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標題: | 建立從周邊血液分離胎兒有核紅血球與偵測胎兒染色體微小結構缺失的新方法 Establishing a New Approach for Isolation of Circulating Fetal Nucleated Red Blood Cell and Detection of Fetal Subchromosome Abnormalities |
作者: | Chen-Wei Hsu 徐臣緯 |
指導教授: | 俞松良 |
關鍵字: | 非侵襲性產前檢查,胎兒有核紅血球,微小染色體缺失,Isoflux,DEParray,multiplex digital PCR, non-invasive prenatal testing,fetal nucleated red blood cells,mircrodeletion,Isoflux,DEParray,multiplex digital PCR, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 近期非侵襲性產前檢查(NIPT)運用孕婦周邊血液游離胎兒DNA (cffDNA)來進行非整倍體遺傳疾病的檢測越來越為準確,甚至能達到目前侵襲性檢查如羊膜穿刺手術或絨毛膜採檢的準確度,雖然目前NIPT已經被臨床所使用且認可,但使用cffDNA的方式依然存在許多限制,最大限制在早期孕期的周邊血液cffDNA含量太少且過於破碎,造成能準確篩檢的疾病項目多半需要有很大的差異性才能被檢測出來,所以像是胎兒的微小染色體缺失等等皆很難做很準確的篩檢。 運用孕婦周邊血液中的胎兒有核紅血球(fnRBC)完整的DNA非常有潛力可以突破這瓶頸,甚至更能準確檢測胎兒的狀況,fnRBC已經被證實是可以成為另外一個可以做NIPT的來源,而目前分離fnRBC的方法很多,科學家不管從物理或是生物的角度分離fnRBC目前結果都不盡理想,如果分離出來的fnRBC多,那純度結果都不高,反之亦然,所以到現在使用細胞做非侵襲性產前檢查的方式一直尚未成熟。 我們希望透過兩個分離系統的搭配,達到分離出高數量且高純度的fnRBC。第一步是透過Isoflux 純化系統去從血液中分離fnRBC,第二步是把分離後的樣本用DEParray做細胞純化。但fnRBC數量甚少,所以建立適合的分析方也非常重要。digital PCR(dPCR) 是目前檢測微量樣本精準度很高的平台,而我們希望可以利用dPCR來檢測微量的fnRBC。 本實驗使用K562細胞作為建立分離平台的體外測試對象,K562 cell line與fnRBC有著相似的特性,所以被廣為使用在建立fnRBC-base NIPT平台的體外測試,在此我們成功了使用K562細胞建立了兩步的純化細胞系統,且建立了目前較常見但NIPT較難檢測的七個微小染色體缺失疾病的8-plex digital PCR 平台。 Recently, analysis of cell-free fetal DNA (cffDNA) in maternal blood for non-invasive prenatal testing (NIPT) has been shown to be highly accurate in the detection of common fetal autosomal trisomies. Several studies indicated the accuracy of NIPT in trisomy detection was comparable to the invasive procedures such as chorionic villus sampling or amniocentesis. Incorporating the new non-invasive technologies into clinical practice will impact the current prenatal screening paradigm for fetal aneuploidy. Although the accuracy of NIPT was well-developed and reached clinical diagnostic levels, certain limitations were arose by using cffDNA especially in subchromosome abnormalities detection due to low yield in early gestation. Hence, fetal nucleated red blood cells (fnRBCs) in maternal peripheral blood might eliminate this bottleneck in NIPT development. fnRBCs have been demonstrated as being proof of concept for NIPT in early gestation. Various isolation and enrichment methods based on the physical and biologic features of the fnRBCs have been developed while the purity and amount of isolated fnRBCs from maternal peripheral blood are unfavorable for NIPT applications yet. We would like to establish a two-step purification method for fnRBCs-based NIPT through isolating high-purity fnRBCs from maternal peripheral blood by IsoFlux and DEParray followed by ultra-sensitive digital PCR (dPCR). In this study, we successfully used the K562 cell model system to establish our two-step purification method and have established 8-multiplex dPCR assay for the seven common subchromosome abnormalities in fetus |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53793 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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