤,以及肝癌 Hep 3B 細胞混合骨髓間葉幹細胞 (BMSCs) 腫瘤之 c-Myc mRNA 表現量 39
圖十一、 肝癌細胞 Hep 3B 腫瘤、肝癌細胞 Hep 3B 混合胎盤絨毛膜蜕膜間葉幹細胞 (pcMSCs) 腫瘤,以及肝癌細胞 Hep 3B 混合骨髓間葉幹細胞 (BMSCs) 腫瘤之幹細胞特性轉錄因子 OCT4 及 NANOG mRNA 表現量 40
圖十二、肝癌細胞 Hep 3B 腫瘤、肝癌細胞 Hep 3B 混合胎盤絨毛膜蜕膜間葉幹細胞 (pcMSCs) 腫瘤,進行免疫化學染色,偵測肝癌癌幹細胞幹細胞表面標記Ep-CAM 及間葉幹細胞之細胞表面標記 CD44 之表現情形 41
圖十三、乳癌細胞 MDA MB-231 腫瘤、乳癌細胞 MDA MB-231 混合胎盤絨毛膜蜕膜間葉幹細胞 (pcMSCs)、乳癌細胞 MDA MB-231 混合骨髓間葉幹細胞(BMSCs) 及乳癌細胞 MDA MB-231 混合脂肪間葉幹細胞 (ADMSCs) 所形成腫瘤大小、腫瘤生長曲線、腫瘤體積與腫瘤重量圖 42
圖十四、乳癌細胞 MDA MB-231 腫瘤與乳癌細胞 MDA MB-231 混合胎盤絨毛膜蜕膜間葉幹細胞 (pcMSCs) 腫瘤組織切片,進行免疫化學染色偵測癌幹細胞之幹細胞特性轉錄因子 OCT4 表現情形 44
圖十五、胎盤絨毛膜蜕膜間葉幹細胞於傷口癒合之情形及人類第三型、第一型膠原蛋白PCR之表現情形 45
圖十六、傷口處組織切片經由原位雜合實驗 (in situ hybridization) 偵測人類DNA 之表現情形 46
圖十七、傷口處組織切片經由蘇木素-伊紅染色 (H&E stain) 之結果 47
表一、驗證胎盤絨毛膜蜕膜間葉幹細胞之安全性 49
表二、肝癌細胞 Hep 3B 腫瘤、肝癌細胞Hep 3B混合胎盤絨毛膜蜕膜間葉幹細胞 (pcMSCs)、肝癌細胞Hep 3B混合骨髓間葉幹細胞 (BMSCs) 以及肝癌細胞Hep 3B混合脂肪間葉幹細胞 (ADMSCs) 所形成之腫瘤比例表 50
表三、乳癌細胞 MDA MB-231 腫瘤、乳癌細胞 MDA MB-231 混合胎盤絨毛膜蜕膜間葉幹細胞 (pcMSCs)、乳癌細胞 MDA MB-231 混合骨髓間葉幹細胞 (BMSCs) 以及乳癌細胞MDA MB-231混合脂肪間葉幹細胞 (ADMSCs) 所形成之腫瘤比例表 51
表四、抗體使用表 52
表五、引子序列表 53
附錄 54
動物實驗申請表暨同意書 55
附圖一、間葉幹細胞促進或抑制腫瘤生長表格 56
附圖二、間葉幹細胞促進傷口癒合之機制 57
七、參考文獻 58
1.James Till, Erenest McCulloch. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiation Research. 1961. 14: 213-222
2.Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001. 414:105-111
3.Scadden DT. The stem-cell niche as an entity of action. Nature. 2006. 441: 1075-1079
4.Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998. 282:1145-1147
5.Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006. 4: 663-676
6.Chamberlain G, Fox J, Ashton B, Middleton J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells. 2007. 25:2739-2749
7.Dominici M, Blanc KL, Mueller I, Slaper-Cortenbach I, Marini F, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006. 8: 315-317
8.Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood. 2005. 105: 1815-1822
9.U.S. National Institutes of Health, ClinicalTrials.gov Identifier: NCT01302015
10.U.S. National Institutes of Health, ClinicalTrials.gov Identifier: NCT01873625
11.Kaplan HS, Brown MB, Paull J. Influence of bone-marrow injections on involution and neoplasia of mouse thymus after systemic irradiation. Journal of the National Cancer Institute. 1953. 14: 303-316
12.Horowitz MM, Gale RP, Sondel PM, Goldman JM, Kersey J, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood. 1990. 75: 555-562
13.Ehninger A, Trumpp A. The bone marrow stem cell niche grows up: mesenchymal stem cells and macrophages move in. The Journal of Experimental Medicine. 2011. 208: 421-428
14.Osiris therapeutics announces preliminary results for Prochymal phase III GvHD trials. 2009.
15.U.S. National Institutes of Health, ClinicalTrials.gov Identifier:NCT01210950
16.U.S. National Institutes of Health, ClinicalTrials.gov Identifier:NCT00850187
17.U.S. National Institutes of Health, ClinicalTrials.gov Identifier: NCT001499056
18.Stenderup K, Justesen J, Clausen C, Kassem M. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells. Bone. 2003. 33:919-926
19.Jo JY, Kang SK, Choi IS, Ra JC. Comparison of neural cell differentiation of human adipose mesenchymal stem cells derived from young and old ages. Journal of Reproduction and Development. 2009. 13: 227-237
20.Ra JC, Shin IS, Kim SH, Kang SK, Kang BC. Safety of intravenous infusion of human adipose tissue-derived mesenchymal stem cells in animals and humans. Stem Cells and Development. 2011. 20:1297-1308
21.U.S. National Institutes of Health, ClinicalTrials.gov Identifier: NCT0 1302015
22.Kern S, Eichler H, Stoeve J, Kluter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells. 2006. 24:1294-1301
23.M Kondo, S Kojima, K Horibe, K Kato, T Matsuyama. Risk factors for chronic graft-versus-host disease after HLA-identical sibling bone marrow transplantation. Bone Marrow Transplantation. 2001. 27:727-730
24.Jeffrey M. Gimble, Adam J. Katz, Bruce A. Bunnell. Adipose-derived stem cells for regenerative medicine. Circulation Research. 2007. 100: 1249-1260
25.Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord. Stem Cells. 2003. 21:105-110
26. In''t Anker PS, Scherjon SA, Kleijburg-van der Keur C, de Groot-Swings GM, Claas FH, et al., Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells. 2004. 22:1338-1345.
27.Makrigiannakis A, Zoumakis E, Kalantaridou S, Coutifaris C, Margioris AN, et al. Corticotropin-releasing hormone promotes blastocyst implantation and early maternal tolerance. Nature Immunology. 2001. 2:1018-1024
28.Li C, Zhang W, Jiang X, Mao N. Human-placenta-derived mesenchymal stem cells inhibit proliferation and function of allogeneic immune cells. Cell and tissue research. 2007. 330:437-446
29.Jerzak M, Bischof P. Apoptosis in the first trimester human placenta: the role in maintaining immune privilege at the maternal–foetal interface and in the trophoblast remodelling. European Journal of Obstetrics and Gynecology and Reproductive Biology. 2002. 100:138-142
30.Ryan JM , Pettit AR, Guillot PV, Chan JKY, Fisk NM. Unravelling the pluripotency paradox in fetal and placental mesenchymal stem cells: Oct-4 expression and the case of the emperor''s new clothes. Stem Cell Reviews and Reports. 2013. 9:408-421
31.Makhoul G, Chiu RCJ, Cecere R. Placental mesenchymal stem cells: a unique source for cellular cardiomyoplasty. The Annals of Thoracic Surgery. 2013. 95: 1827-1833
32.Portmann-Lanz CB, Schoeberlein A, Huber A, Sagera R, Malek A. Placental mesenchymal stem cells as potential autologous graft for pre- and perinatal neuroregeneration. American Journal of Obstetrics and Gynecology. 2006. 194:664-673
33.Tsai KS, Yang SH, Lei YP, Tsai CC, Chen HW. Mesenchymal stem cells promote formation of colorectal tumors in mice .Gastroenterology. 2011. 104:1046-1056
34.Wei HJ, Zeng R, Lu JH, Lai WF, Chen WH. Adipose-derived stem cells promote tumor initiation and accelerate tumor growth by interleukin-6 production. Oncotarget. 2015. 10:7713-7726.
35.Daheron L, Opitz SL , Zaehres H, Lensch WM, Andrews PW. LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells. Stem Cells. 2004. 22:770-778
36.Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nature Medicine. 2004. 10:55-63.
37.Chavez KJ, Garimella SV, Lipkowitz S. Triple negative breast cancer cell lines: one tool in the search for better treatment of triple negative breast cancer. Breast disease. 2010. 32:35-48
38.Garlet GP, Horwat R, Ray HL, Garlet TP, Silveira EM. Expression analysis of wound healing genes in human periapical granulomas of progressive and stable nature. Journal of Endodontics. 2012. 38:185-190
39.Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, et al. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature. 2007. 449:557-563
40.Hua Yu, Heehyoung Lee, Andreas Herrmann, Ralf Buettner, Richard Jove. Revisiting STAT3 signalling in cancer: new and unexpected biological functions. Nature Reviews Cancer. 2014.14:736–746
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