本文關(guān)鍵詞： 血型抗原 基因分型 定點誘變 多重聚合酶鏈?zhǔn)椒磻?yīng) i抗原 Ⅰ抗原 IGnTC基因　出處：《華東師范大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：至今為止,在人類紅細(xì)胞表面已經(jīng)發(fā)現(xiàn)300多種可以遺傳的血型抗原。其中,有些個體不表達(dá)其他人通常表達(dá)的血型抗原,如s、Dib、k抗原等,向這些稀有血型患者多次輸注血型不匹配的血液,可產(chǎn)生同種免疫,嚴(yán)重的可危及生命。因此,有必要系統(tǒng)性地篩選供者和受血者的血型,以提供相匹配的血液。血清學(xué)是檢測抗原抗體反應(yīng)的最經(jīng)典方法,但某些稀有抗血清要么短缺、要么效價不高,從而限制了其在這方面的大規(guī)模應(yīng)用。隨著分子生物學(xué)的不斷發(fā)展,大部分血型抗原的分子基礎(chǔ)均已闡明,多數(shù)血型抗原的多態(tài)性是由單個核苷酸替代所造成的,因此本文將研究重點轉(zhuǎn)移至血型抗原的基因檢測方面。 本研究采用多重PCR技術(shù)對血型抗原進(jìn)行基因分型,即在同一PCR體系中同時擴(kuò)增數(shù)個血型等位基因片段,根據(jù)擴(kuò)增條帶的有無推斷高頻抗原是否缺失。同時,本研究利用基于PCR的基因定點誘變技術(shù),成功制備了含有突變SNP位點的陰性質(zhì)粒,以作為Dib、k、Jsb血型抗原基因擴(kuò)增體系的陰性對照。 使用制備的標(biāo)準(zhǔn)質(zhì)粒與部分抗原缺失型基因組DNA驗證我們所建立的多重PCR體系,確定其具有良好的重復(fù)性和穩(wěn)定性后,對4190份隨機(jī)獻(xiàn)血者樣本進(jìn)行Dib、k、Jsb1910、Jsb2019基因分型,發(fā)現(xiàn)Dib、k、Jsb1910、Jsb2019等位基因在該組調(diào)查人群中的頻率皆小于0.1%,共檢出2例Di(b-)樣本,使用血清學(xué)方法驗證其紅細(xì)胞膜上無Dib抗原表達(dá)。測序分析結(jié)果顯示2例Di(b-)樣本均為CT純合突變,由此得出該組調(diào)查人群中由SLC4A1基因CT多態(tài)性而形成的Dia和Dib等位基因的頻率分別為0.0219和0.9781。此外,我們通過流式細(xì)胞術(shù)與測序方法對其中1例標(biāo)本進(jìn)行了家系分析。流式結(jié)果與測序結(jié)果一致,先證者為Di(a+b-)純合表型,父母均為Di(a+b+)雜合表型,符合孟德爾遺傳定律。 i抗原和Ⅰ抗原的決定簇存在于糖脂和糖蛋白攜帶的碳水化合物結(jié)構(gòu)上。i抗原是直鏈型的N-乙酰半乳糖胺重復(fù)單位,Ⅰ抗原為支鏈形式,由p-1,6-N-乙酰糖基轉(zhuǎn)移酶進(jìn)行支鏈化。i向Ⅰ的轉(zhuǎn)變隨紅系分化進(jìn)行,并與網(wǎng)織紅細(xì)胞上轉(zhuǎn)鐵蛋白受體的出現(xiàn)及消失平行,故我們推測正常成人外周血紅細(xì)胞i抗原主要表達(dá)在網(wǎng)織紅細(xì)胞上。本文通過毛細(xì)管離心方法富集隨機(jī)獻(xiàn)血者新鮮外周血中富含網(wǎng)織紅細(xì)胞的近心端以及遠(yuǎn)心端的年老紅細(xì)胞,分別采用血清學(xué)方法和流式細(xì)胞術(shù)檢測近遠(yuǎn)心端紅細(xì)胞上i抗原的表達(dá)量。結(jié)果顯示近遠(yuǎn)心端紅細(xì)胞以及全血與抗-i抗體的凝集強(qiáng)度基本相同(凝集強(qiáng)度評分為3左右),流式結(jié)果也無顯著性差異。因此我們初步證實了正常成人外周血紅細(xì)胞i抗原的表達(dá)量與網(wǎng)織紅細(xì)胞的多少無關(guān)。 接著我們采用流式細(xì)胞術(shù)研究孕婦紅細(xì)胞上i抗原的表達(dá)情況,發(fā)現(xiàn)孕婦紅細(xì)胞上i抗原的表達(dá)量高于正常成人紅細(xì)胞上的表達(dá),平均高出47.56%,且隨著孕周的增加,i抗原的表達(dá)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與Inaba等人報道人類Ⅰ基因座編碼3種IGnT(β-1,6-N-acetylglucosaminyltransferase,IGnT)轉(zhuǎn)錄本,IGnTA、IGnTB、IGnTC,而紅細(xì)胞Ⅰ支鏈的形成由IGnTC決定。我們通過磁珠分選方法分選20、28、36周孕婦的網(wǎng)織紅細(xì)胞,抽提其RNA后進(jìn)行逆轉(zhuǎn)錄,半定量分析IGnTC基因的表達(dá)量,以期探究i抗原與Ⅰ抗原在懷孕期間是否可能存在反向轉(zhuǎn)變。結(jié)果顯示孕20周、孕28周、孕36周時IGnTC/β-actin的凈灰度比值分別為1.42、1.68、1.89,即在孕期20周以后,隨孕期的增加,IGnTC基因的表達(dá)量逐漸增加。說明孕婦在懷孕期間,可能存在Ⅰ到i的反向轉(zhuǎn)變,這些研究結(jié)果有利于我們進(jìn)一步研究直鏈i向支鏈Ⅰ轉(zhuǎn)變的信號通路以及一些存在Ⅰ到i反向轉(zhuǎn)變的血液疾病的病因及病理機(jī)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.

【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.1

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本文編號：1504308