모체 혈액으로부터 태아 유핵 적혈구를 분리함에 있어서 항 감마 헤모글

Dept. of Medical Science/박사[한글] 임신중 모체혈액에서 태아 유핵 적혈구는 비침습적 산전 진단에 가장 많이 이용된다. 태아 적혈구는 항 감마 헤모글로빈이나 CD71에 의해 분리될 수 있다. 현재 가장 보편적인 태아세포 농축방법은 density gradient로 단핵세포층을 분리해내고, 태아헤모글로빈의 구성 물질인 항 감마 chain을 표지자로 이용하여 모체혈액에서 FACS로 태아 유핵 적혈세포를 농축시킨 후 FISH를 이용하여 현미경 관찰을 하는 방법이다. 그러나, FACS는 상당한 숙련도를 요하며 시간이 많이 걸리고 장비도 비싸다. CD71 표면항체는 전혈(whole blood)을 대상으로 MACS를 이용하는 것으로서 항체의 크기 및 농축 방법에 따라 1 단계 ferrofluid 분리인 immunicon, 2단계 ferrofluid 분리인 miltenyi, 그리고, 2단계 dynal bead 분리 등 세 가지가 있다. 본 실험의 일차 목적은 이들 세가지 CD71 항체와 항 감마 헤모글로빈 항체를 FISH, 양적 중합효소연쇄반응, 그리고 FACS후 FISH 결과를 비교하여 모체 혈액에서 태아 유핵 적혈구를 분리해내는 최적의 항체를 선택하는 것이다. 태아혈액을 성인의 혈액에 spike한 샘플과 임신을 종결시킨 모체혈액을 샘플로 하여 실험하였다. 항 감마 헤모글로빈 항체를 가지고 FACS로 sorting한 후 얻어진 슬라이드와 3가지 다른 CD71 항체를 이용하여 얻어진 샘플을 smear하여 얻어진 슬라이드를 XY signal에 근거하여 FISH를 한 결과, 항 감마 헤모글로빈 항체가 태아 적혈구를 가장 높은 yield를 나타내었다. 일부 샘플에서는 Y-specific probe를 가지고, 양적 중합효소 연쇄반응을 하였는데, MACS를 통하여 분리된 CD71 항체가 유핵 적혈구외에 다른 적혈구의 오염이 많이 됨으로써 오히려 떠 높은 분포를 나타내었다. 그러나, 각각의 항체에 의하여 농축된 샘플을 항 감마 헤모글로빈 항체로 염색한 후 FACS를 통하여 cytometry profile과 purity (sorting된 세포 중 태아 유핵 적혈구의 비율)를 비교해 본 결과, 항 감마 헤모글로빈 항체가 다른 CD71 항체보다 높은 purity를 보였다. 결론적으로, 항 감마 헤모글로빈 항체가 태아 유핵 적혈구를 분리해 내는 최적의 항체임을 알 수 있었다. 본 연구는 임상적용이 가능하고 최적의 효과를 얻기 위하여, 위의 실험을 배경으로 하여 우선 임신을 종결시킨 모체 혈액에 적용하였다. 항 감마 헤모글로빈항체를 가지고 모체 혈액을 농축한 후, sorting을 통하여 태아 유핵적혈구를 얻었다. 최근 개발된 태아 유핵적혈구 scoring system을 이용하여 태아 유핵적혈구의 가능성이 높은 세포를 미세조작을 이용하여 회수하고, 회수된 세포가 실제로 태아 기원의 세포인지를 확인하기 위하여, 3가지 STR primer를 이용하여 DNA 다형성의 차이를 분석할 수 있는 형광 중합효소연쇄반응을 실시하였다. 두 명의 21 삼염색체 증후군, 각각 한 명의 18과 13 삼염색태 증후군 태아를 가진 산모 혈액을 대상으로 실험하였다. 염색체 18에 있는 marker D18S535는 4명 모두에서 정확한 정보를 나타내었으며, 특히 18 삼염색체 증후군 태아 혈액에서는 3개의 peak를 보여주었다. 21번 염색체에 있는 D21S1411는 4명중 3명, 그리고, D21S11는 4명중 2명에서 정보를 나타내었고, 따라서 다운증후군임을 증명할 수 있었다. 결론적으로, 본 연구에서 확인된 방법은 향후 염색체 이상을 가진 태아를 산전 진단하는 비침습적 방법으로 이용 가치가 높고, 기존의 FISH를 이용한 방법을 대체할 수 있으며, 앞으로 유전학적 산전진단 방법에 있어서 많은 DNA source를 제공 할 수 있을 것으로 사료된다. [영문] Intact fetal nucleated red blood cells (NRBCs) in maternal blood during pregnancy can serve as a source for prenatal genetic diagnosis. NRBCs are identified by an intracellular marker such as hemoglobin or a cell surface marker such as CD71. The gamma chain of fetal hemoglobin is frequently used as a unique fetal NRBC identifier and CD71 is expressed in most fetal blood cells. In an attempt to further maximize the potential of genetic analysis from fetal cells isolation, FNRBC recovery with direct anti-gamma hemoglobin staining after density gradient and depletion was compared with three different whole blood magnetic separations (1-step from Immunicon and 2-step ferrofluid from Miltenyi, 2-step Dynal beads from Dynal). In model systems such as quantitatively defined spikes of fetal into adult blood, as well as blood samples after surgical termination procedures, fetal cell yield and purity through the results of FISH, quantitative real time PCR, and FACS were calculated. The yield of total number of cells with a XY signal after FISH was the highest on direct anti-gamma hemoglobin staining. After normalizing the results of each experiment to the corresponding result from anti-gamma hemoglobin staining (1), ratio is 0.42 in 1-step ferrofluid, 0.33 in 2-step ferrofluid, and 0.76 in 2-step dynal beads. The fetal cell purity is clearly better in direct anti-gamma hemoglobin staining than that of the magnetic separation from whole blood. The median ratio is 56.3% in anti-gamma hemoglobin staining, 7.7% in 1-step ferrofluid, 6.5% in 2-step ferrofluid, and 31.4% in 2-step dynal beads. We applied fluorescent polymerase chain reaction after fetal cell isolation using the erythroblast scoring system and micromanipulation. In all four cases analyzed the candidate fetal NRBCs were determined to be of fetal origin by at least two sets of STR markers. This determination was made when candidate fetal NRBCs shared one or two allele(s) with the maternal WBCs while the other allele(s) was (were) clearly different from the maternal WBCs. Fetal alleles were detected in four of four cases with the marker D18S535, in three of four with D21S1411, and in two of four with D21S11. This study shows that the direct anti-gamma staining is the best fetal cell recovery system and it is very useful to isolate fetal nucleated red blood cells as a non-invasive genetic source.restrictio

개선된 지역기후모델을 이용한 동아시아 여름 몬순 지역의 지역규모 지면-해양-대기 상호작용에 대한 연구

Thesis(doctors) --서울대학교 대학원 :지구환경과학부,2009.2.Docto

지구온난화로 인한 북서태평양 온도 증가가 한반도 영향 태풍에 미치는 영향

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Future Projection of Temperature over The Korean Peninsula under Global Warming Targets of 1.5 and 2.0℃, Using the Multi-RCM Ensemble in CORDEX-EA Phase 2

This study presents a future projection of temperature over the Korean Peninsula (KP) under the global warming targets of 1.5℃ and 2.0℃ set in the 2015 Paris Agreement. For projection, simulation data from the multi-Regional Climate Model (RCM) ensemble in Coordinated Regional Climate Downscaling Experiment (CORDEX)-East Asia (EA) Phase 2 are used. Under the 2.0℃ (1.5℃) global warming condition, mean temperature over KP would increase 2.13℃ (1.51℃) compared to the present period (1985 ~ 2005 years). Extreme minimum and maximum temperature intensity indices (TNn and TXx) would also increase 2.61℃ (1.91℃) and 2.38℃ (1.58℃). In addition, changes in probabilities of extreme temperature events that occurs once in 20 years during the present period are investigated. Extreme cold events would occur once in 28.9 years (22.6 years) and warm events once in 5.9 years (7.2 years). Meanwhile, there are several noteworthy points about the future projection. The temperature over KP would increase more over the northern KP than the southern KP and this feature is more distinct in TNn. The uncertainties of the projection are higher for the 1.5℃ warming condition than the 2.0℃ warming condition and for extreme temperature versus mean temperature. The spatial pattern of change in extreme temperature frequency is different from that of intensity. In other words, a smaller increase in the frequency of extreme cold events is expected over the inland KP.22Nkc

Evaluation of Performance and Uncertainty for Multi-RCM over CORDEX-East Asia Phase 2 region

This study evaluates multiple Regional Climate Models (RCMs) in simulating temperature and precipitation over the Far East Asia (FEA) and estimates the portions of the total uncertainty originating in the RCMs and the driving Global Climate Models (GCMs) using nine present-day (1981~2000) climate data obtained from combinations of three GCMs and three RCMs in the CORDEX-EA phase2. Downscaling using the RCMs generally improves the present temperature and precipitation simulated in the GCMs. The mean temperature climate in the RCM simulations is similar to that in the GCMs; however, RCMs yield notably better spatial variability than the GCMs. In particular, the RCMs generally yield positive added values to the variability of the summer temperature and the winter precipitation. Evaluating the uncertainties by the GCMs (VARGCM) and the RCMs (VARRCM) on the basis of two-way ANOVA shows that VARRCM is greater than VARGCM in contrast to previous studies which showed VARGCM is larger. In particular, in the winter temperature, the ocean has a very large VARRCM of up to 30%. Precipitation shows that VARRCM is greater than VARGCM in all seasons, but the difference is insignificant. In the following study, we will analyze how the uncertainty of the climate model in the present-day period affects future climate change prospects.22Nkc

Performance Evaluation and Future Projection of East Asian Climate using SSP Scenario-based CORDEX-East Asia Phase 2 Multi-RCM Simulations

Recently, the CORDEX-East Asia team completed climate change simulations based on five regional climate models (RCM) under four Shared Socioeconomic Pathways (SSP) scenarios. In this study, the performance of five RCMs and their ensemble averages for present-day climate simulations are evaluated. All RCMs have relatively high performance for climatology patterns for Korea, East China and Japan, while relatively low performance is observed for the Tibetan plateau. Many RCMs share cold and wet biases. Future climate changes under four SSP scenarios are analyzed for the end of 21st century (2081 ~ 2100) compared to present day (1995 ~ 2014). East Asia is expected to experience temperature increases of 2.4℃ to 6.2℃ and precipitation increases of 6.7% to 12.6%, with stronger changes in higher-emission scenarios. Among the five RCMs, HadGEM3-RA projects the largest increase in temperature while GRIMs is characterized by the strongest increase in precipitation. In line with mean warming rates, warm extreme days (TX90p) are projected to increase by 35.7 ~ 93.3 days and cold extreme days (TN10p) are projected to decrease by 23.4 ~ 35.2 days. The results of this study can be used as a reference for future detailed analyses of East Asian climate change and its impacts as well as for emphasizing the importance of carbon neutrality.22Nkc

Future Changes in Photovoltaic Potential Using CORDEX-East Asia Phase II Multi-Regional Climate Models

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Future Projection of Extreme Climate over the Korean Peninsula Using Multi-RCM in CORDEX-EA Phase 2 Project

This study presents projections of future extreme climate over the Korean Peninsula (KP), using bias-corrected data from multiple regional climate model (RCM) simulations in CORDEX-EA Phase 2 project. In order to confirm difference according to degree of greenhouse gas (GHG) emission, high GHG path of SSP5-8.5 and low GHG path of SSP1-2.6 scenario are used. Under SSP5-8.5 scenario, mean temperature and precipitation over KP are projected to increase by 6.38oC and 20.56%, respectively, in 2081~2100 years compared to 1995~ 2014 years. Projected changes in extreme climate suggest that intensity indices of extreme temperatures would increase by 6.41oC to 8.18oC and precipitation by 24.75% to 33.74%, being bigger increase than their mean values. Both of frequency indices of the extreme climate and consecutive indices of extreme precipitation are also projected to increase. But the projected changes in extreme indices vary regionally. Under SSP1-2.6 scenario, the extreme climate indices would increase less than SSP5-8.5 scenario. In other words, temperature (precipitation) intensity indices would increase 2.63oC to 3.12oC (14.09% to 16.07%). And there is expected to be relationship between mean precipitation and warming, which mean precipitation would increase as warming with bigger relationship in northern KP (4.08% oC1) than southern KP (3.53% oC1) under SSP5-8.5 scenario. The projected relationship, however, is not significant for extreme precipitation. It seems because of complex characteristics of extreme precipitation from summer monsoon and typhoon over KP.22Nkc

Changes of precipitation extremes over South Korea projected by the 5 RCMs under RCP scenarios

The change of extreme precipitation is assessed with the HadGEM2-AO - 5 Regional Climate Models (RCMs) chain, which is a national downscaling project undertaken cooperatively by several South Korean institutes aimed at producing regional climate change projection with fine resolution (12.5 km) around the Korean Peninsula. The downscaling domain, resolution and lateral boundary conditions are held the same among the 5 RCMs to minimize the uncertainties from model configuration. Climatological changes reveal a statistically significant increase in the mid-21st century (2046- 2070; Fut1) and the late-21st century (2076-2100; Fut2) precipitation properties related to extreme precipitation, such as precipitation intensity and average of upper 5 percentile daily precipitation, with respect to the reference period (1981-2005). Changes depending on the intensity categories also present a clear trend of decreasing light rain and increasing heavy rain. In accordance with these results, the change of 1-in-50 year maximum precipitation intensity over South Korea is estimated by the GEV method. The result suggests that the 50-year return value (RV50) will change from -32.69% to 72.7% and from -31.6% to 96.32% in Fut1 and from -31.97% to 86.25% and from -19.45% to 134.88% in Fut2 under representative concentration pathway (RCP) 4.5 and 8.5 scenarios, respectively, at the 90% confidence level. This study suggests that multi-RCMs can be used to reduce uncertainties and assess the future change of extreme precipitation more reliably. Moreover, future projection of the regional climate change contains uncertainties evoked from not only driving GCM but also RCM. Therefore, multi-GCM and multi-RCM studies are expected to provide more robust projection.clos

E-03 Future Changes in East Asian Summer Monsoon Rainy Season Characteristics in a Multi-GCM Driven Multi-RCM Ensemble

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