類別:http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/1292026-03-09T05:11:51Z2026-03-09T05:11:51Z降低有毒醛類以達到治療小鼠急性腎損傷的效果Zhi-Zhong DING丁致中http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/691392021-06-17T03:09:36Z2020-01-01T00:00:00Z標題: 降低有毒醛類以達到治療小鼠急性腎損傷的效果
; Reducing toxic aldehydes for treating acute kidney injury in mice
作者: Zhi-Zhong DING; 丁致中
摘要: 日常生活中常存在有醛類的危害，這些有害醛類可能來自於酒精、二手菸、工業廢氣、食物腐壞等外在因子或者是經由體內代謝反應所產生。具高反應活性的醛類如4-hydroxynonenal（4-HNE）會與蛋白質產生共價鍵交互結合，進而影響正常蛋白質的構型並損害其功能。
在人體中，位於細胞粒線體內的乙醛脫氫酶(acetaldehyde dehydrogenase 2, ALDH2)主要負責將酒精的下游產物乙醛代謝為乙酸。除此之外，ALDH2同時也負責代謝其他有害的醛類如4-HNE。然而，在東亞地區有超過40％的人群帶有ALDH2基因的錯義性點突變(missense mutation, Glu487Lys)，使得這個酵素在異合子結構時活性失去60-80%，同合子結構時活性失去約90%，在後續的全基因組相關研究中也發現這個東亞人特有的點突變與腎功能有所關聯。
在我們的研究中，我們建立了缺血-再灌流的急性腎損傷小鼠模型，試圖用以探討ALDH2活化劑AD9308是否能應用於治療急性腎損傷。結果顯示，在小鼠體內產生急性腎損傷的前後施以ALDH2活化劑AD9308治療，可以有效改善腎功能並延長小鼠存活率，組織學檢查發現AD9308可以減少小鼠的腎小管損傷以及與4-HNE結合之蛋白堆積並避免腎組織的纖維化病變。
當前急性腎損傷的治療在臨床上以支持性治療為主，本研究開啟了藉由活化ALDH2以治療急性腎損傷的契機。
; In daily life, we are exposed to hazardous aldehydes. These harmful aldehydes may produce from exogenous factors such as alcohol beverage, cigarette smoking, factory exhaust, spoiled fruits or from endogenous intermediate metabolism. The highly active aldehydes such as 4-hydroxynonenal (4-HNE) form covalent binding with proteins, which change their structure resulting in protein dysfunction.
In human, acetaldehyde dehydrogenase 2 (ALDH2) enzymes that locate in cell mitochondria mainly metabolize acetaldehydes into acetic acid in alcohol metabolism pathway. Besides, ALDH2 also metabolizes a variety of toxic aldehydes including 4-HNE. However, approximate 40% of the East Asian population carry a single nucleotide polymorphism (SNP) of ALDH2 (rs671). The inactivating missense mutation is characteristic of the substitution of lysine for glutamate at position 487 (Glu487Lys) within the catalytic site of ALDH2. The ALDH2 enzymatic activity decreases by 60 to 80 % in heterozygotes (ALDH2*1) and 90 % in homozygotes (ALDH2*2). A large-scale meta-analysis of genome-wide association studies has reported that this polymorphism is significantly associated with renal function.
In this study, we established the mice acute kidney injury (AKI) model induces by ischemia-reperfusion-injury (IRI). ALDH2 activator AD9308 improved renal function and prolonged survival in AKI mice. Histological examination also showed that AD9308 reduced renal tubular damage, accumulation of 4-HNE-adducted proteins, and renal tissue fibrosis.
Present clinical treatments for AKI adopt supportive therapies. Our findings herald a new approach for treating AKI by activating ALDH2.2020-01-01T00:00:00Z開發研究人類疾病的新免疫基因組學平台Yu-Chun Lin林郁鈞http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/768172021-07-10T21:37:42Z2020-01-01T00:00:00Z標題: 開發研究人類疾病的新免疫基因組學平台; Development of new immunogenomic platforms to study human disease
作者: Yu-Chun Lin; 林郁鈞
摘要: 許多例子表明，HLA-B 基因的種系變異可能會影響個體的免疫反應和疾病發 生。據此，許多相關的基因轉殖鼠模型已被建立以研究相應的疾病。然而，大多數 小鼠模型都屬於“基因轉殖鼠”，它們將所需基因“隨機插入”到宿主基因組中， 並可能引起一些非預期的副作用。此外，目前為止還沒有 Graves’disease 的相關 HLA-B 小鼠模型。因此，本研究旨在建立具有重組酶介導的盒式交換（RMCE）系 統的 hHLA-B 外顯子 4 KI （knock-in）H-2D1 小鼠，它能方便地根據所需產生不同 的 HLA-B 等位基因。建立此小鼠後，我們將在小鼠中插入 Graves’disease 的風險 等位基因 HLA-B*46:01，並將其發展為 Graves’disease 小鼠模型。另外，我們還在 本研究中建立了人源化的β2 微球蛋白（hB2M）和 CD8 敲入小鼠，它們在抗原呈 現過程中均扮演重要角色。我們採用了 CRISPR-Cas9 或 ES 細胞靶向策略，將目標 基因插入小鼠體內，藉此將特定的突變精確地引入內源基因中，並建立小鼠品系。 我們的最終目標是配種這三種小鼠，以獲得具有三重敲入基因型小鼠。
目前我們只握有 hB2M KI 小鼠，其純系個體中 DNA、mRNA 和蛋白質的表現 已得到驗證。此外，我們發現 hB2M KI 小鼠無法表達 H-2K b 分子，但可以正常表達 H-2Db ，這足以滿足 CD4- CD8 + T 細胞的發育，但是這些細胞的功能需要進一步的實 驗來評估。總體而言，該小鼠品系似乎沒有任何異常或過早死亡的跡象，將有助於 研究免疫相關問題。
B 細胞受體（BCR）和 T 細胞受體（TCR）的集合形構了適應性免疫受體庫 （AIRR）。 BCR 和 TCR 的抗原結合位點由可變（V），多樣性（D）和連接（J） 片段組成。當發生 V（D）J 重組時，V（D）J 各自會有一個等位基因參與連接過 程，造就適應性免疫的極端多樣性。儘管已經提出了許多方法來識別免疫庫，在這 當中很少方法適合用於個人免疫受體庫的評估。在本篇研究中，我們建立了一種基 於探針捕獲的基因組 DNA（gDNA）次世代定序（NGS）方法，並結合了基於 BLASTn 的管線以辨別適應性免疫受體庫。我們根據 ImMunoGeneTics（IMGT）資料庫為 BCR 和 TCR 基因的每個已知的 V 和 J 等位基因設計了探針。 經由 Roche / NimbleGen 的客製化服務，針對每個 V 等位基因生成了三個探針、每個 J 等位基 因生成了一個探針，其中每個探針都是由連續的 60 bps 所組成。由於沒有標準可 以評估這項新提出的流程，因此我們將 IMGT 的 BCR / TCR 等位基因與 NA12878 和 NA24385 的全基因定序的 de-novo 組裝數據進行比對 [1]，並將那些完全匹配 的等位基因視為標準。 我們通過測試這兩種來自 Genome In A Bottle（GIAB, https://jimb.stanford.edu/giab）的參考材料（Reference Materials, RM）來驗證我們的 方法。結果顯示，對於 TCR 基因，NA12878 的所有 160 個 V 等位基因和 83 個 J 等位基因以及 NA24385 的所有 157 個 V 等位基因和 83 個 J 等位基因可以通過這 種方法精確分析，但是當使用源自 Epstein-Barr virus 轉化細胞株的基因組 DNA 時， 該分析流程無法鑑別 BCR 的所有 V 等位基因。
綜上所述，我們開發了一種新穎可靠的方法來分析 TCR 的 V 和 J 等位基因同 時標記了 NA12878 和 NA24385 的 TCR 等位基因。基因組 DNA 具有製備方便以 及易於保存的特點，並可隨時獲得大量樣品。我們的方法將來可以應用於人群研究 或個人精準醫療。; Numerous examples imply that germline variations of HLA-B genes may impact individuals’ immune response and diseases. Thus, many transgenic mouse models have been established to study corresponding diseases. However, most of the mice models belong to “transgenic mice” which “randomly integrates” the desired gene into the host genome and may cause some unexpecting side-effects. Moreover, no Graves’ diseaserelated HLA-B mouse model is available until this point. Accordingly, this study aims to establish an hHLA-B exon 4 KI (knock-in) H-2D1 mouse that carries a recombinasemediated cassette exchange (RMCE) system, which will enable convenient generation of different HLA-B alleles when needed. After this mouse is established, we will insert the risk allele of Graves’ disease, that is, HLA-B*46:01, into the mouse and develop it into Graves’ disease mouse model. Besides, we also want to establish humanized beta 2 microglobulin (hB2M) and CD8 KI mice, which do play important roles in the antigenpresenting process, in this study. We adopt CRISPR-Cas9 or ES-cell targeting strategy to specifically knock our desired gene into mice, which can precisely introduce specific mutations into endogenous genes and transmit them through the mouse germline. Our final goal is to breed these three strains of mice to get a mouse with triple knock-in genotype.
We only have hB2M KI mice in hand at this moment, whose expressions of DNA, mRNA, and protein in the homozygous strain have been verified. In addition, we find that hB2M KI mice fail to express H-2K b molecule but can normally express H-2Db , which is enough for CD4- CD8 + T cells development, but the functionality of these cells requires further experiments to evaluate. Overall, this mouse strain does not appear to have any abnormalities or signs of premature death and will be helpful in studying immune-related issues.
The collection of B cell receptor (BCR) and T cell receptor (TCR) form the adaptive immune receptor repertoire (AIRR). Antigen-binding sites of BCR and TCR are composed of variable (V), diversity (D), and joining (J) segments. When V(D)J recombination occurs, each gene in genomic DNA (gDNA) will have one allele participating in the joining process, which causes the extreme diversity of the adaptive immunity. Although many approaches have been proposed to identify immune repertoires, few of them are trustworthy for personal germline evaluation. In this study, we established a probe captured-based next-generation sequencing (NGS) method for genomic DNA (gDNA) combined with the BLASTn-based pipeline to determine the AIRR profiles. We designed probes for every identified V and J allele of BCR and TCR genes based on the ImMunoGeneTics (IMGT) database. Each probe is a continuous 60 bps sequence customized from Roche/NimbleGen, and each V allele is covered by three probes, while each J allele is covered by one probe. Since there is no benchmark to evaluate the newly proposed AIRR sequencing pipeline, we aligned the BCR/TCR alleles from IMGT to the whole-genome sequencing de-novo assembly data of reference materials (RMs) NA12878 and NA24385 [1] and annotate those perfectly matched alleles as standards. We verified our approach by testing these two RMs from the Genome In A Bottle (GIAB, https://jimb.stanford.edu/giab). For TCR genes, all V alleles and J alleles of NA12878 and NA24385 can be precisely assigned through this approach. However, this pipeline cannot call all V alleles of the BCR when the gDNA is originated from Epstein-Barr virus-transformed cell lines.
To sum up, we develop a novel and reliable method to profile the V genes and J genes of TCR and annotate the TCR alleles of NA12878 and NA24385, which has not been achieved before. Genomic DNA has characteristics of handy preparation and convenient storage, with numerous samples readily available. Our approach can be applied in population studies or personal precision medicine in the future.2020-01-01T00:00:00Z透過高通量化合物篩選人類前列腺素還原酶-2小分子抑制劑Meng-Lun Hsieh謝孟倫http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/590922021-06-16T09:15:56Z2017-01-01T00:00:00Z標題: 透過高通量化合物篩選人類前列腺素還原酶-2小分子抑制劑; Identification of small-molecule human PTGR-2 inhibitor through high-throughput compound screening
作者: Meng-Lun Hsieh; 謝孟倫
摘要: 過氧化物酶體增殖物活化受體γ(PPARγ)主要表現在脂肪組織中，透過與配體結合，在全身能量代謝、葡萄糖恆定以及胰島素阻抗性中扮演重要的調節功能。由於它在脂肪及葡萄糖中重要的代謝功能，所以PPARγ被視為治療疾病的重要標的，例如:第二型糖尿病、脂肪肝等疾病。目前廣泛使用的抗糖尿病藥物，是一種稱為thiazolidinedione(TZD)藥物，此藥物是人工合成之PPARγ強力配體。然而，目前人工合成的PPARγ配體，會有水分滯留、體重增加、及骨質疏鬆的副作用。因此需要開發更有效能的藥物去調控PPARγ活化以調控脂肪及葡萄糖之代謝。現今，PPARγ天然的內生性配體仍不確定。我們團隊過去研究發現15-keto-PGE2為PPARγ內生性的配體, 吾人更盡一步發現前列腺素還原酶-2(PTGR2)是一種可將15-keto-PGE2代謝成為無活性13,14-dihydro-15- keto-PGE2的酵素。Ptgr2基因剔除鼠餵食在高脂高糖飲食之下，相較於對照組，變得更瘦、胰島素敏感性增加、葡萄糖耐受性增加，但沒有水分滯留及骨質疏鬆的副作用，證實抑制PTGR2為治療糖尿病與肥胖的重要新途徑。本研究，目的為開發PTGR2的小分子抑制劑，吾人利用理性藥物設計及高通量化合物篩選方式以篩選出PTGR2小分子抑制物，並進一步在細胞株透過驗證其抑制PTGR2酵素活性、活化PPARγ的能力、以及其細胞毒性。綜合以上分析，找尋最適合的抑制物以進行日後藥物最佳化與活體實驗。; Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of whole-body energy metabolism, glucose homeostasis, and insulin resistance mainly expressed in adipose tissue. PPARγ acts through transcriptional regulation of genes involved in glucose and energy homeostasis upon ligand binding. The widely used anti-diabetic agent thiazolidinediones (TZD) are potent synthetic PPARγ agonist. However, most PPARγ agonists are associated with significant side effects, such as water retention, increased adiposity, and osteoporosis. There is an urgent need to develop effective way to modulate PPARγ activity without unwanted side effects. Nowadays, the natural ligands for PPARγ are still not certain (mostly oxidized fatty acids). Our team previously found prostaglandin reductase-2 (PTGR2) can catalyze15-keto-PGE2 to 13,14-dihydro-15-keto-PGE2, and provided further evidence that15-keto-PGE2 is an endogenous PPARγ ligand. Our team further found that Ptgr2 knockout mice are leaner, more insulin sensitive, and more glucose tolerant than control in high-fat high-sucrose (HFHS) diet without fluid retention and osteoporosis, indicating PTGR2 inhibition is a novel therapeutic approach for treating type 2 diabetes and obesity.
In this study, we sought to identify PTGR2 small-molecule inhibitor through rational drug design and high-throughput compound screening. We further validated the enzymatic inhibitory activities, the PPARγ transactivating activities, and the cytotoxicity of these hits. Based on this study, we identified potential PTGR2 inhibitors for further optimization and animal experiment.2017-01-01T00:00:00Z臺灣族群個人之人類白血球抗原與殺手細胞類免疫球蛋白受體基因體組成分析莊惠文Hui-Wen Chuanghttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/918222024-02-22T16:52:55Z2024-01-01T00:00:00Z標題: 臺灣族群個人之人類白血球抗原與殺手細胞類免疫球蛋白受體基因體組成分析; Analysis of Personal Human Leukocyte Antigen (HLA) and Killer-cell Immunoglobulin-like Receptor (KIR) Genomic Profiles in the Taiwanese Population
作者: 莊惠文; Hui-Wen Chuang
摘要: 人類基因組中的殺手細胞類免疫球蛋白受體（KIR）基因很重要，但研究起來因為基因的複雜性所以具有挑戰性。 這些基因透過與人類白血球抗原 (HLA) 相互作用來影響自然殺手 (NK) 細胞的活性。 這種相互作用調節免疫反應並影響各種健康狀況，例如自體免疫疾病和器官移植結果。 然而，KIR基因的遺傳結構複雜，且受遺傳因素影響個體間拷貝數（CN）差異較大，使得KIR分型變得複雜。 台灣族群特有的個體層級 KIR-HLA 組合資料缺乏歷史資料，影響了我們的研究方向。
我們的研究利用了各種工具，即 HLA-VBseq、HISAT-genotype、T1K、DRAGEN、Qztype、graphKIR 和 capKIR，來解釋來自各種來源的KIR 和 HLA 基因資料，例如、合成資料、 panel、 全基因體定序（WGS） 、人類泛基因組參考聯盟 (HPRC)資料庫、台灣人體生物資料庫 (TWB)，以及結合爬蟲收集的資料進行分析。 由於每種工具的性能因資料集而異，因此對其功能進行了仔細評估。在 HLA 方面，T1K 和 HISAT-genotype 基因分型因其卓越的準確性和全面的分型功能而成為 HLA 分型性能較好的工具。 在 KIR 方面，graphKIR 最適合 WGS 資料類型，capKIR 適合 panel 資料類型。
使用 graphKIR 工具，我們詳細了解了台灣族群中 KIR 基因變異和不同單倍型的分佈。 對 TWB 的 1,492 個樣本的分析揭示了固有的遺傳異質性，其特徵是 A 單倍型的頻率異常高，其中 AA 單倍型是最常見的(49.7%)，BB(4.62%)，BX(45.7%)。
隨後，我們利用等位基因頻率網路資料庫（AFND）對台灣幾個亞人群的 KIR 基因頻率進行了全面評估。 我們觀察到特定基因的差異，是有關於人口差異和研究方法差異引起的變異的問題，展示了樣本人群內 KIR 基因的複雜動態。例如，我們看 AFND 數據，會發現一些顯著的頻率差異。以 K96 資料庫中的 KIR2DL1 為例。這個基因在台灣 KIR（K96）亞族群中只出現在約 65.6% 的情況下，這一比例顯著低於其他資料庫。這種差異可能源於每個研究中樣本收案或 PCR-SSP primer 設計的差異。這些因素突顯了基因頻率差異如何為我們提供有關 KIR 基因動態性質的重要性。
為了加深我們對 KIR-HLA 交互作用對多種疾病易感性的潛在影響的了解，我們對 TWB 的參與者資料進行了一項關聯性研究。 我們的研究揭示了 KIR-HLA(T1K) 組合與多種表型的關聯，並透過邏輯迴歸分析進一步驗證。 儘管存在年齡和性別等潛在限制因素，但我們的觀察有助於了解 KIR 基因和 KIR-HLA 交互作用對台灣免疫相關疾病易感性的影響。; The human genome''s Killer-cell Immunoglobulin-like Receptor (KIR) genes are significant yet challenging to study due to their gene complexity. These genes modulate the activity of natural killer (NK) cells through interactions with Human Leukocyte Antigens (HLA), impacting various health conditions like autoimmune diseases and organ transplantation outcomes. KIR gene complexity arises from their varied copy number (CN) amongst individuals due to genetic factors, complicating KIR typing. Due to a lack of KIR-HLA-related data for the Taiwanese population, we see an opportunity to focus our research in this direction.
Our research utilized various tools, namely HLA-VBseq, HISAT-genotype, T1K, DRAGEN, Qztype, graphKIR, and capKIR, to interpret KIR and HLA gene data from diverse sources. These sources included synthetic data, panel, whole-genome sequencing (WGS), the Human Pangenome Reference Consortium (HPRC), Taiwan Biobank (TWB) data sets, and data collected through web crawling for analysis. Since every tool''s performance shifts according to the data set employed, we meticulously evaluated their capabilities. T1K and HISAT-genotyping emerged as high-performing tools for HLA due to their superior precision and comprehensive genotyping features. Regarding KIR, graphKIR is most suited for WGS data types, while capKIR is befitting panel data types.
Using graphKIR, we have gained a clear and detailed picture of the variation in KIR genes and the distribution of different haplotypes within the Taiwanese population. Our analysis of 1,492 samples from TWB revealed a significant diversity in the genetic makeup. Specifically, the AA haplotype was most common, seen in 49.7% of the population, followed by BB and BX haplotypes at 4.6% and 45.7%, respectively.
Subsequently, we conducted an exhaustive assessment of KIR gene frequencies within several Taiwanese subpopulations using the Allele Frequency Net Database (AFND). We observed the differential occurrence of specific genes that raised questions about variants due to population differences and methodological disparities, illustrating complex dynamics of KIR genes within sampled populations. For instance, if we look at the AFND data, we find some notable frequency discrepancies. Take the KIR gene 2DL1 in the K96 database as an example. This gene shows up in only about 65.6% of the Taiwanese KIR(K96) subpopulation, a percentage significantly lower than in other databases. This discrepancy could stem from differences in the enrollment of samples or variations in the design of PCR-SSP primers used in each study. These factors accentuate how gene frequency divergence can provide important insights into the dynamic nature of KIR genes.
To deepen our understanding of the potential influence of KIR-HLA interactions on susceptibility to multiple diseases, we conducted an association study on participant data from TWB. Our research uncovered associations of KIR-HLA(T1K) combinations with numerous phenotypes, further confirmed by logistic regression analysis. Despite prevailing potential limiting factors like age and gender, our observations contribute significantly to comprehending the influence of KIR genes and KIR-HLA(T1K) interactions on susceptibility to immune-related diseases in Taiwan.2024-01-01T00:00:00Z