利用免疫組庫定序技術探討在免疫球蛋白A腎病和敗血症相關急性腎損傷的T細胞組庫圖譜變化

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題名:	利用免疫組庫定序技術探討在免疫球蛋白A腎病和敗血症相關急性腎損傷的T細胞組庫圖譜變化 Exploration of T-cell Repertoire with Kidney Disease-Immunoglobin A Nephropathy and Sepsis Associated Acute Kidney Injury by Immunosequencing
作者:	何思瑩 HO, SZU-YING
貢獻者:	藥學系碩士班 KUNAL NEPALI 張偉嶠
關鍵詞:	免疫組庫定序;T 細胞組庫;T 細胞受器;免疫球蛋白A 腎病;敗血症相關急性腎損傷 Immunosequencing;T-cell repertoire;T cell receptor;Immunoglobin A nephropathy;Sepsis associated acute kidney injury
日期:	2022-06-20
上傳時間:	2023-01-18 10:34:27 (UTC+8)
摘要:	腎臟是我們人體中很重要的器官之一，它負責代謝我們身體中的水分和廢物以及調節電解質的平衡。然而隨著飲食習慣的改變、其他疾病的影響、基因和自體免疫等問題，造成腎臟急性或慢性的損傷。節自2020年，臺灣腎臟病的死亡率排名第九名，其中因腎病而洗腎的病人更是年年攀升，所造成健保財政上的負擔更是不可計數。目前在現今的診斷技術仍必須仰賴組織切片或是生物標誌物來診斷病人，然而組織切片的限制性與不便性和生物標誌物的偽陽性往往也是造成診斷的上困難。因而，我們希望透過T細胞免疫組庫的圖譜變化開發出一個相對非侵入性、專一性高且能長期追蹤病人狀況的生物標誌物來診斷病人，快速及準確的診斷病人有助於及早治療並延緩病情的惡化。本文主要利用次世代高通量定序的方式，針對個體的T細胞受器進行定序並進行後續相關的分析。我們主要透過T細胞免疫組庫的圖譜變化，探討了兩種腎臟疾病分別為免疫球蛋白A腎病(IgAN)和敗血症相關急性腎損傷(SA-AKI)。在IgAN中，我們分別納入了IgAN、non-IgAN和健康對照組比較T細胞組庫的變化。然而，雖然最後分析的結果中三組間並沒有觀察到顯著的差異，但仍然可以觀察到以下趨勢: (1)在repertoire space的分析圖中，我們觀察到了IgAN組別中擁有較多的hyperexpanded clone size。(2)透過Renyi entropy分析，整體的多樣性由高到低分別為健康對照組、non-IgAN和IgAN組。(3)在基因的分析中，找出了潛在有可能成為指標的基因，分別為TRBV4-1、TRBV6-4、TRBV27和TRBJ2-5基因。在SA-AKI中，我們收入了敗血症的病人並分成AKI和non-AKI兩組。透過分析結果，我們發現兩組間某些T細胞組庫的分析具有顯著差異: (1)根據repertoire space分析，non-AKI組有較高比例的small clone size。相反地，在hyperexpanded clone size分類中，AKI的比例則較高，雖然結果無顯著差異。(2)利用Shannon index公式計算兩組間的多樣性，結果發現non-AKI組別的多樣性高於AKI組。此外，多樣性會隨著AKI的嚴重程度逐漸下降。(3)透過PCA分析CDR3長度，發現其中non-AKI組中病人CDR3長度多為14、15和17胺基酸。(4)另外，經過校正後，雖然基因分析結果中TRBV7-7和TRBV27基因在兩組間並沒有顯著差異，但根據其他文獻報導TRBV7-7和TRBV27基因與其他疾病之間的關聯性，我們還是認為這兩個基因值得進一步做討論。總結來說，我們利用次世代定序技術來分別探討在兩種腎臟T細胞組庫的變化。結合先前的相關研究，我們認為T細胞組庫的變化能夠有機會成為一種診斷指標。 Kidney is responsible for the metabolism of water and waste and the balance of electrolytes. However, the change in eating habits, other comorbidities, genes, and autoimmunity cause acute or chronic kidney damage. In 2020, the mortality of kidney diseases ranked 9th in Taiwan. Especially, the population of dialysis has gradually increased over the decades and has caused a heavy burden on national health insurance care. Currently, the diagnosis of kidney diseases still largely relies on renal biopsy and biochemical data. Nevertheless, the inconvenience of biopsy and the false positivity of biochemical data might limit the diagnosis for kidney diseases. Therefore, we expected to develop relatively non-invasive, highly specific and long-term following-up biomarkers by immunosequencing platform. In our study, we applied next-generation sequencing to investigate the diversity of the T cell receptors in individuals. We mainly explored the characteristics of T-cell repertoire in two project 1.) Immunoglobin A Nephropathy (IgAN) and Sepsis Associated Acute Kidney Injury (SA-AKI). In the first project, IgAN, non-IgAN and healthy control groups were recruited to analyze the characteristics of T-cell repertoire. Although no significant difference was detected, we still observed the trends as below: (1) Based on repertoire space, we observed that IgAN patients had the highest proportion of hyperexpanded clone size in the three groups. (2) According to the diversity analysis, healthy controls exhibited the highest diversity in the three groups, non-IgAN and IgAN patients, sequentially. (3) In gene usage analysis, resulted from TRBV4-1, TRBV6-4, TRBV27 and TRBJ2-5 genes are promising as potential indicators for disease. In the second project, sepsis patients were recruited as two groups 1.) AKI and 2.) non-AKI groups. Through a comprehensive analysis for T cell repertoire data, we obtained interesting findings as follows: (1) Repertoire space analysis showed that non-AKI group exhibited a higher proportion of small clone size than AKI group. Conversely, AKI group exhibited a higher proportion of hyperexpanded clone size than non-AKI group although there was no significant difference. (2) Diversity measurement by Shannon index indicated that non-AKI group had a higher diversity than AKI group. Notably, the diversity was gradually decreasing as the severity of AKI patients was. (3) The PCA plot for CDR3 length indicated that non-AKI patients tended to have CDR3 lengths of 14, 15, and 17 amino acids. (4) No significant difference in gene usage was detected between the two groups after correction. However, we still considered that TRBV7-7 and TRBV27 genes are worth further understanding their functions because other research has already reported the association between the expression of these genes and diseases. In the conclusion, we respectively investigate the characteristics of T-cell repertoire in IgAN and SA-AKI by using next-generation sequencing. Although the results need to be further validated in a larger sample size, combined with previous studies, we still believe that characteristics of T-cell repertoire could serve as potential biomarkers for kidney diseases.
描述:	碩士指導教授：KUNAL NEPALI 共同指導教授：張偉嶠委員：KUNAL NEPALI 委員：張偉嶠委員：張建國委員：陳沛隆委員：卓爾婕
資料類型:	thesis
顯示於類別:	[藥學系] 博碩士論文

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