http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/91 2026-03-12T10:31:21Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63700 標題: 骨骺來源間葉幹細胞之建立與特色分析及其治療應用潛能; Establishment and Characterization of a Novel Mesenchymal Stem Cell Derived from Epiphysis and Their Potentials for Therapeutic Applications 作者: Chun-Chun Cheng; 鄭淳淳 摘要: 間葉幹細胞 (mesenchymal stem cell; MSC) 已知兼具自我更新 (self-renewal) 和複能性分化潛能 (multipotency) 等特性，因此在細胞治療與再生醫學，分別富具應用潛力。根據先前研究，骨髓中富含間葉幹細胞，而小鼠為接近人類之哺乳類動物且具有詳盡的基因背景資訊，因此多以此物種為廣泛研究之人類疾病模式動物。然而小鼠骨髓間葉幹細胞相較於其他物種而言，屬較難以分離與培養者，因而導致研究材料取得之不易，研究也因此易受侷限。本研究旨在由長骨之骨髓外組織來源分離具高增殖及分化能力之可替代骨髓間葉幹細胞的幹細胞。試驗首先自6至8週齡小鼠之骨骺端 (epiphysis) 成功分離並完成建立一新型之間葉幹細胞，命名為EMSC (epiphysis-derived MSC)。此等EMSC於培養過程中，不僅充分具備骨髓來源間葉幹細胞 (bone marrow-derived MSC; BMMSC) 固有之貼附性，且其群落形成 (clonogenic formation) 與細胞增生 (cell proliferation) 等能力，分別皆優於BMMSC者。此外，在誘導分化之特定培養條件下，此等EMSC充分具備能力分化成脂肪細胞、硬骨細胞與軟骨細胞，顯示其具備複能分化潛能。進一步針對細胞表面抗原分析結果顯示，此等EMSC不僅帶有包括CD29, CD44, CD73, CD105, CD166, Sca-1與SSEA-4等表面抗原；且不帶有CD11b, CD31, CD34, 與CD45等表面抗原。此外，在前述培養條件下，此等EMSC不表現主要組織相容性複合體I (MHC I) 與 MHC II，並有效抑制因異體免疫細胞刺激與刀豆素A刺激所導致之免疫細胞增生現象； 且能有效抑制IL-1, IL-6, 與TNF-alpha 等發炎細胞激素之產生，遂亦具有抗發炎之能力。而於細胞移行 (migration) 相關之化學激素 (chemokine) 受體表現分析試驗中顯示，EMSC主要為表現CXCR7，而BMMSC主要表現為CXCR4。又此等EMSC於低氧環境培養之下，可增強血管新生相關因子之表現能力，包括VEGF、Flk-1與PDGF。因受損組織為一低氧環境，藉由此等細胞之此特性，將可提升移植細胞治療時血管新生能力，以促進組織之修復過程。因此，進一步藉由活體內動物模式測試研究結果，證明EMSC在骨折試驗中，確能有效提升骨再生修復效果；於背部皮膚缺血 (skin flap) 之試驗結果，證明EMSC不僅可以顯著降低皮膚壞死區域，且有顯著提升皮膚血管新生之能力；並於後肢缺血試驗中且證明，EMSC可藉由幫助此處血管之再生，從而有效促進了血流的恢復。綜合上述試驗結果，顯示源自骨骺端所建立之新型間葉幹細胞– EMSC，細胞增生與分化能力均遠優於源自BMMSC者。此外，進一步體內活體試驗結果，亦證實其充分具備治療疾病之潛能。本研究結果顯示，此等源自骨骺端之間葉幹細胞殊具發展成為BMMSC替代來源之潛力，未來將有利於小鼠間葉幹細胞相關研究之推進。; While bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find stem cells of high proliferation and differentiation capacity as an alternative murine MSCs source from long bone except marrow tissue. In this study, we described a novel type of MSCs that migrates directly from the mouse epiphysis in culture. Epiphysis-derived MSCs (EMSCs) could be extensively expanded in plastic adherent culture, and they had a greater ability for clonogenic formation and cell proliferation than BMMSCs. Under specific induction conditions, EMSCs demonstrated multipotency through their ability to differentiate into adipocytes, osteocytes and chondrocytes. Immunophenotypic analysis demonstrated that EMSCs were positive for CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, while negative for CD11b, CD31, CD34 and CD45. Notably, EMSCs did not express major histocompatibility complex class I (MHC I) or MHC II under our culture system. EMSCs also successfully suppressed the proliferation of splenocytes triggered by concanavalin A (Con A) or allogeneic splenocytes, and decreased the expression of IL-1, IL-6 and TNF-alpga in Con A-stimulated splenocytes suggesting their anti-inflammatory properties. For chemokine receptors analysis, EMSCs expressed CXCR7 while BMMSCs expressed CXCR4. Furthermore, EMSCs significantly enhanced VEGF, Flk-1 and PDGF expression under hypoxia condition. The results showed that EMSCs enhanced fracture repair, ameliorated necrosis in ischemic skin flap, and improved blood perfusion in hindlimb ischemia in the in vivo experiments. These results indicate that EMSCs, a new type of MSCs, are a preferable alternative for murine MSCs due to their better proliferation and differentiation potentialities. 2012-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69562 標題: 馬利乳酸桿菌APS1對高脂飼糧誘導肥胖與非酒精性脂肪肝疾病動物模式之腸道菌相調節與機理研究; The mechanistic study of Lactobacillus mali APS1 on manipulation of gut microbiome in high-fat diet-induced obesity and non-alcoholic fatty liver disease animal model 作者: Yung-Tsung Chen; 陳詠宗 摘要: 肥胖在已開發國家及開發中國家為常見之疾病。許多疾病常伴隨著肥胖而發生，例如慢性發炎以及非酒精性脂肪肝。然而，目前對於肥胖及非酒精性脂肪肝之療效仍需更進一步的探討與改善。許多文獻指出於肥胖及非酒精性脂肪肝病症中，腸道菌群扮演著重要的角色，而利用益生菌改變腸道菌相可有效改善肥胖症狀。然而探討益生菌可否藉由改變腸道菌群並減緩非酒精性脂肪肝及其影響之機制仍未清楚了解；除此之外，結合益生菌與控制飲食攝取於調節肥胖腸道菌群及其代謝物影響之研究相當稀少。先前的研究結果已證實，糖液克菲爾所分離之馬利乳酸桿菌APS1具有許多益處，其中包括可有效抑制高脂飼糧誘導肥胖小鼠之影響。因此，本研究目的為探討糖液克菲爾APS1菌株對於高脂飼糧誘導大鼠非酒精性脂肪肝之影響以及探討結合APS1菌株與低脂飲食於已肥胖小鼠是否可促進改善肥胖之影響。於本實驗結果我們發現餵食APS1菌株可調節高脂飼糧誘導肥胖大鼠腸道菌相，減少與非酒精性脂肪肝相關之特定菌群和降低體增重，且可增加肝臟組織中SIRT-1/Nrf-2轉錄因子活性及調控下游與脂質代謝和氧化壓力相關分子路徑，降低肝臟脂質累積及增加肝臟抗氧化活性。而結合APS1菌株與低脂飲食可改善肥胖腸道菌群，促進增加已肥胖小鼠之體減重及降低體脂肪堆積。此外，餵食APS1菌株可調節脂質代謝相關之血清代謝物和腸道荷爾蒙表現以及增加糞便中短鏈脂肪酸丁酸表現量。本研究動物實驗結果可證實，糖液克菲爾分離之APS1菌株可調節宿主腸道微生物體，增加血清中腸道荷爾蒙及糞便中短鏈脂肪酸之表現，促進肝臟組織SIRT-1/Nrf-2蛋白質活性表現而降低肝臟脂質形成與堆積，且可改善宿主血清中與脂質代謝相關之代謝物表現。最後，本研究結果提供科學證據證明馬利乳酸桿菌APS1具改善代謝症候群之潛力益生菌，未來可應用於機能性食品開發。; To date, obesity is a widespread disease in developing and developed countries. Some syndromes are accompanied with obesity, such as low grade inflammation and non-alcoholic fatty liver disease (NAFLD). Currently, there are no approved clinical treatment for non-alcoholic fatty liver disease and the difficulty of long-term management has produced a high rate of failure for obesity patients. Therefore, improving the efficacy of obesity and NAFLD treatment is a significant goal. A number of studies indicate that gut microbiota dominates and plays an important role in obesity and non-alcoholic fatty liver disease. Using probiotics to manipulate the gut microbiota has been as the potential approach for improving obesity. However, it remains unclear whether the probiotic can ameliorate the non-alcoholic fatty liver through manipulating the gut microbiota, in addition, the study on the effect of combination of probiotics and diet control in the regulation of obesity-related gut microbiota and metabolites is quite rare. In our previous studies, Lactobacillus mali APS1 (APS1), which is isolated from sugary kefir, has been demonstrated to confer several health benefits in vivo, including amelioration of high-fat diet (HFD)-induced obesity in mice. Therefore, this study aimed to investigate the effect of APS1 on high-fat diet-induced NAFLD and the efficacy of a combination of APS1 and dieting on improvement of obesity in vivo. The results showed that APS1 manipulated the gut microbiota, resulting in reducing the abundance of specific NAFLD-associated bacteria, and significantly reduced hepatic lipid accumulation and increased hepatic antioxidant activity by regulating SIRT-1/Nrf-2 signaling pathway in HFD-fed rats. The combination of APS1 and dieting accelerated body weight loss and reduced fat accumulation though manipulating obesity-associated gut microbiota in preexisting obese mice. Additionally, APS1 intervention modulated the lipid metabolism-associated metabolites, appetitive hormones and increased fecal butyric acid concentration. In conclusion, this study highlighted that APS1 strain isolated from sugary kefir regulating the host gut microbiota and inducing the expressions of short-chain fatty acids and intestinal hormones resulted in reduction of body fat accumulation and hepatic steatosis in vivo. This study provided scientific evidences to show that Lactobacillus mali APS1 can be the potential probiotic to improve metabolic disorder syndrome in the application of functional food. 2018-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/95483 標題: 類3號DNA甲基轉移酶成年睪丸異構體對小鼠雄性生殖細胞發育的重要性; DNA Methyltransferase 3-Like Adult Testis Isoform (DNMT3L_AT) Is Essential For Mouse Male Germ Cell Development 作者: 吳佳勳; Chia-Hsun Wu 摘要: 男性生殖細胞的分化成熟受到許多分子機制的精細調控。生殖顆粒，是在生殖細胞的細胞質中不斷變化的生物分子凝聚物，其對於生殖細胞的發育和物種的生育能力扮演舉足輕重的角色。在小鼠中，其中一種生殖顆粒為粒線體間水泥（intermitochondrial cement, IMC），也稱為pi-body，是PIWI-piRNA途徑和piRNA生成的主要場所，特別是在減數分裂的精母細胞 (spermatocyte) 中。piRNA是一類與PIWI蛋白結合的小非編碼RNA，主要功能是抑制轉座子（transposable element, TE）的表達。此外，piRNA也通過降解mRNA或啟動轉譯起始來調控編碼蛋白基因。在我們的研究中，我們發現了由Dnmt3l較短的轉錄變體編碼的成年睾丸異構體 (DNA methyltransferase 3-like adult testis isoform, DNMT3L_AT），主要定位於精母細胞和精子細胞 (spermatid) 的細胞質中。隨後，DNMT3L_AT與多餘的細胞質殘餘物一起包裹在殘留體 (residual body) 中並脱去。值得注意的是，DNMT3L_AT與粒線體及粒線體間水泥組成成分中的MILI（PIWI-like 2）和VASA共定位。透過對DNMT3L_AT鄰近的蛋白質分析，我們假設其可能參與piRNA生物合成及其他次細胞RNA富集複合物 (subcelluler RNA-enriched complex) 中的功能。為了進一步探討這一點，我們使用條件式基因剔除 (conditional knockout, cKO) 的方式在減數分裂期間剔除Dnmt3l-at 第11號外顯子，儘管我們懷疑條件式基因剔除中因FVB 品系的Stra8-Cre在 C57BL/6JNarl 背景品系中有不完全剔除的現象，但仍導致小鼠睾丸和精子發育受到缺陷，並且其生殖細胞轉座子失去抑制，也表現出較高的細胞凋亡信號。初步的精子品質檢測顯示，Dnmt3l 條件式剔除附睾中的精子活動力和數量均低於對照組，且形態異常。此外，我們也使用另一種敲除方法是透過刪除Dnmt3l-at特有的外顯子和其潛在的啟動子和增強子，也改變其第11號外顯子中的潛在起始密碼子。在這樣的基因編輯小鼠中，我們發現其睾丸比對照組小，並在組織切片中觀察到生精小管中的生殖細胞耗盡。這些結果表明DNMT3L_AT對小鼠出生後的精子發育至關重要。; Male germ cell differentiation is intricately regulated by multiple biochemical processes. Germ granules, dynamic biomolecular condensates in the cytoplasm of germ cells, are crucial for germ cell development and fertility. In mice, a germ granule–intermitochondrial cement (IMC), also known as pi-body, serves as a primary site where the PIWI-piRNA pathway takes place and piRNAs are generated, especially in meiotic spermatocytes. In our study, we identified DNMT3L_AT (adult testis isoform), encoded by the shorter transcript variants of Dnmt3l, predominantly localized within the cytoplasm of spermatocytes and spermatids. Subsequently, DNMT3L_AT, along with cytoplasmic remnants, is encapsulated within residual bodies and released. Notably, DNMT3L_AT is highly colocalized with mitochondria and the IMC components MILI (PIWIL2), and VASA. Through the analysis of proteins proximal to DNMT3L_AT, we hypothesized its potential involvement in piRNA biogenesis and other functions within subcellular RNA-rich complexes. To investigate this further, the conditional knockout (cKO) method was employed by removing loxP-flanked exon 11 of Dnmt3l during meiosis to nullify Dnmt3l-at translation. This resulted in Dnmt3l cKO mice having smaller testes and spermatogenic defects with LINE-1 derepression and high apoptotic signals, even though incomplete recombination was suspected as the Stra8-Cre transgene generated from the FVB/NJ taking effects in the C57BL/6JNarl background. Under the regional Dnmt3l knockout model, we still demonstrated lower sperm motility and quantity, as well as abnormal sperm morphology such as abrupt bending, in the Dnmt3l cKO epididymis compared to the control littermate. Additionally, an alternative knockout method was applied by removing Dnmt3l-at-specific exons and its potential promoters and enhancers, as well as by editing eight potential start codons in exon 11. In this exon 8~11 fusion model, a complete germ cell depletion in seminiferous tubules of the much smaller testes was observed. These results suggest that DNMT3L_AT is critical for mouse postnatal spermatogenesis. 2024-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67494 標題: 類3號DNA甲基化酶監管表觀基因體抵抗細胞衰老 ; DNMT3L Reinforces Chromatin Surveillance to Resist Senescence Progression 作者: Chih-Yun Yu; 游芷芸 摘要: 細胞增生與衰老之間的平衡，與器官生成、老化、再生醫學及癌症發生有密切關係。隨著細胞衰老，細胞核發生結構重整以及表觀基因體重塑，而衰老細胞中異染色質比例下降，會漸漸失去對反轉錄跳躍子的抑制。活化的反轉錄跳躍子將威脅基因體完整性。為維持細胞分裂複製基因模板的正確性，無法修復的細胞將進入「不分裂程序」，我們稱這樣的細胞作「殭屍細胞 (senescence cell)」。這些不分裂的殭屍細胞並未被修復，在被清除前仍為不穩定之存在。本研究中意外發現，表現外源類三號DNA甲基化因子(DNA methylatransferase 3-like,DNMT3L) 可以在分裂代數較晚的小鼠胚胎纖維母細胞 (mouse embryonic fibrobalsts, MEFs) 中，透過細胞核結構重整以及表觀基因修飾重塑，召集表觀基因調控蛋白以靜默跳躍子，顯著延緩老化的細胞進入不分裂程序。類三號DNA甲基化因子相對顯著表現於生殖細胞與幹細胞中，作用為協助抑制跳躍子及建立親緣特異性之基因組印痕標記。完整的DNMT3L蛋白在體細胞內難以測得存在。本研究發現僅需「暫時性」給予老化的小鼠纖維母細胞外源性DNMT3L即可修復大半老化細胞中監管異常的表觀基因，使整體基因表現趨勢相對受控。反觀現有的細胞抗老化研究模式：多強行使衰老細胞重新進入細胞周期，而未修復可能具癌化風險的老化表觀基因體，本研究利用表觀基因組及轉錄基因組的分析，解構DNMT3L造成抗老化現象之分子機轉，並鑑定參與抗老化的因子，提供一新穎的角度開發相對安全的抗老化因子─利用DNMT3L短暫表現模式，緩解基因體不穩定的危險因子，開啟抗老化相關研究的新方向並提供降低癌症的風險的線索，希冀在未來應用於開發老化相關疾病的預防及治療。; Loosening epigenetic control during cellular aging increases chromatin instability. To mitigate damage, cells with irreparable damage of all kinds would enter senescence. However, senescence only blocks cell proliferation for these damaged cells without fixing their aberrant chromatin signatures, which remain unstable and could be cancer-prone. As a serendipitous finding via studying retroviral silencing activities, we discovered that the transient ectopic expression of DNA methyltransferase 3-like (DNMT3L) was sufficient to drive late-passage mouse embryonic fibroblasts (MEFs) to halt senescence progression. DNMT3L promotes repression of some endogenous transposable elements and de-repressed coding genes in old MEFs. While DNMT3L repressed endogenous retroviruses by attracting H3K9me3 modification, the coding genes re-repressed in old MEFs by DNMT3L were mostly Polycomb Repressive Complex 2 (PRC2) targets in young MEFs. The up-regulation of those genes in old MEFs was associated with loss of the PRC2 mediated repressive mark, H3K27me3. After transient ectopic DNMT3L expression, a panel of PRC2 modulated genes regained repressive chromatin features. We demonstrated the interaction between DNMT3L and PRC2 in our system. Our data suggest that ectopic DNMT3L may guide PRC2 to redress certain loosened chromatin regions in aging cells. This study opens perspectives in the development of an epigenetic reinforcement strategy to overcome aging-associated epimutation and senescence. 2020-01-01T00:00:00Z