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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 江伯倫(Bor-Luen Chiang) | |
dc.contributor.author | Yi-Shan Huang | en |
dc.contributor.author | 黃意珊 | zh_TW |
dc.date.accessioned | 2021-06-17T08:42:23Z | - |
dc.date.available | 2019-08-27 | |
dc.date.copyright | 2019-08-27 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-07 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74554 | - |
dc.description.abstract | 實驗室先前的研究發現,B細胞可以做為抗原呈現細胞,並藉由刺激T細胞活化的模式讓這兩群細胞以互相接觸的方式,誘使T細胞轉變成一群具有免疫調控功能的T細胞,我們便將這群細胞命名為B細胞誘發的調節性T細胞 (Treg-of-B)。我們發現這群誘導出的Treg-of-B細胞具有免疫調節的功能,且和天然調節性T細胞一樣會表達相關的調節性分子至細胞表面;但和天然調節性T細胞最大的不同是,Treg-of-B細胞並不會表現轉錄因子Foxp3,我們便以此為特性進一步研究這群細胞。Treg-of-B細胞具有抑制細胞增生的能力,文獻也指出在小鼠模式中,Treg-of-B細胞可以有效改善發炎疾病的症狀。
小分子核糖核酸 (microRNA) 為片段長約22至25個核苷酸,具有調控功能的非編碼核糖核酸分子。由DNA序列轉錄而來的microRNA,會經由不同酵素的切割,使最後成熟的microRNA為單股序列,在細胞質中與RNA誘導沉默複合體 (RISC) 結合,進而執行其基因調控的功能。成熟的單股microRNA利用和目標基因相互配對的程度,進而達到抑制轉譯進行,或是以基因降解的方式執行其調控基因的功能。 在目前研究中,先確認了B細胞誘發的調節性T細胞特性後,經由小分子核糖核酸微陣列分析,我們也發現Treg-of-B細胞相對於天然調節性T細胞有高表現的microRNA-210,並且在Treg-of-B細胞進行誘導及再刺激的條件下,此microRNA-210的表現量會隨之上升。而藉由下游基因的預測,也發現Treg-of-B細胞相關基因的表現也相較天然調節性T細胞為低。除此之外,研究指出microRNA-155存在於天然調節性T細胞,並且具有維持天然調節性T細胞的免疫功能,但我們觀察到Treg-of-B細胞也有microRNA-155的表現。為了確認microRNA在Treg-of-B細胞所扮演的角色,我們利用microRNA 抑制劑去模擬microRNA-210及microRNA-155在Treg-of-B細胞的表現量降低時,Treg-of-B細胞的免疫調控功能皆有減弱的現象,並且在抑制microRNA-210的表現後有更明顯的差異。 從實驗初步結果得知,microRNA-210及microRNA-155或許參與調控Treg-of-B細胞的免疫抑制功能,未來也會更進一步探討其所參與的詳細機制及扮演的角色,期許能將這群Treg-of-B細胞有更明確的定義,並在臨床應用有更完整的研究。 | zh_TW |
dc.description.abstract | In our previous studies, we found that splenic B cells could induce naïve CD4+CD25- T cells into CD4+CD25+Foxp3- regulatory T cells in a cell-cell contact manner, termed Treg-of-B cells. Treg-of-B cells could express regulation-related surface markers and secrete regulatory cytokine. Unlike natural regulatory T cells, Treg-of-B cells did not express Foxp3, but they could also exert the suppressive function like Foxp3+ natural regulatory T cells. In addition, several studies revealed that Treg-of-B cells could alleviate some inflammatory diseases.
MicroRNAs are small endogenous noncoding RNAs that are approximately 21 to 22 nucleotides in length which are transcribed from DNA. Finally, the functional miRNAs single strand is loaded into the RNA-induced silencing complex (RISC) to complementary target genes and represses gene expression by destabilizing the target mRNA or by repressing translation. In this study, after establishing the Treg-of-B cells model, the TaqMan low-density array real-time PCR assay was performed and we found that microRNA-210 was highly expressed in Treg-of-B cells than that of Foxp3+ natural regulatory T cells. To address the expression of microRNA-210 in more detail, we also found that the highest increase in microRNA-210 was observed in Treg-of B cells during induction and different time points activation. Also, the downstream target genes expression of microRNA-210 was lower in Treg-of-B cells compared to that of Foxp3+ natural regulatory T cells. In addition, we also found that microRNA-155 reported to be controlled by Foxp3 and related to regulate Foxp3+ natural regulatory T cells function was also expressed in both Treg-of-B cells and Foxp3+ natural regulatory T cells. In order to clarify the miRNA function, the miRNA inhibitors transfection into Treg-of-B cells was performed to further investigate the effects of miRNA on Treg-of-B cells. The result showed that the suppressive ability to inhibit responder T cells was decreased after inhibiting microRNA-210 and microRNA-155 expression in Treg-of-B cells, and the cells transfected with microRNA-210 had less suppressive function than microRNA-155 transfected Treg-of-B cells. Here, we aim to further study the microRNAs profile of Treg-of-B cells and identify significant microRNA that might be engaged in the suppressive function or development of Treg-of-B cells. In this study, we found that the expression of microRNA-210 and microRNA-155 might be the factors involved in the suppressive ability of Treg-of-B cells. Also, the mechanisms and the impacts of these microRNAs are needed to be clarified in detail. With our efforts, we hope to get more information on the function and development of Treg-of-B cells and contribute to Treg-based therapies which might be applied for the treatment of immunological diseases. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:42:23Z (GMT). No. of bitstreams: 1 ntu-108-R06449006-1.pdf: 3333853 bytes, checksum: 14e210b41e2bb34f80ebaf8a78108024 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 致謝 i
中文摘要 ii Abstract iv Contents vii Chapter I 1 Introduction 1 1. Background 2 1.1 T cells differentiation 2 1.2 Regulatory T cells (Treg) 2 1.3 Regulatory T cells population 4 1.4 The immune function of B cells 5 1.5 Regulatory T cells induced by B cells 5 1.6 MicroRNAs 6 1.7 MicroRNAs and regulatory T cells 7 1.8 MicroRNA-210 8 1.9 MicroRNA-155 9 2. Hypothesis and specific aims 10 Aim 1: Characterization of Treg-of-B cells 10 Aim 2: MiRNAs microarray was initially used to screen the miRNAs profile in Treg-of-B cells 10 Aim 3: Study on the effect and the role of the microRNAs in Treg-of-B cells 11 Chapter II 12 Materials and Methods 12 1. Materials 13 1.1 Animals 13 1.2 Culture medium 13 1.3 Buffer 13 1.4 EasySepTM system 14 1.5 BD IMagTM system 15 1.6 Monoclonal antibodies (mAbs) 15 1.7 Flow cytometry 15 1.8 [3H]-incorporation assay 16 1.9 Enzyme-linked immunosorbent assay(ELISA) 16 1.10 mRNA and microRNA extraction 17 1.11 Reverse transcription polymerase chain reaction (RT-PCR) for mRNA 17 1.12 Reverse transcription polymerase chain reaction (RT-PCR) for microRNA 17 1.13 Real-time polymerase chain reaction (quantitative PCR, Q-PCR) for mRNA 18 1.14 Real-time polymerase chain reaction (quantitative PCR, Q-PCR) for microRNA 20 1.15 microRNA inhibitor transfection 20 2. Methods 21 2.1 Preparation of splenocytes 21 2.2 B220+ and CD4+CD25- T Cell isolation 21 2.3 In vitro generation of Treg-of-B cells 22 2.5 Flow cytometry analysis 23 2.6 [3H]-incorporation assay 24 2.7 Cytokine analysis 24 2.8 TaqMan low-density array Real-time PCR microarray data 25 2.9 mRNA and microRNA extraction 25 2.10 Reverse transcription polymerase chain reaction (RT-PCR) for mRNA and microRNA 26 2.11 Real-time polymerase chain reaction (quantitative PCR, qPCR) for mRNA and microRNA 27 2.12 Treg-of-B cells transfection and cell sorting 28 2.13 Statistical analysis 29 Chapter III 30 Results 30 1. The purity of B220+ B and CD4+CD25- T cells 31 2. The surface molecules expression of Treg-of-B cells 31 3. Suppressive capacity of Treg-of-B cells 32 4. Cytokine analysis of Treg-of-B cells 33 5. The miRNAs expression of Treg-of-B cells in resting and stimulated state 33 6. The expression of microRNA-210 in Treg-of-B cells during their induction and different time points activation 34 7. The expression of the predictive mRNAs which were probably targeted by microRNA-210 in Treg-of-B cells 34 8. The expression of microRNA-155 which was chosen as the control of Treg-of-B cells 35 9. microRNA hairpin inhibitor transfection control with Dy547 was used to determine the transfection efficiency of Treg-of-B cells 36 10. The transfection efficiency was analyzed by the use of the fluorescent-labeled miRNA inhibitors such as microRNA-155, microRNA-210, and negative scramble control 36 11. MicroRNA inhibitors transfection into Treg-of-B cells37 12. The effect of microRNAs in the suppressive function of Treg-of-B cells 38 13. The surface marker expression of Treg-of-B cells after microRNA inhibitors transfection 39 Chapter IV 40 Discussion 40 References 48 Figures 60 Figure 1. Purification of B220+ B cells and CD4+CD25- T cells 61 Figure 2. The surface molecules expression of Treg-of-B cells 63 Figure 3. Suppressive capacity of Treg-of-B cells 64 Figure 4. Cytokine analysis of Treg-of-B cells 65 Figure 5. The miRNAs expression of Treg-of-B cells in resting and stimulated state 66 Figure 6. The expression of microRNA-210 in Treg-of-B cells during their induction and different time points activation 68 Figure 7. The expression of the predictive mRNAs which were probably targeted by microRNA-210 in Treg-of-B cells 71 Figure 8. The expression of microRNA-155 which was chosen as the control of Treg-of-B cells 72 Figure 9. microRNA hairpin inhibitor transfection control with Dy547 was used to determine the transfection efficiency of Treg-of-B cells 73 Figure 10. The transfection efficiency was also analyzed by the use of the fluorescent-labeled miRNA hairpin inhibitor such as microRNA-155, microRNA-210, and negative scramble control 75 Figure 11. microRNA inhibitor transfection into Treg-of-B cells 77 Figure 12. The effect of microRNAs in the suppressive function of Treg-of-B cells 78 Figure 13. The surface marker expression of Treg-of-B cells after microRNA inhibitor transfection 79 | |
dc.language.iso | en | |
dc.title | 探討小分子核糖核酸在Treg-of-B細胞中所扮演的角色 | zh_TW |
dc.title | Study on the role of microRNA in the function of Treg-of-B cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 朱清良(Ching-Liang Chu),楊皇煜(Huang-Yu Yang) | |
dc.subject.keyword | 調節性T細胞,B細胞誘發的調節性T細胞,小分子核糖核酸,microRNA-210,microRNA-155, | zh_TW |
dc.subject.keyword | Regulatory T cells,Treg-of-B cells,microRNA,microRNA-210,microRNA-155, | en |
dc.relation.page | 79 | |
dc.identifier.doi | 10.6342/NTU201902720 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-07 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 免疫學研究所 | zh_TW |
顯示於系所單位: | 免疫學研究所 |
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