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研究生:郭卉玲
研究生(外文):Huei-Ling Kuo
論文名稱:探討間葉幹細胞分化過程中脂小體增生活化受體在免疫調控所扮演的角色
論文名稱(外文):Study on the role of Peroxisome Proliferator-Activated Receptors (PPARs) in the immune regulatory activities during mesenchymal stem cell differentiation
指導教授:江伯倫江伯倫引用關係
口試委員:朱清良周秀慧
口試日期:2016-07-08
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:免疫學研究所
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:2016
畢業學年度:104
語文別:英文
論文頁數:73
中文關鍵詞:間葉幹細胞脂肪分化發炎反應脂小體增生活化受體γ介白素1受體拮抗劑
外文關鍵詞:mesenchymal stem cellsadipogenesisinflammationPPAR-γIL-1Ra
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間葉幹細胞(Mesenchymal stem cells, MSCs)是一種具有自我更新能力及高度分化潛能的成體幹細胞,間葉幹細胞能夠進一步分化成脂肪細胞、硬骨細胞和軟骨細胞等,更有研究指出間葉幹細胞具備免疫抑制之調控能力。但是許多肥胖相關的代謝疾病,例如:第二型糖尿病、心血管疾病則是由脂肪細胞所造成的發炎反應,同時有巨噬細胞受到脂肪組織所分泌的賀爾蒙或是趨化因子所吸引而聚集,並在發炎反應中扮演重要的角色。我們假設在間葉幹細胞的脂肪分化過程中有些調控因子產生了變化,其中像是脂小體增生活化受體γ (peroxisome proliferator-activated receptor-γ, PPAR-γ)在發炎及抗發炎的角色也尚未釐清,因此我們的研究希望探討間葉幹細胞在脂肪分化過程中基因及細胞激素的改變。
在間葉幹細胞脂肪分化過程中,會有許多發炎激素的分泌和相關基因表現,包括了介白素-6 (IL-6)、腫瘤壞死因子-α (TNF-α)和單核球趨化蛋白-1 (mcp1),但間葉幹細胞表面抗原的表現與免疫調控能力皆會隨著分化而降低,抗發炎的介白素1受體拮抗劑 (IL-1Ra)亦會與分化呈現負相關趨勢,此外,脂小體增生活化受體γ更能透過抑制介白素1受體拮抗劑來促進脂肪分化的情形。
目前我們發現在間葉幹細胞脂肪分化過程中,加入脂小體增生活化受體γ促進劑能夠增加脂聯素 (adiponectin)的表現,並且使細胞大量表現發炎相關基因;反之,加入脂小體增生活化受體γ抑制劑則造成相反的效果,不過無論促進劑或是抑制劑在細胞的型態以及分化速度上都會造成抑制效果。除此之外,這兩個組別的細胞皆無法有效的抑制T淋巴球增生,代表細胞免疫調控能力皆受到抑制。未來我們將繼續探討是否有其他更重要的免疫調控因子影響間葉幹細胞脂肪分化及免疫抑制能力。


Mesenchymal stem cells (MSCs) are self-renewable multipotent progenitor cells that can differentiate into a variety of cell types including adipocytes, osteocytes and chondrocytes. Recent studies have demonstrated that MSCs could exert an immunosuppressive activity. However, many obesity-related metabolic diseases such as type II diabetes are attributed to adipocyte-induced inflammation. Macrophages recruited by adipose tissue-derived hormones or chemokines, play the key roles in the chronic inflammation. We hypothesized that some mediators might be changed during the MSCs adipogenesis. Especially, the role of PPAR-γ in inflammation and anti-inflammation are still unclear. Hence, this study was performed to examine the gene and cytokine profiles of MSCs in different differential process.
Alone with MSCs adipogenesis, more inflammatory cytokines, including IL-6, TNF-α, and mcp1 were secreted and expressed. But the expression of MSCs surface markers and immune-modulatory function were decreased. Also, the expression of IL-1Ra was negatively correlated with adipogeneic process of MSCs. Furthermore, peroxisome proliferator-activated receptors (PPARs) can promote adipogenesis by inhibiting IL-1Ra expression.
So far, we discovered that adding PPAR-γ agonist could enhance adiponectin and inflammatory genes expression. On the other hand, adding PPAR-γ antagonist resulted in the opposite effects. Further, the cells of PPAR-γ agonist and antagonist group inhibited phenotypic change or adipogenic progressing. Also, agonist and antagonist decreased MSCs immunosuppressive function during adipogenic process. We would like to find out if there is any factor that can directly influence MSCs adipogenesis and their immune-suppressive function.


口試委員審定書………………………………………………………………………..i
致謝……………………………………………………………………………………..ii
中文摘要………………………………………………………………………………..iv
Abstract…………………………………………………………………………………vi
Contents………………………………………………………………………………...vii
Chapter 1 Introduction………………………………………………………………….1
1. Mesenchymal stem cells(MSCs)………………………………………………....2
2. Adipocytes……………………………………………………………………….5
3. IL-1Ra……………………………………………………………………………7
4. PPARs……………………………………………………………………………8
5. Hypothesis and Specific aims…………………………………………………..10
Chapter 2 Materials and Methods……………………………………………………..12
Part1. Materials……………………………………………………………………..13
Cell culture………………………………………………………………………...13
1. Mice…………………………………………………………………………..13
2. MSC culture…………………………………………………………………..13
Characteristics of MSCs…………………………………………………………...14
1. Flow cytometry……………………………………………………………...14
2. T lymphocyte proliferation assay…………………………………………....14
3. MSCs differentiation………………………………………………………...15
ELISA (Enzyme-linked immunosorbent assay) …………………………………...16
mRNA expression……………………………………………………...…………...16
1. RNA extraction………………………………………………...………...…...16
2. Reverse transcription-polymerase chain reaction (RT-PCR) ...………....…...16
3. Quantitative real-time PCR (qPCR) …………………………...…...…...…...17
Reagents……………………………………………….……………...…...…...…...17
Part2. Methods………………………………………….……………...…...…...…...18
Cell culture………………………………………….………...……...…...…...…...18
Characteristics of MSCs…………………………….………...……...…...…...…...19
1. Flow cytometry…………………………….………...……...….......…...…...19
2. T lymphocyte proliferation assay………….………...……...…........…...…...20
3. MSC differentiation………….………...……...…........…........................…...20
ELISA………….………...……...…........…......................................................…...21
mRNA Expression……...……...…........…........................................................…...21
1. RNA extraction…...……...…........…........................................................…...21
2. RT-PCR…...……...…........…...................................................................…...22
3. qPCR…...……...…........….......................................................................…...23
Statistical analysis…...…........….......................................................................…...23
Chapter 3 Results…...…........…...............................................................................…...25
1. Experimental design………………………………………………………………26
2. MSCs could express specific surface markers, differentiated into multiple kinds of cells, and suppress T lymphocyte proliferation…………………………………...26
3. MSCs adipogenesis……………………………………………………………….27
3.1 MSCs progressively differentiated into adipocytes…………………………...27
3.2 MSCs changed their characteristics during adipogenesis……………………..27
3.3 Adipocytes expressed higher level of inflammatory genes comparing with anti-inflammatory genes…………………………………………………………...28
3.4 Anti-inflammatory IL-1Ra secretion decreased during adipogenesis………...28
3.5 Adipocytes had different expression degree of three PPARs isotypes during adipogenesis…………………………………………………………………..28
4. Treatment of PPAR-γ agonist and antagonist…………………………………….29
4.1 The appropriated dose of PPAR-γ agonist and antagonist for MSCs adipogenesis were both 50 μg/ml……………………………..………………29
4.2 The inflammatory and anti-inflammatory genes profile didn’t show significantly change between PPAR-γ agonist and antagonist group………...30
4.3 Adipocytes showed higher level of the adipocyte-related genes, pparγ2, and cebpα in agonist group than antagonist group………………………………...30
4.4 Anti-inflammatory IL-1Ra decreases during adipogenesis and il1ra expression was low after adipogenesis…………………………………………………....31
4.5 Both PPAR-γ agonist and antagonist group suppressed MSCs adipogenesis while control group gradually differentiated into adipocytes………………....32
4.6 PPAR-γ agonist, antagonist, and control group showed no significance difference in suppressing CD4+ T lymphocytes proliferation at MSCs adipogenesis day 5.……………………………………………………...…….32
Chapter 4 Discussion and Conclusion.....................................................................…....34
References…...…........…...............................................................................……….....41
Figures…........…................................................................................……………….....51
Tables………………………………………………………………………………......72


1.Birbrair, A., et al., Nestin-GFP transgene reveals neural precursor cells in adult skeletal muscle. PLoS ONE, 2011. 6(2): p. e16816.
2.da Silva Meirelles, L., P.C. Chagastelles, and N.B. Nardi, Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci, 2006. 119(Pt 11): p. 2204-13.
3.Dominici, M., et al., Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006. 8(4): p. 315-7.
4.Klingemann, H., D. Matzilevich, and J. Marchand, Mesenchymal stem cells – sources and clinical applications. Transfusion Medicine and Hemotherapy, 2008. 35(4): p. 272-277.
5.Bartholomew, A., et al., Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol, 2002. 30(1): p. 42-8.
6.Djouad, F., et al., Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood, 2003. 102(10): p. 3837-44.
7.Di Nicola, M., et al., Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood, 2002. 99(10): p. 3838-43.
8.Tse, W.T., et al., Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation, 2003. 75(3): p. 389-97.
9.Maccario, R., et al., Interaction of human mesenchymal stem cells with cells involved in alloantigen-specific immune response favors the differentiation of CD4+ T-cell subsets expressing a regulatory/suppressive phenotype. Haematologica, 2005. 90(4): p. 516-25.
10.Corcione, A., et al., Human mesenchymal stem cells modulate B-cell functions. Blood, 2006. 107(1): p. 367-72.
11.Deng, T., et al., Class II Major Histocompatibility complex plays an essential role in obesity-induced adipose inflammation. Cell Metabolism. 17(3): p. 411-422.
12.Glennie, S., et al., Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells. Blood, 2005. 105(7): p. 2821-7.
13.Pradier, A., et al., Human bone marrow stromal cells and skin fibroblasts inhibit natural killer cell proliferation and cytotoxic activity. Cell Transplant, 2011. 20(5): p. 681-91.
14.Rasmusson, I., et al., Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells. Transplantation, 2003. 76(8): p. 1208-13.
15.Sotiropoulou, P.A., et al., Interactions between human mesenchymal stem cells and natural killer cells. Stem Cells, 2006. 24(1): p. 74-85.
16.Nauta, A.J. and W.E. Fibbe, Immunomodulatory properties of mesenchymal stromal cells. Blood, 2007. 110(10): p. 3499-506.
17.Ryan, J.M., et al., Mesenchymal stem cells avoid allogeneic rejection. J Inflamm (Lond), 2005. 2: p. 8.
18.Liu, W.H., et al., Novel mechanism of inhibition of dendritic cells maturation by mesenchymal stem cells via interleukin-10 and the JAK1/STAT3 signaling pathway. PLoS One, 2013. 8(1): p. e55487.
19.Soleymaninejadian, E., K. Pramanik, and E. Samadian, Immunomodulatory properties of mesenchymal stem cells: cytokines and factors. Am J Reprod Immunol, 2012. 67(1): p. 1-8.
20.Prasanna, S.J., et al., Pro-inflammatory cytokines, IFNgamma and TNFalpha, influence immune properties of human bone marrow and Wharton jelly mesenchymal stem cells differentially. PLoS One, 2010. 5(2): p. e9016.
21.Ren, G., et al., Species variation in the mechanisms of mesenchymal stem cell-mediated immunosuppression. Stem Cells, 2009. 27(8): p. 1954-62.
22.Polchert, D., et al., IFN-gamma activation of mesenchymal stem cells for treatment and prevention of graft versus host disease. Eur J Immunol, 2008. 38(6): p. 1745-55.
23.Nagura, I., et al., Characterization of progenitor cells derived from torn human rotator cuff tendons by gene expression patterns of chondrogenesis, osteogenesis, and adipogenesis. J of Orthop Surg Res, 2016. 11(1): p. 1-8.
24.Nishimoto, S., et al., Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance. Sci Adv, 2016. 2(3): p. e1501332.
25.Greenberg, A.S. and M.S. Obin, Obesity and the role of adipose tissue in inflammation and metabolism. Am J Clin Nutr, 2006. 83(2): p. 461s-465s.
26.Garg, A., Adipose tissue dysfunction in obesity and lipodystrophy. Clin Cornerstone, 2006. 8 Suppl 4: p. S7-s13.
27.Zhang, Y., et al., Positional cloning of the mouse obese gene and its human homologue. Nature, 1994. 372(6505): p. 425-32.
28.Trujillo, M.E. and P.E. Scherer, Adipose tissue-derived factors: impact on health and disease. Endocr Rev, 2006. 27(7): p. 762-78.
29.Murano, I., et al., Dead adipocytes, detected as crown-like structures, are prevalent in visceral fat depots of genetically obese mice. J Lipid Res, 2008. 49(7): p. 1562-8.
30.Weisberg, S.P., et al., Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest, 2003. 112(12): p. 1796-808.
31.Lumeng, C.N., J.L. Bodzin, and A.R. Saltiel, Obesity induces a phenotypic switch in adipose tissue macrophage polarization. J Clin Invest, 2007. 117(1): p. 175-84.
32.Stefanovic-Racic, M., et al., Dendritic cells promote macrophage infiltration and comprise a substantial proportion of obesity-associated increases in CD11c+ cells in adipose tissue and liver. Diabetes, 2012. 61(9): p. 2330-9.
33.Winer, S., et al., Normalization of obesity-associated insulin resistance through immunotherapy. Nat Med, 2009. 15(8): p. 921-929.
34.Yang, H., et al., Obesity increases the production of proinflammatory mediators from adipose tissue T cells and compromises TCR repertoire diversity: implications for systemic inflammation and insulin resistance. J Immunol, 2010. 185(3): p. 1836-45.
35.Nishimura, S., et al., CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity. Nat Med, 2009. 15(8): p. 914-920.
36.Ahima, R.S., Revisiting leptin''s role in obesity and weight loss. J Clin Invest, 2008. 118(7): p. 2380-3.
37.Lord, G.M., et al., Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression. Nature, 1998. 394(6696): p. 897-901.
38.De Rosa, V., et al., A key role of leptin in the control of regulatory T cell proliferation. Immunity, 2007. 26(2): p. 241-55.
39.Ohashi, K., et al., Adiponectin promotes macrophage polarization toward an anti-inflammatory phenotype. J Biol Chem, 2010. 285(9): p. 6153-60.
40.Wilk, S., et al., Adiponectin is a negative regulator of antigen-activated T cells. Eur J Immunol, 2011. 41(8): p. 2323-32.
41.Herder, C., M. Carstensen, and D.M. Ouwens, Anti-inflammatory cytokines and risk of type 2 diabetes. Diabetes Obes Metab, 2013. 15 Suppl 3: p. 39-50.
42.Arend, W.P., et al., Interleukin-1 receptor antagonist: role in biology. Annu Rev Immunol, 1998. 16: p. 27-55.
43.Otto, T.C. and M.D. Lane, Adipose development: from stem cell to adipocyte. Crit Rev Biochem Mol Biol, 2005. 40(4): p. 229-42.
44.Michalik, L., et al., International Union of Pharmacology. LXI. Peroxisome proliferator-activated receptors. Pharmacol Rev, 2006. 58(4): p. 726-41.
45.Bougarne, N., et al., PPARalpha blocks glucocorticoid receptor alpha-mediated transactivation but cooperates with the activated glucocorticoid receptor alpha for transrepression on NF-kappaB. Proc Natl Acad Sci U S A, 2009. 106(18): p. 7397-402.
46.Bishop-Bailey, D. and J. Bystrom, Emerging roles of peroxisome proliferator-activated receptor-beta/delta in inflammation. Pharmacol Ther, 2009. 124(2): p. 141-50.
47.Pascual, G., et al., A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma. Nature, 2005. 437(7059): p. 759-63.
48.Buler, M., et al., Energy-sensing factors coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1alpha) and AMP-activated protein kinase control expression of inflammatory mediators in liver: induction of interleukin 1 receptor antagonist. J Biol Chem, 2012. 287(3): p. 1847-60.
49.Chong, H.C., et al., Regulation of epithelial-mesenchymal IL-1 signaling by PPARbeta/delta is essential for skin homeostasis and wound healing. J Cell Biol, 2009. 184(6): p. 817-31.
50.Meijer, K., et al., Human primary adipocytes exhibit immune cell function: adipocytes prime inflammation independent of macrophages. PLoS ONE, 2011. 6(3): p. e17154.
51.Tseng, T.C. and S.H. Hsu, Substrate-mediated nanoparticle/gene delivery to MSC spheroids and their applications in peripheral nerve regeneration. Biomaterials, 2014. 35(9): p. 2630-41.
52.Mathiasen, A.B., et al., Bone marrow-derived mesenchymal stromal cell treatment in patients with severe ischaemic heart failure: a randomized placebo-controlled trial (MSC-HF trial). Eur Heart J, 2015. 36(27): p. 1744-53.
53.Hayes, M., et al., Mesenchymal stromal cells are more effective than the MSC secretome in diminishing injury and enhancing recovery following ventilator-induced lung injury. Intensive Care Med Exp, 2015. 3(1): p. 29.
54.Resnick, I.B., et al., Treatment of severe steroid resistant acute GVHD with mesenchymal stromal cells (MSC). Am J Blood Res, 2013. 3(3): p. 225-38.
55.Kucerova, L., et al., Long-term efficiency of mesenchymal stromal cell-mediated CD-MSC/5FC therapy in human melanoma xenograft model. Gene Ther, 2014. 21(10): p. 874-87.
56.Amable, P.R., et al., Gene expression and protein secretion during human mesenchymal cell differentiation into adipogenic cells. BMC Cell Biol, 2014. 15: p. 46.
57.Danobeitia, J.S., et al., Donor pretreatment with IL-1 receptor antagonist attenuates inflammation and improves functional potency in islets from brain-dead nonhuman primates. Cell Transplant, 2015. 24(9): p. 1863-77.
58.Lin, J., et al., The multifaceted effects of polysaccharides isolated from Dendrobium huoshanense on immune functions with the induction of interleukin-1 receptor antagonist (IL-1ra) in monocytes. PLoS One, 2014. 9(4): p. e94040.
59.Keshet, R., et al., c-Abl tyrosine kinase promotes adipocyte differentiation by targeting PPAR-gamma 2. Proc Natl Acad Sci U S A, 2014. 111(46): p. 16365-70.
60.Watt, J. and J.J. Schlezinger, Structurally-diverse, PPARgamma-activating environmental toxicants induce adipogenesis and suppress osteogenesis in bone marrow mesenchymal stromal cells. Toxicology, 2015. 331: p. 66-77.
61.He, K., et al., Effect of peroxisome proliferator-activated receptor gamma on the cholesterol efflux of peritoneal macrophages in inflammation. Mol Med Rep, 2014. 10(1): p. 373-8.
62.Lin, C.F., et al., Rosiglitazone regulates anti-inflammation and growth inhibition via PTEN. Biomed Res Int, 2014. 2014: p. 787924.
63.Motoki, T., et al., PPAR-gamma agonist attenuates inflammation in aortic aneurysm patients. Gen Thorac Cardiovasc Surg, 2015. 63(10): p. 565-71.


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