【摘要】： 目的：RhD--缺失型是Rh血型系統(tǒng)一種極為罕見的變異體,血清學(xué)特征表現(xiàn)為紅細胞上RhC／c、E／e抗原的完全缺乏和(或)D抗原的過量表達。目前RhD--缺失型的分子生物學(xué)機制尚不明確,國外的研究發(fā)現(xiàn)了兩種情況：一種是RHCE基因完整但無表達活性,另一種是RHCE基因部分缺失。前者考慮是由于mRNA的轉(zhuǎn)錄活性降低所致,后者考慮是外顯子部分缺失和基因重組兩種機制。國內(nèi)還沒有相關(guān)報道。由于同一種血型表型,可以對應(yīng)多種基因型,而且在不同種族和同一種族的不同群體中,同一種表型所對應(yīng)的基因型也不盡相同。Rh血型系統(tǒng)的多態(tài)性一直是阻礙基因定型技術(shù)普及的障礙。目前有關(guān)血型基因結(jié)構(gòu)的資料,大多數(shù)來源于白人,不符合我國的種族群體特點。中國人群民族多,數(shù)量大,遺傳雜合性大。本研究通過對兩例確證為RhD--個體RH基因結(jié)構(gòu)和表達的深入研究,明確中國部分漢族人群的RhD--缺失型個體可能存在的新的發(fā)生機制。這對搞清中華民族的血型基因結(jié)構(gòu),廣泛開展血型基因分型技術(shù)是十分必要的,還可能發(fā)現(xiàn)新的RH等位基因。 方法：(1)從正常人外周血中網(wǎng)織紅細胞中提取總RNA逆轉(zhuǎn)錄RHD基因；(2)構(gòu)建RHD基因克隆載體,轉(zhuǎn)染大腸桿菌；(3)地高辛標記PCR法擴增RHcDNA特異性三段探針和Exon1特異性探針,對兩例RhD--個體采用HindⅢ和SphⅠ限制性內(nèi)切酶酶切血液基因組DNA并與特異性探針進行Southern blot,并采用PCR-SSP方法擴增兩例RhD--個體RHCE基因多個外顯子和內(nèi)含子；(4)采用RT-PCR方法檢測RhD--個體是否有RHCE剪接體轉(zhuǎn)錄。 結(jié)果：(1)獲得RHD基因；(2)構(gòu)建RHD基因克隆成功；(3)Southern blot發(fā)現(xiàn)兩例RhD一個體RHCE基因均存在exonl,但存在中間段缺失(exon4-exon7缺失),進一步采用PCR-SSP方法特異性擴增RHCE基因外顯子及內(nèi)含子,證實RhD--個體RHCE intro、exon4-7基因片段丟失,但exon3、exon9及3’端基因序列均存在。(4)未檢測到兩RhD--個體RHCE基因轉(zhuǎn)錄產(chǎn)物表達。 結(jié)論：(1)中國部分漢族人群的RhD--缺失型個體的產(chǎn)生原因是RHCE基因exon4-7缺失,其截短的RHCE基因不表達相關(guān)活性產(chǎn)物,這與國外學(xué)者對白人、黑人RhD--缺失型個體發(fā)生的分子機制研究結(jié)果不同。
[Abstract]:Objective: the Rh D- deletion is an extremely rare variant of the Rh blood group system. The serological features are the complete absence of RhC / cr / e antigen and / or the over-expression of D antigen on the erythrocytes. At present, the molecular biological mechanism of RhD- deletion type is still unclear. Two situations have been found in foreign studies: one is complete RHCE gene but no expression activity, the other is partial deletion of RHCE gene. The former is due to the decrease of transcriptional activity of mRNA, while the latter is due to two mechanisms: partial deletion of exon and gene recombination. There are no reports at home. Because of the same blood type phenotype, it can correspond to multiple genotypes, and in different groups of different races and the same race, The polymorphism of the Rh blood group system is an obstacle to the popularization of gene typing technology. Most of the information on genetic structure of blood group is white, which does not accord with the ethnic group characteristics of our country. There are many ethnic groups, large numbers and large genetic heterozygosity in Chinese population. In this study, we studied the structure and expression of RH gene in two RhD- individuals confirmed as RhD- individuals, and clarified the possible new mechanism of RhD- deletion type in some Han population in China. It is necessary to understand the blood group gene structure of the Chinese nation and widely develop the blood group genotyping technique, and a new RH allele may also be discovered. Methods: (1) Total RNA reverse transcriptional RHD gene was extracted from reticulocyte of normal human peripheral blood; (2) RHD gene clone vector was constructed and transfected into Escherichia coli; (3) digoxin labeled PCR was used to amplify RHcDNA specific three-segment probes and Exon1 specific probes. Two RhD- individuals were digested with Hind 鈪，

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