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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 江伯倫 | |
| dc.contributor.author | Hsin Ho | en |
| dc.contributor.author | 何欣 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:05:26Z | - |
| dc.date.copyright | 2018-08-01 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-07-27 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22154 | - |
| dc.description.abstract | 間葉幹細胞（Mesenchymal stem cells, MSCs）因具有分化和免疫抑制之特性而已被用於治療許多疾病，而代謝調節可增強間葉幹細胞之特性，但對於免疫調節抑制之特性的影響卻鮮少被探討。第二型糖尿病（Type 2 diabetes）同時視為代謝和發炎疾病，並可以間葉幹細胞治療，因此適合探討代謝對於間葉幹細胞的特性影響。高血糖會誘導發炎反應和破壞檸檬酸循環（TCA cycle），導致琥珀酸（Succinate）的堆積，而琥珀酸在免疫反應上扮演著重要的角色。二甲雙胍（Metformin）為糖尿病的代謝調節藥物，但在機制上還有許多未知的地方，NAD+依賴性的去乙醯酶sirtuin 1（SIRT1）為二甲雙胍潛在的調控因子，因此若結合二甲雙胍與間葉幹細胞或許可作為第二型糖尿病的新療法。本次研究中，我們主要探討二甲雙胍和琥珀酸對於間葉幹細胞功能的影響，包括了解SIRT1所扮演的角色。我們將間葉幹細胞分別與二甲雙胍和琥珀酸作處理，並偵測對於CD4+ T細胞增生的抑制功能、脂肪細胞和硬骨細胞的分化能力以及調控因子的基因表現，研究結果顯示，二甲雙胍和琥珀酸可以提升間葉幹細胞抑制CD4+ T細胞增生的功能，並且二甲雙胍會增強間葉幹細胞分化成脂肪細胞的能力，然而琥珀酸卻會降低脂肪分化的能力，而兩者也會降低間葉幹細胞分化成硬骨細胞能力。在間葉幹細胞免疫抑制作用之調控因子基因表現上，發現二甲雙胍和琥珀酸會提升iNOS和IL-6的基因表現，間葉幹細胞免疫抑制功能需藉由IFN-γ活化，但再給予IFN-γ刺激後iNOS基因的表現量則會下降，此外，IFN-γ刺激前後，二甲雙胍和琥珀酸亦會改變代謝基因的表現量，並呈現相反的效果；而收集經過二甲雙胍或琥珀酸處理後的間葉幹細胞之上清液，也發現到具有增強的抑制CD4+ T細胞增生之效果。根據目前的研究結果，二甲雙胍和琥珀酸可以提升間葉幹細胞對於CD4+ T細胞的免疫抑制效果，對於脂肪和硬骨分化以及代謝基因表現上也有相反的影響，而二甲雙胍和琥珀酸對於間葉幹細胞的確切的潛在調節機制則需要更進一步探討。 | zh_TW |
| dc.description.abstract | Mesenchymal stem cells (MSCs) have been used for many diseases because of their multipotency and immunomodulatory function. Metabolic regulation has indicated to enhance MSCs properties, however, the underlying mechanisms remain unclear. Type 2 diabetes (T2D) is considered as a metabolic and inflammatory disease and it could be treated by MSCs. Thus it is worthy to study the effect of metabolic regulation immunomodulatory function of MSC. Hyperglycemia induces inflammation, impairs TCA cycle and lead to the accumulation of succinate, while succinate plays a vital role in immunity and inflammation. Metformin as a metabolic modulator is the first-line medication for T2D patients, and NAD+-dependent deacetylase sirtuin 1 (SIRT1) has been suggested as new mediator of metformin, which plays a vital role in its metabolism. Therefore, the combination of metformin and MSCs may serve as a novel therapeutic strategy for T2D. In our study, we investigated the immunomodulatory effect of metformin and succinate on the MSCs, including clarifying the role of SIRT1 in the underlying mechanisms. We treated MSCs with metformin or succinate, and detected the immunomodulation, differentiation and genes expression of treated-MSCs. The results showed that metformin and succinate could increase suppressive function of MSCs on CD4+ T cell proliferation. Also, metformin treatment promoted the differentiation into adipocyte, whereas succinate non-interacted this effect. In contrast, both metformin and succinate interfered the differentiation of MSCs into osteocyte. We also found metformin and succinate treatment increased mRNA levels of iNOS and IL-6 of MSCs but iNOS decreased from IFN-γ treated MSCs, as IFN-γ was required for MSC-mediated immunosuppression. In the presence or absence of IFN-γ, metformin and succinate seemed to display opposite effects on glycolysis, oxidative metabolism, fatty acid oxidation and mitochondrial biogenesis. In addition, soluble factors secreted by treated MSCs significantly enhanced the suppressive function of MSCs. The underlying mechanisms of metformin and succinate effects of MSCs functions await further investigation. | en |
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| dc.description.tableofcontents | Contents
口試委員審定書.............................................................................................................. I 致謝................................................................................................................................. II 摘要................................................................................................................................ III Abstract .......................................................................................................................... V Contents ....................................................................................................................... VII Figure Contents ...........................................................................................................XIII Table Contents ............................................................................................................. XV Chapter I Introduction .............................................................................................. 1 1. Overview of Mesenchymal Stem/Stromal Cells ...................................................... 2 1.1 Characterization of MSCs .................................................................................. 2 1.2 Immunoregulatory Activities of MSCs .............................................................. 3 1.3 Therapeutic Applications of MSCs .................................................................... 5 2. Metabolic Regulation of MSCs in Therapeutic Applications .................................. 7 2.1 Strategies to Enhance MSCs Properties by Metabolic Regulation .................... 7 2.2 Overview of Metabolic Syndrome ..................................................................... 9 2.3 Type 2 Diabetes：Metabolic Disease and Inflammatory Disease ................... 10 2.4 Abnormal Succinate Accumulated in T2D mice................................................11 2.5 Succinate in Immunity and Inflammation .........................................................13 3. Metformin is the First-line Medication for T2D .................................................... 14 3.1 Metfomin: A Metabolic Modulator .................................................................. 14 3.2 Metformin can Mediate Its Anti-diabetic Activity through SIRT1 .................. 15 4. Hypothesis and Specific Aims ................................................................................ 17 Chapter II Materials and Methods ......................................................................... 18 Part1 Materials ......................................................................................................... 19 1. Reagents ................................................................................................................. 19 2. Cell Culture ............................................................................................................ 19 2.1 Animals ............................................................................................................ 19 2.2 MSCs culture medium and buffer..................................................................... 19 3. Characterization of MSCs ...................................................................................... 20 3.1 Flow cytometry ................................................................................................ 20 3.2 T lymphocyte proliferation assay ..................................................................... 21 3.3 MSCs differentiation ........................................................................................ 22 4. Detection of RNA expression ................................................................................ 23 4.1 Extraction of RNA ........................................................................................... 23 4.2 Reverse transcription-polymerase chain reaction (RT-PCR) .......................... 24 4.3 Quantitative real-time polymerase chain reaction (qPCR) .............................. 24 Part2 Methods .......................................................................................................... 25 1. Cell Culture ............................................................................................................ 25 1.1 Preparation of bone marrow derived MSCs .................................................... 25 1.2 Cell passage ..................................................................................................... 25 1.3 Isolation of CD4+ T lymphocytes .................................................................... 26 1.4 MTT cytotoxicity assay ................................................................................... 26 2. Characterization of MSC ....................................................................................... 27 2.1 Flow cytometry ................................................................................................ 27 2.2 T lymphocyte proliferation assay .................................................................... 27 2.3 MSC differentiation assay ............................................................................... 28 2.4 Oil Red O staining and quantification ............................................................. 29 2.5 Alizarin Red S staining and quantification ...................................................... 29 3. Detection of RNA expression ................................................................................ 30 3.1 Extraction of RNA ........................................................................................... 30 3.2 Reverse transcription-polymerase chain reaction (RT-PCR) .......................... 31 3.3 Quantitative real-time polymerase chain reaction (qPCR) ............................. 32 4. Statistical analysis ................................................................................................. 33 Chapter III Results .................................................................................................. 34 1. Experimental Designs ............................................................................................ 35 2. MSCs can express specific surface markers, differentiate into various kinds of cells, and suppress CD4+ T-lymphocyte proliferation ......................................................... 37 3. Metformin or succinate treatment increases the immunosuppressive function of MSCs on activated CD4+ T cells................................................................................. 37 4. Metformin increases adipocyte differentiation from MSCs, whereas succinate displays opposite effect ................................................................................................. 38 5. Both metformin and succinate treatment inhibit osteoblast differentiation from MSCs ............................................................................................................................. 39 6. Metformin and succinate at the concentration of 1mM express the higher mRNA level of iNOS than other immunoregulatory factors, and 1mM metformin increases the mRNA level of IL-6 ..................................................................................................... 39 7. The changes of metabolic genes in metformin- and succinate-treated MSCs .......... 40 8. Metformin- or succinate-treated MSCs decreases the mRNA level of iNOS after treating with IFN-γ, which is required for MSC-mediated immunosuppression activation ....................................................................................................................... 41 9. The changes of metabolic genes in metformin- and succinate-treated MSCs after treating with IFN-γ ........................................................................................................ 42 10. The potential role of SIRT1 in the MSCs, which has suggested a newly molecular mechanism for metformin ............................................................................................. 43 11. The supernatant of treated-MSCs demonstrated enhanced immunosuppressive function compared to cell-cell contact between MSCs and T cells .............................. 44 Chapter IV Discussion and Conclusion .................................................................... 45 References ................................................................................................................... 55 Figures ......................................................................................................................... 71 Tables ........................................................................................................................... 96 Figure Contents Figure 1. Experimental design .................................................................................... 72 Figure 2. MSCs can express specific surface markers, differentiate into various kinds of cells, and suppress CD4+ T-lymphocyte proliferation .............................................. 74 Figure 3. Metformin or succinate treatment increases the immunosuppressive function of MSCs on activated CD4+ T cells ............................................................................... 76 Figure 4. Metformin increases adipocyte differentiation from MSCs, whereas succinate displays opposite effect ................................................................................. 78 Figure 5. Both metformin and succinate treatment inhibit osteoblast differentiation from MSCs .................................................................................................................... 80 Figure 6. Metformin and succinate at the concentration of 1mM expressed the higher mRNA level of iNOS than other immunoregulatory factors, and 1mM metformin increases the mRNA level of IL-6 ................................................................................ 82 Figure 7. The changes of metabolic genes in metformin- and succinate-treated MSCs ............................................................................................................................ 84 Figure 8. Metformin- or succinate-treated MSCs decreased the mRNA level of iNOS after treating with IFN-γ, which is required for MSC-mediated immunosuppression activation ....................................................................................................................... 86 Figure 9. The changes of metabolic genes in metformin- and succinate-treated MSCs after treating with IFN-γ ............................................................................................... 88 Figure 10. The potential role of SIRT1 in the MSCs, which has suggested a newly molecular mechanism for metformin ........................................................................... 90 Figure 11. The supernatant of treated-MSCs demonstrated enhanced immunosuppressive function compared to cell-cell contact between MSCs and T cells ............................................................................................................................... 92 Figure 12. The schematic of the conclusion .............................................................. 94 Table Contents Table 1. Primer sequences ......................................................................................... 97 | |
| dc.language.iso | en | |
| dc.subject | 第二型糖尿病 | zh_TW |
| dc.subject | 間葉幹細胞 | zh_TW |
| dc.subject | 二甲雙胍 | zh_TW |
| dc.subject | 琥珀酸 | zh_TW |
| dc.subject | 代謝 | zh_TW |
| dc.subject | Type 2 diabetes | en |
| dc.subject | Succinate | en |
| dc.subject | Mesenchymal stem cells | en |
| dc.subject | Metformin | en |
| dc.subject | Metabolism | en |
| dc.title | 二甲雙胍和琥珀酸對間葉幹細胞分化能力與功能影響之探討 | zh_TW |
| dc.title | Study on the Effect of Metformin and Succinate on the Differentiation and
Functions of Mesenchymal Stem Cell | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 莊雅惠,孫昭玲 | |
| dc.subject.keyword | 間葉幹細胞,二甲雙胍,琥珀酸,代謝,第二型糖尿病, | zh_TW |
| dc.subject.keyword | Mesenchymal stem cells,Metformin,Succinate,Metabolism,Type 2 diabetes, | en |
| dc.relation.page | 98 | |
| dc.identifier.doi | 10.6342/NTU201802011 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2018-07-30 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 口腔生物科學研究所 | zh_TW |
| 顯示於系所單位： | 口腔生物科學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-107-1.pdf 未授權公開取用 | 7 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。