【摘要】：背景:Rh血型系統(tǒng)是所有血型系統(tǒng)中最復(fù)雜的血型系統(tǒng),包含了 50多種抗原,在胎母免疫和新生兒胎兒溶血病(HDFN)中,是最具臨床價值的血型系統(tǒng)。RHD、RHCE基因編碼決定了 Rh血型抗原。D--變異體是Rh血型系統(tǒng)中一種非常罕見的Rh基因變異型,其紅細(xì)胞膜上只有D抗原,而C、c、E、e均不能正常表達(dá)。本文對D--變異體綜合進(jìn)行RHD、RHAG、RHCE基因、血清學(xué)以及家系分析。目的:研究D--變異體的分子遺傳背景,以期能夠發(fā)現(xiàn)新的Rh血型變異體發(fā)生機(jī)制,同時初步探索了中國人RHAG血型系統(tǒng)中RHAG高頻抗原,以及RhD、RhCE抗原表達(dá)的影響因素。方法:1.研究對象實(shí)驗(yàn)樣本來源于甘肅省血液中心的1個D--個體樣本及家系成員(其弟弟,父親,母親),所有實(shí)驗(yàn)樣本均是經(jīng)過血液中心法定檢測項(xiàng)目的合格血液。2.實(shí)驗(yàn)方法(1)血清學(xué)Rh表型檢測:分別用鹽水試管法、微柱凝膠卡法和間接抗人球蛋白試驗(yàn)鑒定D--個體樣本及家系成員RhD、C、c、E和e抗原。(2)序列特異性PCR法(PCR-SSP)檢測RHCE基因:設(shè)計(jì)4對序列特異性引物,根據(jù)體系內(nèi)擴(kuò)增產(chǎn)物的有或無來判斷RHCE基因型。(3)測序:依據(jù)RHD、RHCE基因序列的特異性,分別擴(kuò)增這3個基因的10個外顯子,確認(rèn)產(chǎn)物條帶符合后將擴(kuò)增產(chǎn)物送公司純化測序,測序序列與標(biāo)準(zhǔn)基因序列進(jìn)行比對。(4)RHD合子型分析:采用雙管PCR方法擴(kuò)增融合Rh盒子和RHD基因第一外顯子,通過擴(kuò)增產(chǎn)物的條帶一次性判斷3種RHD合子型。(5)家系分析:通過RHCE基因測序結(jié)果結(jié)合RHD合子型分析繪制D--個體的家系譜圖。結(jié)果:1.血清學(xué)測定個例為弱D--表型;2.RHC 基因測定結(jié)果為DC-表型;3.RHD、RHAG、基因編碼區(qū)第1-10外顯子測序,結(jié)果與標(biāo)準(zhǔn)序列一致。RHAG基因未觀察到形成Oldeide和Duclos-Like抗原的堿基變異,亦未見形成高頻抗原Duclos的RHAG316CG突變,所有序列均未見雜合峰;4.RHCE基因編碼區(qū)第1-10外顯子測序,發(fā)現(xiàn)該個例第3-5外顯子缺失,其它外顯子正常,第1-2外顯子為RHC基因型,確定個體攜帶RHCE(e)-D(3-5)-CE(e)等位基因,因此血清學(xué)檢測為D--表型,而基因檢測為DC-表型;5.先證者父母和其弟的RHAG、RHD基因編碼區(qū)序列與先證者完全一致,父母Rh血清學(xué)表型分別為DCCee和DccEE,父親的PCR-SSP和測序結(jié)果一致,母親PCR-SSP結(jié)果DCcEE與測序結(jié)果一致,而與血清學(xué)結(jié)果不符(DccEE),弟弟血清學(xué)表型、PCR-SSP和測序結(jié)果均與先證者一致。結(jié)合RHD合子型分析結(jié)果,該家系父母分別為DCe/DC-和DcE/DC-基因型,先證者和兄弟為DC-/DC-。結(jié)論:1.RHCE(e)-D(3-5)-CE(e 等位基因形成D--表型并穩(wěn)定遺傳;2.RHAG Duclos高頻抗原在中國人群中需要觀察更多標(biāo)本才能認(rèn)可。3.本研究未發(fā)現(xiàn)RHAG和RHD基因編碼區(qū)變異。
[Abstract]:Background: the Rh blood group system is the most complex blood group system of all blood groups. It contains more than 50 antigens. It is the most clinically valuable blood group system in fetal immunity and neonatal fetal hemolytic disease (HDFN). RHD, RHCE gene encodes Rh blood group antigen. D- variant is a very rare variant of Rh gene in Rh blood group system. The RHD,RHAG,RHCE gene, serology and pedigree analysis of D-variant were studied. Objective: to study the molecular genetic background of D- variant in order to find out the new mechanism of Rh blood group variant, and to explore the high frequency antigen of RHAG and the factors influencing the expression of RhD,RhCE antigen in Chinese RHAG blood group system. Methods: 1. The experimental samples were collected from a D- individual sample and a family member (brother, father, mother) from the blood center of Gansu province. All the samples were qualified blood from the blood center. 2. Methods (1) serological Rh phenotype detection: D- individual samples and family members RhD,C,c, were identified by saline tube test, microcolumn gel calorie assay and indirect antihuman globulin test, respectively. E and e antigens. (2) sequence-specific PCR assay (PCR-SSP) for detection of RHCE genes: design 4 pairs of sequence-specific primers to determine RHCE genotypes according to the presence or absence of amplified products in the system. (3) sequencing: based on RHD, The specificity of RHCE gene sequence, 10 exons of the three genes were amplified, the products were confirmed to match the bands, and the amplified products were sent to the company for purification and sequencing. (4) RHD zygotypic analysis: the fusion Rh box and the first exon of RHD gene were amplified by double-tube PCR. (5) Family analysis: RHCE gene sequencing and RHD zygote analysis were used to draw the family pedigree of D- individuals. Results: 1. The results of serological analysis were weak D- phenotype, 2.RHC gene was DC- phenotype. 3. RHD-RHAG, exon 1-10 of gene coding region, was sequenced and the results were consistent with the standard sequence. The RHAG gene did not observe the base mutation to form Oldeide and Duclos-Like antigens, nor did RHAG316CG mutation to form high frequency antigen Duclos. No heterozygous peak was found in all sequences. The sequence of exon 1-10 in the coding region of 4.RHCE gene showed that the deletion of exon 3-5 was found in this case, the other exons were normal, and exon 1-2 was RHC genotype. RHCE (e) D (3-5)-CE (e) alleles were identified in individuals, so serological tests showed D- phenotypes, while genes were detected as DC- phenotypes. 5. The sequence of RHAG,RHD gene coding region of parent and younger brother of proband was identical with that of proband. The serological phenotype of parent Rh was the same as that of DCCee and DccEE, father, and the result of DCcEE from mother PCR-SSP was consistent with the result of sequencing. The results of PCR-SSP and sequencing were consistent with those of the proband. Combined with the results of RHD zygote analysis, the parents of the family were DCe/DC- and DcE/DC- genotype, and the proband and brother were DC-/DC-.. Conclusion: 1.RHCE (e) D (3-5)-CE (e alleles form D- phenotypes and be stably inherited, and 2.RHAG Duclos high frequency antigens need to be observed in more samples in order to be recognized. No variation in the coding region of RHAG and RHD genes was found in this study.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R457.11

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