The Research Of Prenatal Gene Diagnosis Of DMD Using Fetal Erythro Blasts From Maternal Peripheral Blood

Background:There are fetal blood cells in pregnant women blood, which for the use of fetal cells for prenatal diagnosis of a non-invasive diagnostic methods, can not interfere with fetal growth under the prerequisite of obtaining fetal cells, which can be avoided Amniotic puncture, villus biopsy, fetal blood, and other methods of pregnant women and fetal harm, has taken convenient, small risk, pregnant women, aged from history and so on the merits. In recent years, the study found that fetal red blood cells is the ideal place for prenatal diagnosis of fetal cells. However, due to fetal nucleated red blood cells in maternal blood in less (1:107 ~ 108), so their enrichment, the purification is carried out the sex of the foetus and prenatal diagnosis of hereditary diseases of the pre-conditions. Purification of the current common methods: magnetic activated cell selection method (MACS), flow cytometry, fluorescence cell selection method (FACS), and so on. However, these methods require specific antibodies and fetal red blood cell nuclear absolutely no specific antibody, and the high cost, not in clinical. Density gradient centrifugation is based on cells in size, density, the settlement coefficient, osmotic pressure, and other aspects of the different design a simple and effective, more practical methods of enrichment and separation, suited to domestic conditions, it can from the maternal blood cells From mononuclear cells of the (nuclear containing fetal red blood cells), mononuclear cells from the suspension to further separation and purification, a high purity of the fetal red blood cells, while the experimental use of microsurgical operation from the pick-morphological Identification of nucleated red blood cells, received a red blood cell nuclear higher purity than other methods and at lower cost. Will be received by the nucleated red blood cells used for X-linked recessive genetic diseases (DMD) in the fetal prenatal diagnosis for non-invasive prenatal genetic diagnosis to provide a reliable basis and experimental methods. Objectives:Exploration of density gradient separation of microscopic separation operation in the peripheral blood of pregnant women fetal red blood cells in the conduct of Duchenne muscular dystrophy (DMD) of the fetus for non-invasive prenatal genetic diagnosis is feasibleMethods:The blood samples from the pregnant women in DMD family was collected at 15 to 16 weeks of gestation .The mononuclear cells were gained from maternal peripheral blood by the density gradient centrifuge ,then smeared and stained. We identified the nuclear red blood cells and collected it undermicromanipulator.And DNA from the pregnant women,their husbands,the patients and the NRBCs were extracted. Whole genome amplification to increase the quantity of the DNA of the NRBCs. Then we used the DNA for linkage analysis of the STR locis (intron44, 45, 49and5,- DYS-II) in the dystrophin gene.Results :1. The number of nucleated red blood cells found under microscope: family 1,8; family 2,6; family 3,7.2. By random primer after the pre-amplification DNA,theDNA of nucleated red blood cells are enough for diagnosis3. Three out of 3 fetuses was successfully determined.Using FNRBCs by the means of linkage analysis,prenatal gene diagnosis could distinguish normal female(1 cases)and female carrier(1 case)and DMD patient(1 case). And the use of the results of amniotic fluid for prenatal diagnosis confirmed unanimously.Conclusions :1.High purity of fetal nuclear red blood cells can be gained by gradient centrifugation separation and microscopic methods of operation2. Nucleated red blood cells of the DNA primer by random pre-amplification can gain the enough template3. Separation of fetal nuclear red blood cells can be used for DMD prenatal diagnosis of hereditary diseases.