微环境的改变对富集有核红细胞影响的实验研究

发布时间：2018-10-11 08:04

【摘要】： 目的 1．对脐血中有核红细胞性状进行研究，探讨从孕妇外周血中富集胎儿有核红细胞的有效方法，为进行无创性产前诊断提供科学依据。 2．探讨利用孕妇尿液中游离DNA进行性连锁遗传病产前诊断的可行性研究。方法 1．对脐血进行液体、半固体培养，以期获得脐血中胎儿有核红细胞(Fetal Nuclear Red Blood Cells，FNRBC)进行细胞基本性状研究；用聚偏二氟乙烯(Polyvinylidene Difluoride，PVDF)膜对淋巴细胞、单核细胞、有核红细胞进行分离；脐血单个核细胞微环境改变对PVDF膜富集胎儿有核红细胞效率的影响。 2．将42例孕妇尿液进行离心富集后提取游离DNA作为模板，以Y染色体短臂上的特异序列(SRY基因)为扩增对象，用套式聚合酶链反应(Nested-PCR)技术对其进行扩增，产物经琼脂糖凝胶电泳分析。结果 1．联苯胺染色和K-B染色法都能够将NRBC同其他单个核细胞区分开来，而K-B染色可以进一步将NRBC和FNRBC进行区分；71份样本中NRBC的占有比例不均，范围在1％～54％，平均在8％左右；脐血的液体培养经流式细胞仪检测，得率最大为20％，细胞培养效果有待探讨。 2．在检测的42例孕妇尿液中32例孕妇尿液中检出SRY基因片段，与其相对应的血浆及血细胞亦检出SRY基因片段。10例未检出SRY基因片段，，与其相对应的血浆及血细胞也未检出SRY基因片段。结论 1．聚偏二氟乙烯膜可用来分离脐血中淋巴细胞、单核细胞、有核红细胞、红细胞。脐血中单个核细胞微环境的改变可以提高有核红细胞通过聚偏二氟乙烯膜的效率， 2．孕妇尿液中确实存在胎儿游离DNA，经富集提取DNA，通过套式PCR法进行胎儿性别判定，为非侵入性早期产前诊断性连锁遗传病及其它疾病奠定了基础。
[Abstract]:Objective 1. To study the characteristics of nucleated red blood cells in umbilical cord blood, and to explore an effective method for enriching fetal nucleated red blood cells from maternal peripheral blood. To provide scientific basis for noninvasive prenatal diagnosis. 2. To explore the feasibility of prenatal diagnosis of progressive DNA in pregnant women's urine. Methods 1. Umbilical cord blood was cultured in liquid and semisolid medium to obtain fetal nucleated red blood cell (Fetal Nuclear Red Blood Cells,FNRBC). Lymphocytes, monocytes and nucleated erythrocytes were separated by polyvinylidene fluoride (Polyvinylidene Difluoride,PVDF) membrane. Effects of changes in microenvironment of umbilical cord blood mononuclear cells on the efficiency of PVDF membrane enrichment of fetal nucleated red blood cells. 2. Free DNA was extracted from urine of 42 pregnant women after centrifugation. The specific sequence on the short arm of Y chromosome (SRY gene) was amplified by nested polymerase chain reaction (Nested-PCR). The product was analyzed by agarose gel electrophoresis. Results 1. Both benzidine staining and K-B staining could distinguish NRBC from other mononuclear cells, but K-B staining could further distinguish NRBC from FNRBC. The proportion of NRBC in 71 samples was uneven, ranging from 1 to 54, with an average of about 8%. The effect of cell culture needs to be explored. 2. SRY gene fragment was detected in urine of 32 pregnant women out of 42 pregnant women. SRY gene fragments were also detected in plasma and blood cells, and SRY gene fragments were not detected in 10 cases and SRY gene fragments were not detected in corresponding plasma and blood cells. Poly (vinylidene fluoride) membrane can be used to isolate lymphocytes, monocytes, nucleated erythrocytes and erythrocytes from cord blood. The change of microenvironment of mononuclear cells in umbilical cord blood can improve the efficiency of nucleated erythrocytes passing through polyvinylidene fluoride (PVDF) membrane. 2. Fetal migration does exist in pregnant women's urine. Extracted DNA, from isolated DNA, by nested PCR method was used to determine the sex of fetus. It lays a foundation for non-invasive early prenatal diagnosis of genetic linkage disease and other diseases.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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