多重置换扩增方法在无创性产前基因诊断中的应用

目的探讨多重置换扩增（multiple displacement amplification，MDA）方法应用到杜氏肌营养不良（Duchenne muscular dystrophy，DMD）的无创性产前基因诊断中的可行性。方法对12例孕7～25周的孕妇外周血用Pcreoll不连续密度梯度离心初步富集、甩片后，用Kleihauer抗酸染色法标记胎儿有核红细胞。显微操作法获取阳性细胞后，进行多重置换扩增，得到的全基因组扩增产物直接作为模板进行性别鉴定及短串联重复序列连锁分析检测，验证有核红细胞的来源，同时进行DMD的无创性产前基因诊断。结果经多重置换扩增后的产物进行琼脂糖电泳，显示片段长度大于15kb。应用多重置换扩增方法对12例孕有DMD高风险患儿的孕妇进行了无创性产前基因诊断，诊断结果与引产或产后反馈相一致。结论多重置换扩增能从微量模板中扩增出大量均一、完整的胎儿基因组序列，确保了无创性产前基因诊断结果的准确性。     

杜氏肌营养不良症的无创性产前基因诊断研究

目的 探讨杜氏肌营养不良症（Duchenne muscular dystrophy,DMD)的无创性产前基因诊断的可行性。方法 用不连续密度梯度离心方法初步富集妊娠9－21周孕妇外周血中的有核红细胞,细胞涂片离心机制片,瑞氏姬姆萨染色标记,显微操作仪获得单个有核红细胞,改良的PEP（primer extension preamplification)方法扩增单个核红细胞的全基因组DNA,在综合性别和DMD基因内的数个STR位点连锁分析进行DMD诊断诊断的同时,鉴定单个有核红细胞的来源,再应用标记聚合酶链反应扩增9个微卫星片段,进行基因型分析,进一步判定单个核红细胞来源。结果 成功诊断了1例DMD男性患病胎儿。结论 初步建立了DMD的无创性产前基因诊断的方法。     

Duchenne肌营养不良(DMD)的无创伤性产前基因诊所

目的:对DMD进行无创伤性产前基因诊断.方法:采用孕妇外周血,ZFY基因及DMD基因多重PCR反应体系,对3例进行性肌营养不良DMD患者的母亲再次怀孕时的胎儿进行无创伤性产前基因诊断.结果:1例女性胎儿,两例男性胎儿.男性胎儿中1例与其先证者哥哥同为外显子48缺少,另1例同为基因突变型.结论:无创伤性产前基因诊断X-连锁隐性遗传病DMD是可行的.     

非创伤性产前基因诊断胎儿DNA比较研究

目的：探讨非创伤性产前基因诊断胎儿DNA的最适方法。方法：用聚合酶链反应技术对40例8-12孕周人工流产胎儿性别产前检测,以三种不同取材途径：外周血;血浆游离DNA;孕妇宫颈胎儿脱落细胞;制备相应模板DNA。以绒毛组织染色体直接分析为对照。结果：40例上述三种取材途径总检出符合率分别为90%、80%、82 。5%,Y阳性检出符合率分别为81.8%、63.6%、68.2%。结论：外周血细胞DNA模板取材方法是非创伤性产前基因诊断胎儿DNA的最适方法。     

Duchenne肌营养不良（DMD）的无创伤性产前基因诊断

目的：对DMD进行无创伤性产前基因诊断。方法：采用孕妇外周血，ZFY基因及DMD基因多重PCR反应体系，对3例进行性肌营养不良DMD患者的母亲再次怀孕时的胎儿进行无创伤性产前基因诊断。结果：1例女性胎儿，两例男性胎儿，男性胎儿中1例与其先证者哥哥同为外显子48缺少，另1例同为基因突变型。结论：无创伤性前前基因诊断X－连锁隐性遗传病DMD是可行的。     

F8基因倒位检测及其在甲型血友病基因诊断中的应用

目的建立F8基因第22内含子倒位突变检测新方法,应用于甲型血友病(hemophilia A)基因诊断。方法应用长距离PCR(long distance-polymerase chain reaction,LD-PCR)、倒位PCR(inversion-PCR,IPCR)技术检测31例甲型血友病患者F8基因22内含子倒位;对于倒位突变阳性患者的母亲应用上述两种方法进行携带者诊断;而对倒位携带者孕妇于孕中期抽取羊水,进行产前基因诊断。结果31例甲型血友病患者中查出7例存在倒位突变;4例倒位突变阳性患者的母亲有3例为倒位携带者;对1例倒位携带者孕妇进行了产前诊断,确定其胎儿无倒位突变。结论LD-PCR、I-PCR技术可快速检测F8基因22内含子倒位突变,可应用于患者及携带者基因诊断;I-PCR可用于F8基因22内含子倒位的产前基因诊断。     

磁激活细胞富集孕妇外周血中胎儿有核红细胞行产前诊断

目的：研究磁激活细胞分离富集孕妇外周血胎儿有核红细胞进行产前诊断的可行性。方法：２１名孕１３－２９周孕妇及５名未妊娠妇女外周血４－６ｍｌ,经单密度梯度离心和激活细胞分离,收集阳性组分抽提ＤＮＡ,经ＰＣＲ扩增预测胎儿性别,并以出生后婴儿性别验证预测性别的准确性。     

孕妇外周血中SRY基因检测在产前诊断中的应用

目的：本文旨在探索使用套式PCR的方法，对通过密度梯度离心所得母体外周血中的胎儿细胞SRY基因片断进行检测，用以评估基因检测在早期判定胎儿性别方面的准确性，从而为利用孕妇血中胎儿细胞进行无创性产前基因诊断奠定基础。方法：采取正常妊娠10－20周孕妇外周血，经密度梯度离心后收集各层细胞，并分别提取其中的DNA，然后使用套式PCR的方法对SRY基因进行检测，并将结果与胎儿性别进行比较，用以评估套式PCR检测SRY基因在早期判定胎儿性别中的准确性和可行性。结果：共检测10例孕妇，有7例套式PCR扩增出SRY,其中有6例证实是男胎，男性性别预测符合率为85.7%(6/7)；未扩增出SRY基因的3例均证实为女性，女性预测符合率为100％（3／3）。在套式PCR扩增第一轮中，除阳性对照外所有细胞均未扩增出SRY基因，而第二轮PCR后，部分细胞扩增出SRY基因。结论：（1）孕妇外周血中存在有胎儿细胞，密度梯度离心法可对其中的胎儿细胞起一定的分离富集作用，富集到的胎儿细胞主要集中在中层，但含量较少，要用于临床检测，还需进行进一步的富集和纯化。（2）应用套式PCR能够检测到从母体外周血中所富集到胎儿细胞中的SRY基因，且对预测胎儿性别有较高的准确度，尤其是女性胎儿的判定，而在对男性胎儿的判定方面应注意外源性男性DNA的污染。     

泰国缺失型α地中海贫血1的产前基因诊断

目的对携带泰国缺失型α地中海贫血1,可能生育α地中海贫血高危胎儿的夫妇进行产前基因诊断。方法应用聚合酶链反应、DNA测序等方法对夫妇及胎儿DNA进行基因分析。结果4个家系的孕妇均为泰国缺失型α地中海贫血1复合α地中海贫血2的Hb H病患者,5个家系的孕妇或丈夫为泰国缺失型α地中海贫血1杂合子;其配偶为东南亚缺失型α地中海贫血1杂合子。他们的胎儿检出2例Hb Bart’s胎儿水肿综合征,1例Hb H病,4例α地中海贫血杂合子,2名正常。DNA测序分析证实PCR的检测结果。结论泰国缺失型α地中海贫血1的产前基因诊断研究,对遗传咨询和地中海贫血产前基因诊断具有重要意义。     

孕妇血中胎儿细胞在产前诊断中的初步应用—胎儿SR…

以Ｎｙｃｏｄｅｎｚ和Ｐｒｅｃｏｌｌ作为分离介质，对２３名孕龄为６￣４１周，年龄为２１￣２８岁的孕妇外周血中胎儿细胞进行了密度梯度离心富集。其中１５名怀男胎孕妇外周血富集前后各类细胞ＳＲＹ基因的ＰＣＲ扩增检查表明，大部分孕妇外周血多核细胞及低密度单个核细胞扩增结果为阳性，与胎儿实际性别符合率分别为８０．０％和９３．３％。这一结果提示：（１）ＰＣＲ分析母血中胎儿细胞单拷贝基因应采用低密度细胞多核细胞，     

应用原位PCR检测母体循环中胎儿遗传信息HbF γmRNA的表达

目的原位PCR是一种直接在细胞或组织材料标本上原位扩增目的DNA或RNA片段,并在原位检测扩增产物,从而进行细胞内特定核酸序列检出及定位的分子技术。胎儿血红蛋白(fetal hemoglobin,HbF)(α2γ2)是胎儿红细胞中一种主要的胞浆蛋白,依据其特有的发育规律,拟从转录水平的角度,采用高灵敏度的反转录聚合酶链反应(RT-PCR)结合原位杂交技术,深入探讨胎儿血红蛋白γmRNA在孕妇外周血中的表达机理。方法取EDTA抗凝的孕妇静脉血5 ml,PBS稀释后,经淋巴细胞分离液分离获取单个核细胞层。PBS洗3次,离心后将沉淀涂片在经多聚赖氨酸处理的载玻片上,10%缓冲中性甲醛固定,PBS洗3次,蛋白酶K消化,PBS洗3次,气干。用地高辛标记的脱氧三磷酸尿苷掺入的原位RT-PCR的方法进行检测,经25轮PCR循环后,用碱性磷酸酶标记的抗地高辛抗体检测扩增产物,显色镜检,分析36例孕妇外周血中胎儿血红蛋白γ基因的表达情况。结果靶序列的原位RT-PCR结果显示,36例孕妇外周血中有30例标本中可定位检测到胞核、胞浆内的蓝紫色的阳性信号,符合率为83.33%,在孕妇外周血和初产妇的脐血的有核红细胞的胞浆内均可检测到DIG标记的HbF-γmRNA蓝紫色信号,而正常人外周血则无此信号。结论原位RT-PCR将PCR技术的高效扩增与细胞定位相结合,在细胞原位检测低拷贝mRNA,具有敏感性高、特异性强的特点,能从分子水平上定位定量检测HbF-γmRNA的表达,该基因仅在孕妇外周血中表达,未孕女性未见其表达,提示HbF-γmRNA可能为孕妇外周血中的一种新的靶标,为无创性产前诊断提供线索和思路。     

孕妇外周血胎儿有核红细胞的分离与检测

目的从孕妇血循环中分选出有核红细胞（ＮＲＢＣｓ），并证实其是胎儿来源的。方法选用红细胞特异单克隆抗体ＧｌｙｃｏｐｈｏｒｉｎＡ（ＧＰＡ）结合流式细胞术成功地从３例妊娠１６～２４周的孕妇血中分离出ＮＲＢＣｓ，并用Ｙ－染色体特异重复序列ＤＮＡ探针（ＰＹ３．４）荧光原位杂交及聚合酶链反应技术检测Ｙ－染色体特异序列（ＳＲＹ）结果ＦＩＳＨ结果，３例分选出的ＧＰＡ阳性细胞中ＰＹ３．４杂交阳性率分别是３１．４％、０、３８％；其ＰＣＲ结果，分别为阳性、阴性、阳性，并在引产后得到性别证实。结论孕妇外周血中确实存在胎儿有核红细胞，采用非创伤性方法可以分离并检测它们     

Phenotypic and functional analysis of human T lymphocytes in early second- and third-trimester fetuses

This study was undertaken to investigate the phenotypic and functional status of T lymphocytes of human fetuses from early second- to third-trimester. Cord blood samples were obtained from 19 healthy human fetuses (gestation weeks: 18-36), by cordocentesis, and 16 term newborns (gestation weeks 37-42). Maternal and unrelated male blood samples were also taken as controls. Percentage of lymphocytes in fetal white blood cells was 79.3%, reducing to 40% by term birth, much higher than that of adults. Cord blood mononuclear cells (CBMC), prepared by density gradient centrifugation followed by lysis of erythrocytes, were stained using PE- or FITC-labelled monoclonal Abs and analysed by flow cytometry. The frequencies of CD3+ T cells in fetal (40.1%) and neonatal (42.4%) CBMC were significantly lower than that of men (59.6%) and pregnant women (53.6%). Proportions of CD8+ T cells (9.5%), gammadelta-T cells (0.5%) and NK cells (4.8%) in fetal CBMC were also lower than that of neonates (except gammadelta-T cells) and adults. A negative linear correlation (r = -0.609) between the ratio of CD4+/CD8+ T cells in fetal blood and gestation age could also be established. Fetal CBMC showed vigorous spontaneous proliferation but failed to respond to mitogen (PHA) or allogeneic stimulation in vitro. The fetal mononuclear cells were unable to produce IL-2, IL-4 or IFN-gamma, but spontaneously secreted IL-10, IL-6 and TNF-alphain vitro. Stimulation with PHA up-regulated the production of IL-10, IL-6 and TNF-alpha substantially.     

长距离PCR在血友病A携带者检测和产前诊断中的应用

目的　建立一种重型血友病 A(hemophilia A,HA)携带者检测和产前诊断方法。方法　选择有重型 HA家族史的 2 5例 HA携带者 ,采用长距离 DNA扩增 (long distance- PCR,L D- PCR)技术 ,直接检测是否存在凝血因子 (factor ,F )基因倒位。对 L D- PCR基因诊断阳性携带者于妊娠 2 0～ 2 4周抽取夫妻静脉血和胎儿脐静脉血 ,应用 Bigg's一期法检测血浆凝血因子 F 活性 (F ∶C) ,EL ISA法检测血浆血管性血友病因子 (von Willbrand factor,v WF)浓度 ,进一步应用 L D- PCR技术进行产前诊断。应用DNA测序技术验证 L D- PCR结果。结果　在 2 5个无亲缘关系的重型 HA家系中查出 F 基因倒位携带者 8例 ,其中对 4例 L D- PCR基因诊断阳性的携带者进行了产前诊断 ,2例确诊胎儿为 HA患者而建议终止妊娠 ,另 2名为正常胎儿 ,随访 1年 ,小儿发育正常。结论　应用 L D- PCR技术结合携带者外周静脉血及胎儿脐血血浆 F ∶ C和 v WF∶Ag浓度可准确、快速进行重型 HA患者的诊断及其携带者的产前诊断 ,防止血友病患儿的出生     

Fetal plasma carbonic anhydrase III in prenatal diagnosis of Duchenne muscular dystrophy

Carbonic anhydrase III (CAIII), a skeletal-muscle-specific enzyme which is elevated in the plasma of Duchenne muscular dystrophy (DMD) patients, was measured by radioimmunoassay in fetal plasma in order to evaluate its application to prenatal diagnosis of DMD. Using fetoscopy, pure fetal blood samples were taken at 17-24 weeks gestation from 25 fetuses at risk for DMD and from 78 control fetuses. Care was taken in the handling and storage of all samples. Normal sons were born in eight cases at risk for DMD. The CAIII levels in the infants were not significantly different from those of the control infants. Pregnancies were terminated in the remaining 17 at-risk cases. The CAIII levels in the fetuses were significantly different (p = 0.0034) from those of the control fetuses, although the distributions overlapped. Based on prior maternal risk, seven affected fetuses were expected in the terminated group; five had CAIII levels at or above the 95th centile of the control range. It is suggested that measurement of CAIII achieves partial discrimination between affected fetuses and their normal at-risk brethren.     

Isolation of fetal nucleated red blood cell from maternal blood using immunomagnetic beads for prenatal diagnosis

The immunomagnetic beads method for isolation of fetal nucleated red blood cells (FNRBCs) from peripheral blood of 78 pregnant women for prenatal diagnosis was developed. The study subjects were classified into 8-10 and 11-14 weeks of gestation (n = 39 each). Peripheral blood cells were divided into two for the FNRBCs isolation using two protocols, one with anti-CD45 depletion followed by anti-CD71 and anti-GPA monoclonal antibodies and another without CD45 depletion. The use of CD45 depletion gave a slightly higher number of sorted cells but not significantly different (p > 0.05). The percentage of CD71+ and GPA+ cells obtained from 8-10 weeks and 11-14 weeks of gestation was not different (p > 0.05). The sensitivity in determining the sorted FNRBCs for male fetal sex by PCR using 8-10 and 11-14 weeks of gestation was generally 50 and 69%, respectively. The method so developed is simple and cost effective and may thus be applied for prenatal diagnosis.     

产前超声在胎儿染色体异常筛查中的临床价值

目的探讨产前超声在胎儿染色体异常筛查中的临床价值。方法 2008.1～2011.3孕期在我中心行羊水及脐血穿刺3702例病例,诊断染色体核型异常221例,221例孕妇中至少产前在我院接受过1次超声检查者共37例,其中21-三体儿19例,18-三体儿5例,13-三体儿2例,45-XO儿11例。孕妇年龄21～40岁,接受超声检查平均孕周17＋～25＋周,对其超声声像图进行回顾性分析。结果本研究37例胎儿,除了8例21-三体儿超声表现未见异常外,另外29例超声均提示至少一个或一个人以上的染色体软指标或结构异常。结论不同类型的染色体异常有不同的结构畸形谱,了解并掌握不同类型染色体异常各自特定的畸形谱声像图,可以发挥产前超声在胎儿染色体异常筛查中的临床价值。     

Phosphorylation of 3'-azidothymidine in maternal and fetal peripheral blood mononuclear cells during gestation and at term

The present study compared the phosphorylation rate of 3'-azidothymidine (AZT) in isolated maternal and fetal peripheral blood mononuclear cells (PBMCs) with that in amniocytes obtained during gestation and at term. Maternal PBMCs were isolated from venous blood samples obtained from HIV-seronegative pregnant women during delivery. Immediately after delivery, cord blood specimens were collected, and fetal PBMCs were isolated. In a separate set of experiments, maternal and fetal PBMCs and amniocytes were obtained at 17-21 weeks of gestation. The fresh isolated PBMCs and amniocytes were maintained in RPMI 1640 medium until incubation with 10 microM tritiated AZT (10 microCi/mL). Thereafter, methanolic cell extracts were prepared for determination of AZT phosphates by high-performance liquid chromatography. Fetal PBMCs can efficiently convert AZT to its antivirally active metabolite. There were no significant differences after 6 or 12 hours of incubation with AZT between AZT phosphate levels in maternal and fetal PBMCs isolated at term or at 17-21 weeks of gestation: AZT monophosphate was found to be the major metabolite (about 95%). AZT phosphate levels in the amniocytes were up to sevenfold higher than those in the maternal or fetal PBMCs. These results show that during pregnancy and at term, fetal PBMCs-like maternal PBMCs-are able to take up AZT and to efficiently generate the active metabolite AZT triphosphate. These results are of major significance both in enabling efficient treatment of the fetuses of HIV-infected women and in the prediction and understanding of the efficacy and toxicity of AZT in pregnant women and their fetuses.