【作者】 刘丽英；

【导师】 金春莲；

【作者基本信息】 中国医科大学 ， 遗传学， 2004， 硕士

【摘要】 前言 1969年Walknowska等报道在孕妇外周血中发现了核型为46，XY的细胞，为无创性产前基因诊断研究开辟了新的道路。目前，对遗传性疾病的产前诊断，根据诊断方法是否对胎儿构成不良影响而分为有创性的和无创性的。有创性产前诊断包括绒毛活检、羊膜腔穿刺、胎儿宫内取血等，这些手段虽然诊断准确率较高，但对胎儿有一定的危害，且有导致孕妇发生流产的可能。而从孕妇外周血中富集、纯化胎儿细胞或游离DNA，用于产前诊断则属无创性的。由于其具有安全可靠，对母婴无损伤等优点，因此是产前基因诊断研究的必然趋势。它是以孕妇外周血为材料，获取其中微量的胎儿细胞，从而为遗传病的产前诊断提供可靠的胎儿基因组。随着研究的不断深入，学者们发现胎儿有核红细胞(Nucleates Red Bbod Cell，NRBC)是一种较理想的靶细胞。然而目前还存在两个主要问题：(1)靶细胞数目稀少，大约为母体有核细胞的1／10~4～10~6；(2)需要明确的标记检测细胞的胎儿源性。目前用于分离胎儿细胞的方法很多，如流式细胞仪分选和／或磁激活细胞分选法富集等，然而这些富集程序不可避免的导致细胞的丢失。因此准确获取胎儿有核红细胞，简化实验步骤，减少细胞的丢失是无创性产前基因诊断技术的关键。 Duchenne型肌营养不良症(DMD)，是一种严重致死性X连锁隐性遗传病，发病率约为1／3500活男婴，其临床表现以肌肉的进行性萎缩和无力为特征。患者多在3～5岁发病，20岁前死亡。Dystrophin基因定位于Xp21.1～21.3，它是目前已知的人类最大的基因，全长约为2.4Mb。DMD基因突变中以缺失最为常见，约占55％～65％，重复次之，约占6％。有两个缺失热区：即5’端的4～21外显子(占缺失的20％)；另一为第44～52外显子(占缺失的54％～60％)。应用2×9对引物的多重PCR方法能检测到98％的缺失。其他非缺失型可通过利用DMD基因上的一些短串联重复顺序(STR)进行连锁分析。由于目前尚无有效的治疗方法，唯一有效的预防途径是对高风险胎儿进行产前诊断，杜绝患儿的出生。因此探讨DMD的无创性产前基因诊断具有重大的社会和经济效益。实验材料与方法 40例受试者，妊娠7一26周，其中巧例为孕有杜氏肌营养不良(DMD)高风险患儿的孕妇。2例胎儿脐血做为阳性对照;3例正常未孕女性及3例正常男性做为阴性对照。 本实验以不连续密度梯度离心法富集孕妇外周血中的胎儿细胞，细胞涂片离心机制片，然后应用胎儿血红蛋白特异性抗体的免疫组化鉴定、识别胎儿有核红细胞，计数、定位，显微操作法获取HbF阳性细胞，对其进行全基因组扩增。在此基础上，进行性别、多重PCR及STR连锁分析检测，进一步验证有核红细胞的来源，并同时完成DMD的无创性产前基因诊断。实验结果 经免疫组化能检测到胎儿有核红细胞的最早期为孕8周，最适孕周为孕14一18周。40例孕妇中，37例检出有HbF阳性细胞，其余3例未检出。检出数量从2.5/10耐一17.14个/10血不等，平均为9.巧个/10而。另外3例正常未孕女性及3例正常男性的外周血中均未检出有NRBC。巧例孕有DMD高危患儿的孕妇中共检出9例男性胎儿，6例女性胎儿，应用多重PCR方法，对先征者及有核红细胞进行遗传学分析(绒毛或羊水作为对照)，其中5例标本的DMD基因经外显子检测，发现存在缺失，结果与绒毛/羊水对照一致，诊断为DMD男性患儿。另10例标本经外显子检测未发现缺失，进一步经STR连锁分析，其中l例诊断为男性患儿，3例正常男性胎JL;3例诊断为女性携带者，3例正常女性胎儿。结论 1.首次应用胎儿血红蛋白特异性抗体(HbF，链)标记、识别胎儿NR-BC，建立了胎儿NRBC识别与分离一步完成的新技术。 2.经免疫组化能检测到胎儿有核红细胞的最早期为孕8周，且以孕14一18周分离效果最好。 3.本研究进一步完善了全基因组扩增技术，通过获取几个细胞进行PEP扩增弥补了由于单细胞全基因组扩增可能产生的等位基因丢失和假阳性扩增现象，提高了基因诊断的准确率。 4.联合性别检测、多重PCR和grR连锁分析并结合上述识别方法，进一步验证有核红细胞的来源，并完善了DMD的无创性产前基因诊断方法。更多还原

【Abstract】 IntroductionIn 1969 Walknowska et al. identified cell of karyotype 46, XY in the peripheral blood of pregnant woman. This was the first to demonstrate that fetal cells enter the maternal circulation in normal pregnancy and suggested that these cells had potential for use in non - invasive prenatal diagnosis. According to the difference of the dignosis method, Prenatal diagnosis can be divided into two kinds; invasive and non - invasive. Invasive prenatal diagnosis include Chorionic villus sampling^ Amniocentesis et al. Though having a high accuracy , these methods have significant risk of fetal loss. While enrichment and purification fetal cell or free DNA from maternal blood belongs to non - invaisve prenatal diagnosis. Because of many benefits,it has become the major field of prenatal diagnosis. This method takes the fetal cells from peripheral blood of pregnant women as materials, using these cells’ DNA as sample to diagnose the genetic disease prenatally. With the progress of the investigation,nucleated red blood cell (NR-BC) was regard as the candidate for non - invasive prenatal diagnosis. However , two main problems need to be overcome. First, the low frequency of the target cells i. e. 1 in 104 ~ 106nucleated blood cells, depending upon detection method and gestational age , and second, the need for an unequivocal marker for the fetal origin of the cells detected. Several seperated method, such as flow - sorting and magnetic sorting et al, had been used to enrich for NRBCs, However, enrichment procedures inevitably lead to cell loss. So how to get fetal NRBC accurately , simplify the experiment procedure and decrease the loss of fetal cells becomes the most important thing.Duchenne muscular dystrophy is a common motal X - Linked recessive disease, whose incidence of male birth is 1/3500. It characterized by progressivelymuscular atrophy and weakness. The patient commonly appear symptom at 3 ~ 5 years old and die before 20. Dystrophin gene located in Xp21.2 , about 2.4Mb long, the biggest disease - related gene as well - known at present. The main gene disorder of dystrophin is deletion of different regions, account for 55 ~65% or so, repetition about 6%. There are two deletions hot spot regions, with one was exon 4 ~21 in 5’end,and the other was exon 44 ~52. Using multiplex PCR could detected 98% of the detection, other non - deletions have to depend on STR linkage analysis. At present,this disease can not be cured. The only measure is to prevent patient child born by prenatal diagnosis. Therefore non - invasive prenatal diagnosis of DMD is beneficial for society and econimy.Materials and Methods:We obtained maternal blood samples from 40 pregnancies, at 7 - 26 weeks’ gestation, 15 of these pregnancies were at high risk for DMD and were followed up with molecular genetic analysis of chorionic villus or amniotic fluids. Blood from 3 normal non - pregnant women and 3 normal men was used as negative control, and 2 fetal umbilical cord blood was used as positive control.NRBCs were separated with percoll using a discontinuous density gradient method ,and then smeared on microscope slides using cytocentifugation. Slides were stained with antibodies against the - chain of fetal hemoglobin (HbF). NRBC were detected,counted and localized. After this, all positive NRBCs were collected by micromanipulator under microscopic observation, and then amplified them by improved primer extension preamplification(PEP). Sex and DMD genetic diagnosis were determind from a small aliquot of the PEP reaction. This can further verificated the origin of NRBCs and make the prenatal diagnosis of DMD at the same time.Results:NRBCs stained with HbF were found earliest at 8 week of gestation. And the optimum week in which to perform a reliable non - invasive prenatal diagno-sis was around 14 ~ 18 week. The number of detected NRBCs from 10ml of maternal blood ranged from 2. 5 to 17. 14 in each case and average was 9. 15 in 10ml. No HbF positive were found in peripheral blood of normal non - pregnant wo更多还原