孕妇外周血胎儿细胞中巨细胞病毒基因的检测及意义

目的利用孕妇外周血中胎儿细胞进行无创性产前诊断巨细胞病毒宫内感染的新方法。方法(1)采用显微操作技术从273例孕妇外血中分离单个胎儿有核红细胞。(2)应用多重引物原位合成技术(primed in situ labeling，PRINS)检测76例孕妇外周血HCMV-DNA阳性标本的单个胎儿细胞的SRY基因和HCMV-DNA基因。(3)应用引物延伸预扩增法(primed extension preamplification，PEP)及聚合酶链反应(PCR)检测273例孕妇外周血中单个胎儿细胞的SRY基因和HCMV-DNA基因。结果(1)应用显微操作技术分选胎儿细胞的分选率为100％。(2)PRINS技术检测SRY基因的敏感性为97．56％(40／41)，特异性为100％(35／35)。检测HCMV-DNA基因的敏感性为92．68％(38／41)，特异性为100％(35／35)。(3)PEP及PCR方法检测SRY基因的敏感性为97．39％(149／153)，特异性为99．17％(119／120)，检测HCMV-DNA基因的敏感性为95．12％(39／41)，特异性为100％(232／232)。结论应用PRINS技术、PEP方法及PCR技术对孕妇外周血中单个胎儿细胞进行非创伤性产前诊断巨细胞病毒宫内感染是一种敏感性高特异性强的新方法，可能具有广阔的临床应用前景。     

Changes in human umbilical vein endothelial cells induced by endothelial nitric oxide synthase traffic inducer

This study investigated the changes in human umbilical vein endothelial cells (HUVECs) induced by overexpression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) and its role in cellular injury. Recombinant NOSTRIN-expressing and empty vectors were transfected into cultured HUVECs, and factor Ⅷ-related antigen was examined by using immunohistochemical analysis. Growth curves were generated for both transfected and untransfected cells and these indicated that the proliferative ability of cells overexpressing NOSTRIN was significantly decreased. The expression of NOSTRIN and eNOS proteins was detected by using Western blot analysis, endothelial NOS (eNOS) activity was assayed by using spectrophotometry, and NO 2 /NO 3 levels were measured using nitrate reductase. Immunohistochemical analysis demonstrated that all groups expressed NOSTRIN in the plasma membrane and cytoplasm, and Western blot analysis confirmed that NOSTRIN levels were significantly higher in cells transfected with the NOSTRIN plasmid (P<0.01). The activity of eNOS and the levels of NO 2 /NO 3 were significantly decreased in NOSTRIN overexpressing cells as compared with empty vector and untransfected cells (P<0.01 and P<0.01, respectively). Morphological and ultrastructural changes were observed under light and electron microscopy, and it was found that NOSTRIN-overexpressing cells were elongated with deformities of the karyotheca, injury to the plasma membrane, increased lipids in the cytoplasm, and shortened microvilli. This study showed that overexpression of NOSTRIN had a significant effect on eNOS activity in HUVECs and resulted in significant cellular damage.     

三种特异性抗体结合流式细胞术分选母血中胎儿细胞方法的评价

研究证实母血中的游离胎儿细胞可用于无创性单基因病产前诊断。胎儿细胞仍是检测胎儿染色体异常的惟一来源，虽然母血中胎儿有核红细胞（FNRBC）数量很少，但其特异性形态在正常人外周血中极罕见，并可携带完整的胎儿基因。因此，建立富集母血中胎儿细胞的方法则是无创性产前诊断的关键。本研究用3种FNRBC特异性抗体和流式细胞术分选胎儿细胞，并通过计算FNRBC准确率和特异性，以评价分选母血中胎儿细胞的最适宜方法。     

川芎嗪抑制子痫前期脐血清诱导脐静脉内皮细胞中NOSTRIN表达上调的体外研究

目的:探讨子痫前期患者脐血清诱导脐静脉内皮细胞(HUVEC)中人内皮型一氧化氮合酶运输介导物(NOSTRIN)的表达及川芎嗪对其表达的调节作用。方法:采用消化培养法培养正常晚孕妇女的HUVEC,传代后待细胞长满至70%～80%后,加或不加川芎嗪作用30min后分别加入正常妊娠(正常组)及子痫前期(子痫前期组)脐静脉血清,培养48h后,MTT测定细胞活力,逆转录-聚合酶链反应(RT-PCR)测定NOSTRIN的表达。结果:子痫前期患者脐静脉血清培养的HUVEC,其细胞的活力(0.1318±0.031)明显低于正常组(0.1929±0.055),P<0.05,细胞中NOSTRIN mRNA的相对表达量(0.493±0.057)明显高于正常组(0.202±0.031),P<0.01,加川芎嗪预处理后,子痫前期组细胞的活力明显升高(0.1864±0.041),其细胞中NOSTRIN mRNA的相对表达量(0.273±0.048)明显下降(P<0.01)。结论:子痫前期患者脐静脉血清可诱导NOSTRIN表达增加,应用川芎嗪预处理后,可抑制NOSTRIN表达的增加,川芎嗪对子痫前期的防治有一定作用。     

CR16在特发性无精子症患者睾丸组织中的表达

目的:研究糖皮质激素区域表达基因16(corticosteroids and regional expression 16,CR16)在特发性无精子症患者睾丸组织中的表达,探讨CR16在精子发生中的作用。方法:采用免疫组化法和逆转录-聚合酶链反应(RT-PCR)检测CR16蛋白和CR16 mRNA在48例特发性无精子症患者(病例组)和10例正常生育男性(正常组)睾丸组织中的表达情况。结果:CR16蛋白表达于睾丸生精小管上皮,主要分布在支持细胞与生精细胞连接区,在特发性无精子症患者睾丸组织中的表达显著弱于正常生育男性睾丸组织中的表达。CR16 mRNA在病例组睾丸组织中的表达水平显著低于正常组。结论:特发性无精子症患者睾丸组织中CR16表达水平显著降低,这可能与无精子症相关。     

Endothelial nitric oxide synthase traffic inducer in the umbilical vessels of the patients with pre-eclampsia

The expression of endothelial nitric oxide synthase traffic inducer （NOSTRIN） was examined in the umbilical vessels of the patients with pre-eclampsia （PE） to explore its possible role in the pathogenesis of PE. The NOSTR1N rnRNA in umbilical tissues was determined by RT-PCR. The eNOS activity in umbilical vessels was spectrophotometrically detected. NO2 /NO3, the stable metabolic end products of NO, was measured by using nitrate reductase. RT-PCR showed that the expression level of NOSTRIN was significantly higher in women with PE than in the normal group （P〈0.01）. The activity of eNOS was significantly decreased in PE group [（12.83±3.61） U/mg] than in normal group [（21.72±3.83） U/mg] （P〈0.01）. The level of NO2-/NO3- in PE patients （27.53±7.48） pmol/mg was significantly lower than that of normal group （54.27±9.53） μmol/mg （P〈0.01）. The significant negative correlation existed between the expression of NOSTRIN and the activity of eNOS in umbilical vessels of women with PE （r=-0.58, P〈0.01）. It was concluded that the level of NOSTR1N expression was increased in umbilical vessel of women with PE, indicating that it may be involved in the pathogenesis of PE.     

内皮型一氧化氮合酶运输介导物在子痫前期患者胎盘组织中的定位及定量研究

目的探讨内皮型一氧化氮合酶运输介导物(endothelial n itric oxide synthase traffic inducer,NOSTR IN)在子痫前期(pre-ec lampsia,PE)患者胎盘血管内皮细胞中表达的变化及其在子痫前期发病过程中的作用。方法HE染色后镜下观察胎盘组织及血管的病理变化,免疫组织化学方法及W estern b lot检测子痫前期患者胎盘组织中NOSTR IN的表达。结果HE染色显示子痫前期患者胎盘绒毛血管变细,数目减少,血管合体膜增厚,纤维素样坏死明显多于正常妊娠;免疫组织化学显示正常妊娠和子痫前期患者胎盘血管内皮细胞中都有NOSTR IN的表达,但子痫前期患者胎盘血管内皮细胞胞浆染色较正常妊娠明显增强;W estern b lot显示子痫前期患者胎盘组织中NOSTR IN的表达显著高于正常妊娠(P<0.01)。结论胎盘组织中NOSTR IN表达增加可能是子痫前期发病机制的重要环节之一。     

胎盘异铁蛋白的表达异常与妊娠高血压综合征的关系

目的　通过比较正常孕妇与妊娠高血压综合征 (妊高征 )患者胎盘组织中胎盘异铁蛋白的表达 ,从分子免疫学角度探讨妊高征的病理机制。方法　用免疫组化染色法检测 30例妊高征胎盘组织 (妊高征组 )和 10例正常妊娠胎盘组织 (正常组 )中PLF的表达 ,通过高清晰度彩色病理图文分析系统对其定量分析。结果　PLF在中、重度妊高征胎盘组织中的表达明显低于轻度妊高征和正常组 ,两者间差异有非常显著意义 (P <0 .0 1)。结论　胎盘组织中PLF表达降低 ,导致母胎的免疫耐受的破坏 ,其异常的免疫反应可能是妊高征发病的重要机制。诱导PLF的产生或调节母胎的免疫耐受 ,将为临床治疗妊高征提供新的思路和方向。     

对孕妇外周血中胎儿细胞进行基因检测的三种方法比较

目的　建立用孕妇外周血中胎儿细胞进行基因分析的技术。方法　采用引物延伸预扩增(primed extension preamplification,PEP)法加 PCR、多重引物原位合成技术 (primed in situ labeling,PRINS)和巢式聚合酶链反应 (nested PCR)检测孕妇外周血中单个胎儿细胞的性别决定区 Y基因 (sex- de-termination region Y gene,SRY)基因片段。结果　 PEP加 PCR方法检测 SRY基因的敏感性为 97.39%(14 9/ 15 3) ,特异性为 99.17% (119/ 12 0 )。PRINS技术检测 SRY基因的敏感性为 97.5 6 % (40 / 4 1) ,特异性为10 0 % (35 / 35 )。巢式 PCR检测 SRY基因的敏感性为 80 .0 0 % (2 4 / 30 ) ,特异性为 87.5 0 % (14 / 16 )。结论　PEP加 PCR技术和 PRINS技术是对孕妇外周血中单个胎儿细胞进行基因诊断的可靠技术 ,其敏感性高、特异性强 ,有望成为利用孕妇外周血中单个胎儿细胞进行非创伤产前诊断的常规方法。     

快速无创性产前诊断唐氏综合征的初步研究

目的建立快速无创性产前诊断唐氏综合征的有效诊断模式。方法从98名孕妇中筛选出12名高度怀疑唐氏妊娠者,当日流式细胞术分选母血中的胎儿有核红细胞（Fetal nucleated red blood cells,FNRBCs）,制备滴片后,次日进行多重引物原位杂交（muti—primed in situ labeling,multi—PRINS）检测胎儿细胞21号、Y染色体。结果均可在两日内得到诊断结论,诊断10例正常胎儿和两例男性唐氏综合征胎儿,与实际胎儿核型完全一致。结论流式分选母血中胎儿有核红细胞后多重PRINS技术检测细胞染色体可作为快速无创性产前诊断的有效模式,并可为其他非整倍体异常和单基因病的快速无创性产前诊断提供有效参考。