FLOW CYTOMETRY AND MOLECULAR GENETICS OF FETAL CELLS

胎儿细胞的流式细胞术和分子遗传学

• 批准号: 3086858
• 负责人: DIANA W. BIANCHI
• 金额: $ 4.73万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3086858
• 关键词: alpha fetoprotein; chorionic villus sampling; embryo /fetus antigen; embryo /fetus cell /tissue; erythroblastosis fetalis; erythrocytes; flow cytometry; genetic disorder diagnosis; genetic manipulation; genetic mapping; human subject; immunofluorescence technique; male; molecular cloning; pregnancy immunology; prenatal diagnosis; sex chromosomes

Fetal cells of many types cross the placental barrier and persist in the maternal blood throughout gestation. Thus, they represent a reservoir of fetal tissue potentially available for genetic and cell surface analysis. One aim of this project is to detect fetal erythrocytes in the maternal circulation using fluorescently labeled antibodies against uniquely fetal antigens (such as Rh-D, i, glycophorin, and membrane-crosslinked HgB F). The labeled erythrocytes may then be physically sorted, based on combinations of fetal antigens, using a fluorescence-activated cell sorter (FACS IV). Accurate quantification of fetomaternal hemorrhage occurring during gestation will provide insight into the sensitization of Rh negative primigravidae and help evaluate the effectiveness of therapy such as Rh immune globulin. Additionally, it will determine whether or not a correlation exists between the extent of fetomaternal transfusion and otherwise unexplained elevations in maternal serum alphafetoprotein. Importantly, since the majority of the FACS-isolated fetal erythrocytes are nucleated, their DNA may be utilized for molecular probing. As a first approach, Y chromosome-specific probes (highly reiterated, hence requiring relatively small amounts of DNA) will be used as a means of identifying male fetal cells in the blood of pregnant women. As other probes become available and the sensitivity of detecting the sequences hybridizing even with non-reiterated probes increases, the inheritance of specific genes in the fetus will be determined. Prenatal genetic diagnosis on material obtained via maternal venipuncture should thereby be facilitated. Chorionic villus biopsy represents another, new method for obtaining fetal cells during the first trimester, permitting both chromosome and DNA analysis. This procedure, which is undergoing active development and evaluation, may ultimately be subject to complications depending on the extent to which it might cause fetomaternal hemorrhage. Women who are blood group incompatible with their fetuses would then be at risk for sensitization if significant fetomaternal transfusion occurs post biopsy. The current project will specifically address this issue. In addition, recombinant DNA technology will be used to differentiate between maternal and fetal DNA, to monitor possible maternal contamination of tissue samples and to provide prenatal genetic diagnosis. In the course of the experiments proposed, it is anticipated that a general fund of knowledge in molecular biology and flow cytometry will be developed.

许多类型的胎儿细胞穿过胎盘屏障并在胎盘中存活。 整个妊娠期的母血。 因此，它们代表了 胎儿组织可能可用于遗传和细胞表面分析。 该项目的一个目的是检测母体中的胎儿红细胞， 使用针对独特胎儿的荧光标记抗体进行循环 抗原（如Rh-D，i，血型糖蛋白和膜交联HgB F）。 然后可以根据以下条件对标记的红细胞进行物理分选 使用荧光激活细胞分选仪进行胎儿抗原组合 (FACS IV）。 准确定量发生的胎儿-母体出血 在妊娠期间将提供深入了解Rh阴性的致敏性 帮助评估治疗的有效性，如Rh 免疫球蛋白 此外，它还将确定 母胎输血的程度与 母体血清甲胎蛋白的其他原因不明的升高。 重要的是，由于大多数FACS分离的胎儿红细胞是 如果它们是有核的，它们的DNA可以用于分子探测。 作为第一 方法，Y染色体特异性探针（高度重复，因此需要 相对少量的DNA）将被用作识别 孕妇血液中的男性胎儿细胞。 随着其他探测器成为 并且检测杂交序列的灵敏度甚至 随着非重复探针的增加， 胎儿将被确定。 材料上的产前遗传学诊断 因此，应便于通过母体静脉穿刺获得。 绒毛活检代表了另一种获得胎儿 在前三个月的细胞，允许染色体和DNA 分析. 这一程序正在积极发展， 评估，最终可能会出现并发症，这取决于 可能导致母胎出血的程度。 的妇女 与胎儿血型不相容的人， 如果活检后发生显著的母胎输血，则会导致致敏。 目前的项目将专门处理这一问题。 此外，本发明还提供了一种方法， 重组DNA技术将用于区分母体 和胎儿DNA，以监测组织样本可能受到母体污染 并提供产前基因诊断。 在拟议的实验过程中，预计一般 将建立分子生物学和流式细胞术知识基金。