稀有血型基因检测系统的建立及应用和i抗原、I抗原的相关研究

发布时间：2018-02-11 23:57

本文关键词： 血型抗原基因分型定点诱变多重聚合酶链式反应 i抗原 Ⅰ抗原 IGnTC基因　出处：《华东师范大学》2012年硕士论文　论文类型：学位论文

【摘要】：至今为止,在人类红细胞表面已经发现300多种可以遗传的血型抗原。其中,有些个体不表达其他人通常表达的血型抗原,如s、Dib、k抗原等,向这些稀有血型患者多次输注血型不匹配的血液,可产生同种免疫,严重的可危及生命。因此,有必要系统性地筛选供者和受血者的血型,以提供相匹配的血液。血清学是检测抗原抗体反应的最经典方法,但某些稀有抗血清要么短缺、要么效价不高,从而限制了其在这方面的大规模应用。随着分子生物学的不断发展,大部分血型抗原的分子基础均已阐明,多数血型抗原的多态性是由单个核苷酸替代所造成的,因此本文将研究重点转移至血型抗原的基因检测方面。本研究采用多重PCR技术对血型抗原进行基因分型,即在同一PCR体系中同时扩增数个血型等位基因片段,根据扩增条带的有无推断高频抗原是否缺失。同时,本研究利用基于PCR的基因定点诱变技术,成功制备了含有突变SNP位点的阴性质粒,以作为Dib、k、Jsb血型抗原基因扩增体系的阴性对照。使用制备的标准质粒与部分抗原缺失型基因组DNA验证我们所建立的多重PCR体系,确定其具有良好的重复性和稳定性后,对4190份随机献血者样本进行Dib、k、Jsb1910、Jsb2019基因分型,发现Dib、k、Jsb1910、Jsb2019等位基因在该组调查人群中的频率皆小于0.1%,共检出2例Di(b-)样本,使用血清学方法验证其红细胞膜上无Dib抗原表达。测序分析结果显示2例Di(b-)样本均为CT纯合突变,由此得出该组调查人群中由SLC4A1基因CT多态性而形成的Dia和Dib等位基因的频率分别为0.0219和0.9781。此外,我们通过流式细胞术与测序方法对其中1例标本进行了家系分析。流式结果与测序结果一致,先证者为Di(a+b-)纯合表型,父母均为Di(a+b+)杂合表型,符合孟德尔遗传定律。 i抗原和Ⅰ抗原的决定簇存在于糖脂和糖蛋白携带的碳水化合物结构上。i抗原是直链型的N-乙酰半乳糖胺重复单位,Ⅰ抗原为支链形式,由p-1,6-N-乙酰糖基转移酶进行支链化。i向Ⅰ的转变随红系分化进行,并与网织红细胞上转铁蛋白受体的出现及消失平行,故我们推测正常成人外周血红细胞i抗原主要表达在网织红细胞上。本文通过毛细管离心方法富集随机献血者新鲜外周血中富含网织红细胞的近心端以及远心端的年老红细胞,分别采用血清学方法和流式细胞术检测近远心端红细胞上i抗原的表达量。结果显示近远心端红细胞以及全血与抗-i抗体的凝集强度基本相同(凝集强度评分为3左右),流式结果也无显著性差异。因此我们初步证实了正常成人外周血红细胞i抗原的表达量与网织红细胞的多少无关。接着我们采用流式细胞术研究孕妇红细胞上i抗原的表达情况,发现孕妇红细胞上i抗原的表达量高于正常成人红细胞上的表达,平均高出47.56%,且随着孕周的增加,i抗原的表达量下降,孕28周比孕20周下降20.23%,孕36周比28周下降19.60%。Yu与Inaba等人报道人类Ⅰ基因座编码3种IGnT(β-1,6-N-acetylglucosaminyltransferase,IGnT)转录本,IGnTA、IGnTB、IGnTC,而红细胞Ⅰ支链的形成由IGnTC决定。我们通过磁珠分选方法分选20、28、36周孕妇的网织红细胞,抽提其RNA后进行逆转录,半定量分析IGnTC基因的表达量,以期探究i抗原与Ⅰ抗原在怀孕期间是否可能存在反向转变。结果显示孕20周、孕28周、孕36周时IGnTC/β-actin的净灰度比值分别为1.42、1.68、1.89,即在孕期20周以后,随孕期的增加,IGnTC基因的表达量逐渐增加。说明孕妇在怀孕期间,可能存在Ⅰ到i的反向转变,这些研究结果有利于我们进一步研究直链i向支链Ⅰ转变的信号通路以及一些存在Ⅰ到i反向转变的血液疾病的病因及病理机制。
[Abstract]:So far, on the surface of human red cell blood group antigens have been found 300 kinds of heritable. Among them, some individuals do not express antigens, other people are usually expressed as s, Dib, K antigen, to those patients with rare blood repeatedly transfused blood does not match, can produce the same immune, life-threatening serious. Therefore, it is necessary to systematic screening of donors and recipients of blood, to provide matching blood. Serum is the most classic method for detection of antigen antibody reaction, but the shortage of some rare antibodies either, or potency is not high, which limits its large-scale application in this area. With the continuous development of molecular biology, molecular basis of most antigens have been clarified, most of the blood group antigen polymorphisms are caused by a single nucleotide substitution, therefore this paper will study the gene detection focus to antigen Measurement.
This study used multiple techniques of PCR antigen genotyping, i.e. in the same PCR system at the same time a number of alleles amplified fragments according to the amplified bands are inferred high frequency antigen is missing. At the same time, the use of genetic mutagenesis technology based on PCR, was successfully prepared with negative plasmid SNP mutations, as Dib, K, Jsb negative blood type antigen gene amplification system control.
The use of standard plasmid and partial deletion antigen preparation of genomic DNA validation of multiplex PCR system we build, determine its good repeatability and stability, of 4190 random donor samples were Dib, K, Jsb1910, Jsb2019 genotype, Dib, K, Jsb1910, Jsb2019 allele frequency in the group of population is less than 0.1%, there were 2 cases of Di (b-) samples, using serological methods to verify the erythrocyte membrane without Dib antigen expression. Sequencing analysis showed that 2 cases of Di (b-) samples were homozygous for the CT mutation, the group in the survey by the SLC4A1 CT gene polymorphism and the formation of Dia and Dib allele frequencies were 0.0219 and 0.9781. respectively. In addition, we through flow cytometry and sequencing method in 1 specimens of pedigree analysis. Flow cytometry results and sequencing results, the proband was Di (a+b-) homozygous phenotype, parents The Di (a+b+) heterozygous phenotype conforms to the Mendel's law of inheritance.
Determinants of I antigen and 1 antigen carbohydrate structure present in glycolipid and glycoprotein carrying.I antigen is a repeat unit N- galactosamine acetyl chain type, 1 antigen as branched form of branched.I to 1 change with erythroid differentiation by p-1,6-N- acetyl glycosyltransferases, and reticulocytes on the appearance and disappearance of the transferrin receptor in parallel with the network, so we speculate that normal adult peripheral blood erythrocyte I antigen was mainly expressed in reticulocytes. Through capillary centrifugation enriched random donor proximal in reticulocyte cells in peripheral blood and fresh distal of old red blood cells. Respectively by flow cytometry and serological detection of near the distal end of the red blood cells expressing the I antigen. The results showed that nearly distal blood and red blood cells and anti -i antibody agglutination intensity is basically the same (agglutination strength assessment Divided into about 3), flow cytometry results have no significant difference. So we confirmed that normal adult peripheral blood cells expression of I antigen and the amount of reticulocyte.
Then we used flow cytometry to study I antigen on red blood cells of pregnant women, pregnant women found that red blood cells expressing the I antigen expression was higher than that of normal adult red blood cells, an average of 47.56%, and with the increase of gestational age, the expression of I antigen decreased at 28 weeks than 20 weeks of pregnancy decreased 20.23%, at 36 weeks than 28 weeks decreased by 19.60%.Yu and Inaba et al reported human gene encoding a 3 IGnT (beta -1,6-N-acetylglucosaminyltransferase, IGnT) transcripts, IGnTA, IGnTB, IGnTC, and the formation of red blood cells of the branched chain of IGnTC is made. We weave red blood cells by magnetic bead sorting sorting method of 20,28,36 weeks pregnant women, the extraction of RNA after reverse transcription, the expression level of IGnTC gene semi quantitative analysis, in order to explore the I antigen and antigen I might have a reverse change in pregnancy. The results showed that 20 weeks of pregnancy, 28 weeks pregnant, net ash at 36 weeks of gestation IGnTC/ beta -actin The ratio was 1.42,1.68,1.89, namely during pregnancy after 20 weeks, with the increase of pregnancy, the expression of IGnTC increased gradually. During pregnancy, the reverse may have changed to 1 I, the research results are helpful to the signaling pathway we further study the straight chain I to change and some branched I I the etiology and pathogenesis of I reverse transformation of blood diseases.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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