摘要
目的 从母血循环中分离、富集、鉴定和提取胎儿有核红细胞 ,建立遗传病无创性产前基因诊断方法。方法 取 2 5名妊娠 8～ 36周的孕妇静脉血 ,加载在细胞分离液Histopaque 1.0 77上分离单个核细胞 (MNC) ;用包被CD71抗体的DynabeadsM 45 0CD71免疫磁珠或包被CD4 5抗体的DynabeadsM 45 0CD4 5免疫磁珠富集MNC中的胎儿有核红细胞 ;anti ζ biotin或anti γ biotin珠蛋白链抗体和FastRed显色反应识别富集的有核红细胞。在倒置显微镜下刮取 5例重型 β地中海贫血 (地贫 )高危胎儿的有核红细胞进行巢式聚合酶链反应 (PCR) ,扩增产物经DNA逆向斑点杂交法阐明地贫基因突变类型。结果 2 5名孕妇外周血中均发现与anti ζ biotin或anti γ biotin珠蛋白链呈阳性反应的胎儿有核红细胞。 5例重型β地贫高危胎儿中 ,有 3例所刮取的母血胎儿有核红细胞能成功进行巢式PCR ,并鉴定基因突变类型。结论 Histopaque 1.0 77单一密度梯度离心分离MNC ,结合抗CD71免疫磁珠阳性富集或CD4 5免疫磁珠阴性富集法 ,能有效富集母血循环中的胎儿有核红细胞。应用巢式PCR能对 2 0个左右的胎儿有核红细胞DNA进行扩增 ,使遗传病的无创性产前基因诊断成为可能。
Objective In order to develop a non-invasive technique for prenatal gene diagnosis. Methods Peripheral blood mononuclear cells (MNCs)were separated by single density gradient Histopaque 1.077 from 25 pregnant women with gestations between 8～36 weeks. The fetal nucleated red blood cells (NRBCs)were enriched from the MNCs by positive selection using Dynabeads M-450 CD 71 or negative selection using Dynabeads M-450 CD 45 . The enriched NRBCs were identified by anti-γ-biotin or anti-ζ-biotin antibodies. Globin gene of NRBCs from fetuses with risk of β-thalassemia major were amplified with nested PCR followed by reverse dot blot hybridization for gene diagnosis. Results NRBCs stained by anti-γ-biotin or anti-ζ-biotin antibodies could be found in the peripheral blood samples of the 25 pregnant women. Three out of 5 fetuses with risk of β-thalassemia major were successfully diagnosed using these NRBCs. Conclusion Fetal NRBCs in maternal circulation can be isolated and enriched by single gradient density Histopaque 1.077 followed by magnetic activated cell sorting. Nested PCR can amplify DNA for gene diagnosis from no less than 20 NRBCs.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2000年第10期512-516,共5页
Chinese Journal of Hematology
