孕妇外周血中单个胎儿有核红细胞在β地中海贫血无创性产前基因诊断中的应用研究
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摘要
β地中海贫血(地贫)是由于β珠蛋白基因突变导致β珠蛋白的合成部分或完全受抑制而引起的慢性溶血性贫血，是世界上最常见的常染色体遗传病之一，目前治疗费用昂贵、效果不理想，降低地贫发病率的主要手段还是对高危胎儿进行产前诊断。但是常用的产前诊断取样方法具有创伤性，对母亲及胎儿均有一定的危害性，期望能代之以无创性诊断方法。广西是地贫高发省区，无创性产前诊断方法的建立对于我们尤其具有重大意义。本研究采用孕妇外周血中单个胎儿有核红细胞(nucleated red blood cell，NRBC)作为产前诊断的材料，试图建立一条可应用的β地贫无创性产前基因诊断途径。
第一部分 联苯胺染色联合显微操作法分离孕妇外周血中有核红细胞
目的 建立一种简单、快速、经济的孕妇外周血中NRBC的识别方法，便于显微操作法高效、准确地获取单个NRBC进行后续的细胞鉴定及β地贫无创性产前基因诊断。
方法 采集28例孕龄9～34周，孕育β地贫高危胎儿的孕妇外周血各5ml，单密度梯度离心法富集，联苯胺染色识别母血中NRBC，显微操作单个获取。
结果 联苯胺染色能有效识别NRBC，28例样本均能检出NRBC，检出率100％，每例发现NRBC4～13个/5ml，共获取298个。
结论 联苯胺染色后母血中NRBC易于识别，熟练的显微操作能迅速获取单个NRBC，为应用单个胎儿NRBC进行产前基因诊断打下前期基础。
β thalassaemia is a hereditary chronic hemolytic anemia, results from a reduced output of the β-chains of adult haemoglobin. The disorder is characterized by a variety of geng mutations that either reduce or completely abolish the synthsis of β-globin . Since β thalassaemia is one of the most common autosomal recessive single gene disorders, and the treatment required is very expensive and unsatisfactory recently, the effective strategy for controlling the incidence of β thalassaemia is prenatal diagnosis, whose techniques rely on an invasive procedure to obtain fetal tissue . These invasive produres are associated with a small but significant risk to both mother and child, hence, there is a considerable need for a non-invasive alternative. β thalassaemia have particularly high incidences in Guangxi province, the investigation on non-invasive prenatal dignosis for β thalassaemia seems more crucial.
In this study, single fetal NRBC from maternal blood was used for prenatal genetic diagnosis .Our aim is to construct a utility approach for non-invasive prenatal diagnosis of β thalassaemia.
Part one Obtaining nucleated erythrocytes from maternal blood by benzidine staining and micromanipulation
Objective To develop a simple, rapid and lowcost method for detection and isolation nucleated erythrocytes (NRBCs) from maternal blood, that allows the non-invasive prenatal genetic diagnosis for β thalassaemia by using a single fetal NRBC isolated from maternal blood.
第一部分 联苯胺染色联合显微操作法分离孕妇外周血中有核红细胞
目的 建立一种简单、快速、经济的孕妇外周血中NRBC的识别方法，便于显微操作法高效、准确地获取单个NRBC进行后续的细胞鉴定及β地贫无创性产前基因诊断。
方法 采集28例孕龄9～34周，孕育β地贫高危胎儿的孕妇外周血各5ml，单密度梯度离心法富集，联苯胺染色识别母血中NRBC，显微操作单个获取。
结果 联苯胺染色能有效识别NRBC，28例样本均能检出NRBC，检出率100％，每例发现NRBC4～13个/5ml，共获取298个。
结论 联苯胺染色后母血中NRBC易于识别，熟练的显微操作能迅速获取单个NRBC，为应用单个胎儿NRBC进行产前基因诊断打下前期基础。
β thalassaemia is a hereditary chronic hemolytic anemia, results from a reduced output of the β-chains of adult haemoglobin. The disorder is characterized by a variety of geng mutations that either reduce or completely abolish the synthsis of β-globin . Since β thalassaemia is one of the most common autosomal recessive single gene disorders, and the treatment required is very expensive and unsatisfactory recently, the effective strategy for controlling the incidence of β thalassaemia is prenatal diagnosis, whose techniques rely on an invasive procedure to obtain fetal tissue . These invasive produres are associated with a small but significant risk to both mother and child, hence, there is a considerable need for a non-invasive alternative. β thalassaemia have particularly high incidences in Guangxi province, the investigation on non-invasive prenatal dignosis for β thalassaemia seems more crucial.
In this study, single fetal NRBC from maternal blood was used for prenatal genetic diagnosis .Our aim is to construct a utility approach for non-invasive prenatal diagnosis of β thalassaemia.
Part one Obtaining nucleated erythrocytes from maternal blood by benzidine staining and micromanipulation
Objective To develop a simple, rapid and lowcost method for detection and isolation nucleated erythrocytes (NRBCs) from maternal blood, that allows the non-invasive prenatal genetic diagnosis for β thalassaemia by using a single fetal NRBC isolated from maternal blood.
引文
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