【作者】 杜迎亭；

【导师】 潘颖；

【作者基本信息】 吉林大学 ， 妇产科学， 2011， 硕士

【摘要】 目的探讨孕妇外周血中胎儿有核红细胞的富集及分选的方法——抗体偶联磁性纳米复合粒子富集及分离胎儿有核红细胞的可行性,并应用FISH法对无创性产前诊断唐氏综合症进行初步探讨,为从孕妇外周血分选胎儿有核红细胞进行无创性产前诊断积累资料和经验。本实验应用抗体CD71 (transferrin receptor转铁蛋白受体)、CD45 (leukocyte common antigen白细胞共同抗原)及CD14 (Lipopolysacharide白细胞分化抗原)偶联磁性纳米复合粒子,通过阳性细胞分选和阴性细胞分选,富集到研究对象(唐氏筛查风险高于1/270或孕妇年龄大于等于35岁,或彩超筛查提示胎儿异常,或既往有畸形胎儿分娩史)的外周血中胎儿有核红细胞,然后通过荧光原位杂交(FISH)特异性探针CSP18/X/Y进行胎儿性别的鉴定,如果能够成功鉴定出胎儿性别,进而通过GLP13/21探针鉴定21号染色体是否存在异常。通过鉴定胎儿性别来探讨利用这种无创性产前诊断方法富集及分离胎儿有核红细胞的可行性;进一步探讨利用这种方法进行无创性产前诊断唐氏综合症的可行性。方法以羊水穿刺胎儿细胞进行荧光原位杂交(FISH)检测18、X、Y、13、21号染色体是否存在异常作为对照组。抽取32例唐氏筛查高危的或有高风险因素的孕妇的羊水20毫升及外周静脉血10毫升,前者为对照组(羊水组),后者为研究组(母血组),两组均通过CSP18/X/Y及GLP13/21探针进行荧光原位杂交(FISH)检测21号染色体是否存在异常。利用统计学方法计算两组检测胎儿性别的特异度、灵敏度、阳性预测值及阴性预测值。比较两组之间在产前诊断唐氏综合症方面的差异。结果初步建立了抗体偶联磁性纳米复合粒子富集胎儿有核红细胞进行无创性产前诊断唐氏综合征的方法。应用该方法研究组(母血组)产前诊断32例唐氏综合征筛查为高危的或有高风险因素的孕妇,成功富集到胎儿有核红细胞的例数为20例。利用该法从孕妇外周血中富集胎儿有核红细胞的富集率为62.5%。成功检测胎儿性别19例,其中1例因镜下未见Y染色体探针荧光信号,故误诊为女胎。研究组(母血组)中FISH法诊断男胎的灵敏度为94%,特异性为100%,诊断女胎的灵敏度为100%,特异度为94%。检测出2例唐氏综合征胎儿,结果分别为XY,+21和XX,+21,其余标本均未见异常信号。对照组(羊水组)成功抽取了实验对象羊水32例,但其中2例因羊水中混入母血进行了第2次抽取得到满意结果,除研究组(母血组)中1例男性胎儿误诊为女胎外,其余两组结果均相一致。结论本分子生物学方法诊断唐氏综合征为无创性的产前诊断,检测过程快速、准确、高效,较羊水穿刺胎儿细胞进行荧光原位杂交法具有无创及省力等优点,可望用于产前诊断唐氏综合症,是一种值得推广的方法。更多还原

【Abstract】 Enrichment of fetal cells by magnetic nanoparticle in the application of non-invasive prenatal diagnosis Down ’s syndromeObjective: It’s objective is exploring fetal nucleated cells enrichment and sorting method in pregnant women’s peripheral blood cells of the feasibility through antibody coupling magnetic nano particle separation and enrichment of fetal cells. And application the method of FISH noninvasive prenatal diagnosis Down ’s syndrome is discussed,for accumulating datas and experiences about separating the fetal nucleated cells from pregnant women’s peripheral blood cells to non-invasive diagnosis of prenatal diagnosis.The experiment application transferrin receptor antibody CD71 surface(transferrin receptor), leukocyte antigen CD45 (white to common antigen) and CD14 (Lipopo-lysacharide leucocyte differentiation antigen) coupling magnetic nano particle. Through the positive and negative cell sorting enrichments to researchful objects′(Down ’s syndrome screening the risk is higher than 1/270 or pregnant women older than equal to 35, or color dopplar ultrasound screening tip fetal abnormalities, or always have malformation fetus history of childbirth) peripheral blood nucleated cells, then through the fluorescence in situ hybridization (FISH) specific probe CSP18 / X/Y fetus gender identification. If successfully identified fetus gender, then through the GLP13/21 probe appraisal 21 existence abnormal chromosomes. Through using the non-invasive diagnosis of prenatal diagnosis the fetal sex appraisal to explore the method of enrichment and separation fetal nucleated feasibility. Using this method to discussing the feasibility of further noninvasive prenatal diagnosis Down’s syndrome.Methods:With amniotic fluid puncture cell fluorescence in situ hybridization (FISH) detection 18, X, Y, 13, 21 existence abnormal chromosomes as control group.Extraction of 32 cases of screening high-risk borderline pregnant or have high-risk factors of amniotic fluid 20 ml and 10 ml of peripheral blood . The former as control group (amniotic fluid group) , the latter for research group (mother blood group).Both groups through theCSP18 / X/Y and GLP13/21 probe on fluorescence in situ hybridization to detect the 21st chromosome abnormalities and determine whether there exists. Using statistical method to calculate the two groups of testing fetus gender specific degrees,sensitivity,the positie predictie and negatie predictie. Comparison between two groups in prenatal diagnosis Down’s syndrome differences.Results: Established the antibody coupling magnetic nano particle concentration on fetal nucleated cells to noninvasive prenatal diagnosis Down’s syndrome method.Applying this method the research group (mother blood group) prenatal diagnosis 32 cases Down ’s syndrome screening for high-risk or high-risk factors of the pregnant women. Successful enrichment to fetal nucleated for the number 20 patients . Using the method to enrich fetal cells from peripheral blood of pregnant women which enrichment rate is 62.5%. The experiment successfully detect fetus gender (19 cases), including 1 patients with microscopically did not see Y chromosome probe fluorescent signals, the reason misdiagnosed as XX. The research group (mother blood group) FISH method have the sensitivity of diagnostic male fetuses as 94%, and the specific degrees of 100%, but the sensitivity of diagnostic female fetuses as 100%, and the specific degrees of 94%.Detecting for fetuses with Down’s syndrome in 2 cases, there are XX, + 21 and XY, + 21, and the others all have no abnormal signals. The control group (amniotic fluid group) selected a successful experiments amniotic fluid, but 2 in 32 cases of patients with amniotic fluid mix mother performed for the first 2 times blood extraction. Except one example male fetus, the research group (mother blood group) misdiagnosed as female foetus outside, and the remaining results in two groups are in agreement. Conclusion: This molecular biology method for non-invasive diagnostic Down’s syndrome of prenatal diagnosis have a lot of advantages such as experimental process rapid, accurate, efficient. Comparative amniotic fluid puncture fluorescent in situ hybridization method, this experiment is non-invasive and save energy etc. It will be used for prenatal diagnosis Down ’s syndrome.It is a kind of worth popularizing method.更多还原