胎儿细胞及游离DNA穿越胎盘的实验研究

Experimental study in placentas on the transmission approach of fetal nucleated cells and DNA

目的 从胎盘组织中寻找胎儿细胞及其游离DNA穿越胎盘屏障的实验室证据 ,为利用孕妇外周血进行无创性产前基因诊断提供实验依据。方法 分娩胎盘组织 2 2份 (男婴的 12份 ,女婴的 10份 ) ,切片 ,平行分为 2组。一组HE染色 ,光镜观察绒毛中胎儿细胞的分布 ;另一组用Dig 碱性磷酸酶标记男性性别特异性标记物SRY(sexdeterminingregion)基因扩增片断制成探针 ,通过原位杂交鉴定胎盘绒毛内特别是绒毛间隙是否存在相应的游离DNA。阳性对照由男性外周血有核细胞涂片经原位杂交提供。结果 HE染色光镜观察 ,2 2例胎盘组织切片可见胎儿细胞有穿越绒毛毛细血管内皮和滋养层基膜的现象 ;胎盘组织原位杂交结果显示 :分娩男婴的 12份胎盘切片的绒毛毛细血管腔内、绒毛滋养层基膜边缘、以及绒毛间隙 ,可见阳性信号 ;分娩女婴的 10份胎盘切片均无阳性信号出现 ;阳性对照涂片杂交后可见清晰阳性信号。结论 胎盘组织切片HE染色、镜检观察胎儿细胞分布 ,结合原位杂交技术鉴定绒毛内、绒毛间隙游离核酸的性质 。

Objective To investigate the proofs of fetal genetic material in the placentas and their distribution traits, so as to development a series detecting procedures by the non invasive prenatal diagnosis for genetic diseases.Methods In 22 placentas tissues (12 of them are from male fetus labor and 11 are from female fetus labor ) ,HE stain was used to investigate the scattering offetal nucleated cells in chorionic villus; In situ hybridization (ISH) technique with SRY DNA probes was used to identify the characters of free DNA in capillary and the villus gap.Results In all the 22 samples, with HE stain , fetal nucleated cells could be found at the border of capillary and trophoblastic cell;After ISH with SRY DNA probe, positive signals were displayed in 12 male placentas chorionic villus and the gap.But no signals in 10 female placentas.Conclusion This study demonstrated that the traits of the fetal free DNA in the chorionic villus gap could be identified. It means detecting fetal free DNA in maternal circulation is one of the candidate approaches for non invasive prenatal gene diagnosis.