自動化微流體碟盤系統分離血液中稀少細胞並應用於非侵入式產前檢測

Isolation of Rare Cells in Human Peripheral Blood Using an Automated Disk-based Microfluidic System for Non-invasive Prenatal Diagnosis

施沐承(Mu-Cheng Shih)

指導教授 ： 胡文聰

國立臺灣大學/工學院/應用力學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803873

摘要

根據台灣行政院內政部的統計資料，民國105年國人生育第一胎的平均年齡為30.7歲，這顯示許多孕婦已達到高齡產婦的標準(年齡大於34歲)，胎兒異常的機率亦會增加，因此產前檢測變得相對重要。雖然侵入式產前檢測，如:羊膜穿刺與絨毛膜取樣，是目前診斷胎兒唐氏症唯一的確診依據，但侵入式手術對孕婦所造成的不適感，且伴隨著0.25-2%的流產機率，更甚傷及孕婦的生命，使得非侵入式產前檢測 (Non-invasive prenatal diagnosis; NIPD) 倍受矚目。近年來，許多研究團隊皆發展出非侵入式產前檢測技術，大致上可分為分離母血中胎兒游離DNA以及分離母血中胎兒有核細胞(cell-based NIPD; cbNIPD)，由於分離出的游離DNA為不完整的片段，具有偽陰性與偽陽性的缺點，且不易檢測單點基因變異，因此分離母血中胎兒有核細胞較有可能達到產前基因診斷的標準，做為醫師確診之依據。
西元1978年，科學家已在母血中發現胎兒滋胚層細胞 (fetal placental extravillous cytotrophoblast cells; EVTs)，而有核細胞含有完整的胎兒基因訊息，配合適當的基因放大技術與基因分析，可應用於非侵入式產檢。但胎兒細胞在母血中的含量非常稀少，所以需要發展出一高靈敏度、可處理大量血液，與可自動化之技術。近年來，微流體 (microfluidics) 系統因所需檢體少、反應時間快、高分離純度、高精準度、與實驗成本低等優點，被大量應用於稀少細胞之分離。
此研究提出一微流體碟盤系統，利用密度梯度離心法分離胎兒細胞，並整合免疫螢光染色法 (Immunofluorescence Staining)、單細胞抓取技術 (Single Cell Picking)、與基因放大與分析技術。微流體碟盤系統利用細胞株(MCF-7)來驗證其可行性與穩定性，首先將定量過之細胞株放進7.5毫升非孕婦之血液中，以模擬胎兒細胞在母血中之稀少性，經過自動化機台進行細胞分離與染色流程後，細胞株之回收率為98.8%。再經基因分析後，分離出的細胞與細胞株具有相同的基因表現，且異於非孕婦血液中之細胞，此結果顯示本研究之微流體碟盤系統配合自動化離心處理機，可成功回收血液中之稀少細胞。
最後，將微流體碟盤系統應用於非侵入式產前檢測，分離孕婦周邊血液中的胎兒細胞以取得胎兒基因訊息。血液檢體來自台大醫院婦產科，收取14-18毫升孕婦周邊血，孕婦年齡介於34-38歲且孕期為14-24週。結果顯示分離出的細胞與母親細胞之基因型相異，因此確認分離出的細胞為胎兒細胞。故本研究發展出一微流體碟盤系統搭配自動化離心機，可分離母血中之胎兒細胞，未來若能對此細胞進行基因分析，有望取代羊膜穿刺等侵入式產前檢測，成為產前檢測的確診依據。

關鍵字

非侵入式產前檢測 ； 微流體碟盤系統 ； 稀少細胞 ； 胎兒滋胚層細胞

並列摘要

Prenatal genetic diagnosis is becoming an important aspect of prenatal care. Traditional techniques to obtain the fetal sample, e.g. amniocentesis and chorionic villus sampling (CVS), are the gold standard of prenatal diagnosis. However, the procedure are invasive, which carries an abortion rate of 0.25-2%. As a result, non-invasive prenatal testing (NIPT) is highly noticed in recent years. Many groups have developed non-invasive technique, which can be divided into two categories: cell-free fetal DNA (cffDNA) and isolation of circulating fetal nucleated cells (CFNCs). The cffDNA approach to NIPT is limited by the fragmented fetal DNA in maternal circulate, while the cell-based approach has the potential to enable comprehensive fetal diagnosis.
This study proposes an automated disk-based microfluidic system. The fetal placental extravillous cytotrophoblast cells (EVTs) are isolated from maternal peripheral blood by density gradient centrifugation. The viability and stability of the microfluidic system are verified by spiking the MCF-7 cell line into non-pregnant woman blood. To simulate the rarity of fetal cells in the maternal blood, approximately 150 cells are spiked into 7.5 mL blood. After cells enrichment by the system, the recovery rate reaches 98.8%. Fetal cells are also isolated from maternal blood to obtain fetal genomic information. Peripheral blood of 14-18 mL from 34-38 year-old pregnant women between 14-24 weeks of gestation were tested. Taken together, this study introduces a novel automated disk-based microfluidic system to enrich fetal cells from maternal blood and the microfluidic system might provide a mean for NIPD.

並列關鍵字

non-invasive prenatal diagnosis ； microfluidic disk-based system ； rare cells ； fetal placental extravillous cytotrophoblast cells

參考文獻

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