微流體碟盤系統分離母血中胎兒滋養層細胞研究

Isolation and Characterization of Fetal Trophoblasts in Maternal Blood Using a Microfluidic Disk System for Non-invasive Prenatal Diagnosis

呂立國(Li-Kuo Lu)

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(2019年)

https://doi.org/10.6342/NTU201902801

摘要

根據衛生福利部國民健康署統計，2019年35歲以上的「高齡產婦」比例接近3成，此現象會導致先天缺陷兒及死產比例提高，因此產前檢測技術越來越重要。傳統產前檢測方式，像是羊膜穿刺、絨毛取樣術檢查胎兒染色體一直是產前基因篩檢的黃金標準。然而此兩種侵入式檢測方式分別有1.4%以及1.9%的流產風險。因此非侵入式產前檢測(Non-invasive prenatal diagnosis; NIPD)是備受矚目的。此技術大致分為取得母血中胎兒游離DNA以及分離母血中胎兒有核細胞(cell-based NIPD; cbNIPD)兩種。其中游離DNA無法得到胎兒基因完整資訊，且具有偽陰性與偽陽性的缺點。因此發展出分離胎兒有核細胞的技術較有可能成為產前基因診斷的標準。1978年科學家成功檢測和純化循環胎兒細胞。
本論文之目標為利用微流體碟盤系統分離且表徵出胎兒絨毛外滋養層細胞(fetal extravillous trophoblasts; fetal EVTSs)。近年來微流體技術的開發為胎兒細胞的純化提供了更有效的方法。我們開發出一種微流體碟盤系統，利用密度梯度離心法(Density gradient centrifugation)將大量母體血液中分離出胎兒細胞以及其餘母親有核細胞，血液檢體來自台大醫院婦產科，收取16-20毫升孕婦周邊血，孕婦年齡介於31-43歲且孕期為16-19週。利用免疫螢光(Immunofluorescence Staining)染色Hoechst、anti-TBA FITC、anti-cytokeratin PE、anti-CD45 APC、anti-CD66b APC識別出胎兒細胞，接著透過單細胞抓取技術回收目標細胞。並通過下游基因組分析以確認胎兒與母體間的關聯，包括全基因組擴增(Whole genome amplification)、聚合酶鏈反應（Polymerase chain reaction）和短縱列重複序列（Short tandem repeat）。
本研究在PCR過程中成功顯現出SRY，此區域表現出男胎性別Y。來自臨床母體血液的胎兒細胞STR分析結果顯示，與羊膜穿刺STR結果部分基因位點一致，且與母體細胞STR結果的基因位點有差異。因此我們認為通過基於碟盤的微流體系統可以從母體血液中富集胎兒細胞，以及進行下游基因組分析。微流體盤系統對目標胎兒EVT的陽性預測值為53.8％。未來期待能增加陽性預測值，並成為產前檢測的判斷依據。

關鍵字

非侵入式產前檢測 ； 微流體碟盤系統 ； 胎兒滋養層細胞

並列摘要

According to the National Health Insurance Administration Ministry of Health and Welfare, the proportion of pregnant women over 35 years old is close to 30%. This phenomenon leads to an increasing proportion of birth defects and stillbirths, so prenatal testing becomes ever more important. Amniocentesis and chorionic villus sampling(CVS) are considered the gold standard for traditional invasive prenatal genetic testing. However, the miscarriage rates were 1.4% after amniocentesis and 1.9% after CVS. Therefore, non-invasive prenatal diagnosis (NIPD) is highly regarded, which can be divided into two categories: obtaining fetal cell-free DNA in maternal blood and separating fetal nucleated cells (cell-based NIPD; cbNIPD) from maternal blood. The cell-free fetal DNA might not receive the complete set of genetic information about fetal. Therefore, the development of isolating fetal nucleated cells approach has the potential to enable comprehensive fetal diagnosis. In 1978, scientists successfully detect and enrich the circulating fetal cells.
This study proposes to isolate and characterize fetal extravillous trophoblasts (fetal EVTs) using a microfluidic disk system. The development of microfluidic technology has provided a more effective method for the purify of fetal cells in recent years. We describe a microfluidic disk system that uses density centrifugation to separate fetal EVTs and other nucleated blood cells from a large amount of maternal blood. The blood of 16-20 ml from pregnant women were collected from the Obstetrics and Gynecology Department of National Taiwan University Hospital. The pregnant women were between 31-43 years old and 16-19 weeks pregnant. Fetal cells were identified by immunofluorescence staining (Hoechst, anti-TBA FITC, anti-cytokeratin PE, anti-CD45 APC, anti-CD66b APC). The target cells were recovered by single cell picking technique. The retrieved fetal/maternal cells were confirmed by downstream genomic analysis, including whole genome amplification, polymerase chain reaction, and short tandem repeat.
Results show that SRY was successfully demonstrated during the PCR process which enables sex-determining region Y. STR profiling among fetal EVT-derived, maternal cell-derived, and matching amniocentesis STR fingerprints, revealing the feto-maternal relationship between the fetal EVTs and maternal cells. The positive predictive value of the microfluidic disk system for the target fetal EVTs was 53.8%. Future study expects to increase the positive predictive value and provides a solution to researchers and clinicians.

並列關鍵字

non-invasive prenatal diagnosis ； microfluidic disk-based system ； density gradient separation ； fetal extravillous trophoblasts

參考文獻

[1] C. Kantak, C. P. Chang, C. C. Wong, A. Mahyuddin, M. Choolani, and A. Rahman, "Lab-on-a-chip technology: impacting non-invasive prenatal diagnostics (NIPD) through miniaturisation," Lab Chip, vol. 14, no. 5, pp. 841-54, Mar 7 2014.

[2] B. Levy and E. Norwitz, "Non-invasive prenatal aneuploidy testing: technologies and clinical implication," MLO Med Lab Obs, vol. 45, no. 6, pp. 8, 10, 12 passim; quiz 16, Jun 2013.

[3] J. L. Simpson, "Invasive procedures for prenatal diagnosis: any future left?," (in eng), Best Pract Res Clin Obstet Gynaecol, vol. 26, no. 5, pp. 625-38, Oct 2012.

[4] Tabor A, Vestergaard CHF, Lidegaard Ø: Fetal loss rate after chorionic villus sampling and amniocentesis: an 11-year national registry study. Ultrasound Obstet Gynecol 2009;34:19–24.

[5] Zane S, Creanga AA, Berg CJ, et al. Abortion-related mortality in the United States: 1998–2010. Obstet Gynecol 2015; 126: 258–65.