本文選題：RHD合子型 + Rhesus盒　； 參考：《華東師范大學(xué)》2009年碩士論文

【摘要】： Rh血型系統(tǒng)是非常復(fù)雜的血型系統(tǒng),在臨床上的重要性僅次于ABO血型系統(tǒng)。D抗原是所有Rh血型抗原中,免疫原性最為強(qiáng)烈的。由D抗原所引起的輸血反應(yīng)及新生兒溶血癥也較為常見(jiàn)。D抗原由RHD基因編碼,RHD合子型測(cè)定是一項(xiàng)新興技術(shù),其對(duì)診斷、監(jiān)護(hù)和防治Rh同種免疫及新生兒溶血病等有重要臨床價(jià)值,2000年Wagner等建立了限制性片段長(zhǎng)度多態(tài)性(RFLP)方法檢測(cè)RHD基因合子型,而在大多數(shù)中國(guó)人群中,僅使用RFLP一種方法檢測(cè)中國(guó)人RHD合子型,并以此推測(cè)RhD血清學(xué)表型可能會(huì)出現(xiàn)誤判,我們從Rhesus盒子及RHD基因自身兩個(gè)角度出發(fā)進(jìn)行探索。 本研究265例標(biāo)本(93例RhD陽(yáng)性、72例RhD陰性、84例Del、16例其它D變異型)均取自2006年9月至2007年12月期間上海市血液中心健康獻(xiàn)血者及新疆地區(qū)兄弟血站的交流樣品,其中除5名維吾爾族和1名回族獻(xiàn)血者外,大部分為漢族。使用IgM、IgG單克隆抗一D試劑對(duì)樣本進(jìn)行RhD抗原和弱D測(cè)定后,用D表位分型試劑盒測(cè)定不完全D變異型,吸收放散實(shí)驗(yàn)檢測(cè)Del表現(xiàn)型,TIANamp GenomicDNA kit提取試劑盒提取樣本基因組DNA。 通過(guò)3種方法對(duì)標(biāo)本進(jìn)行RHD合子型檢測(cè),包括PCR-RFLP、同時(shí)擴(kuò)增RHD第1外顯子和雜交Rhesus盒的雙重PCR方法、同時(shí)擴(kuò)增RHD第5和第7外顯子的實(shí)時(shí)熒光定量PCR,結(jié)果顯示PCR-RFLP和雙重PCR的檢測(cè)結(jié)果一致,而有9例標(biāo)本(3例RhD陰性、2例Del和4例其它D變異型)與實(shí)時(shí)熒光定量PCR檢測(cè)結(jié)果不一致,對(duì)這9例標(biāo)本進(jìn)行進(jìn)一步的多重PCR和測(cè)序研究發(fā)現(xiàn),這些標(biāo)本的基因型分別為DVIⅢ/RHD-,RHD-CE(2-9)-D/RHD-,RHD1227A/RHD711 del C。本實(shí)驗(yàn)引入實(shí)時(shí)定量PCR技術(shù),通過(guò)檢測(cè)RHD基因外顯子直接判斷RHD合子型,既部分解決了以往各種方法對(duì)“無(wú)效RHD基因”的誤判,又解決了一般PCR技術(shù)只能定性檢測(cè)RHD基因外顯子是否存在而無(wú)法判斷其合子型的問(wèn)題。結(jié)合RFLP檢測(cè)Rhesus盒及定量PCR檢測(cè)RHD基因兩個(gè)不同的外顯子這兩種方法去判斷RHD合子型是可行的。 另外隨機(jī)抽取20例不同RhD表形的標(biāo)本(7例Rh陽(yáng)性,7例Del,3例Rh陰性,3例其它D變異型)對(duì)其Rhesus盒進(jìn)行測(cè)序分析,結(jié)果顯示雜交Rhesus盒沒(méi)有出現(xiàn)多態(tài)性,與國(guó)內(nèi)外前期研究一致。而下游Rhesus盒則發(fā)現(xiàn)7個(gè)變異位點(diǎn),4951G→A突變,5161T→C突變,5222G→A突變,5442G→A突變,5467 G→C突變,7403G→C突變,7443A→T突變。但由于測(cè)序標(biāo)本數(shù)不多,未能涵蓋中國(guó)人群中所有RhD表型,且7個(gè)變異位點(diǎn)分布沒(méi)有規(guī)律,故并沒(méi)有證據(jù)顯示RhD表型同這些變異位點(diǎn)之間具有相關(guān)性。 作為Rh蛋白復(fù)合體組分,CD47的功能研究近年來(lái)成為一個(gè)新的熱點(diǎn),紅細(xì)胞膜上的CD47可作為紅細(xì)胞的一個(gè)自身標(biāo)記,通過(guò)與吞噬細(xì)胞上的SIRPα作用,向吞噬細(xì)胞提供了一個(gè)抑制吞噬的信號(hào),阻止了吞噬細(xì)胞吞噬年輕正常的紅細(xì)胞。因此推斷CD47分子在調(diào)控血液循環(huán)中血細(xì)胞的壽命中具有重要意義。 本研究隨機(jī)抽取上海市血液中心18例獻(xiàn)血者新鮮血樣,取壓積紅細(xì)胞通過(guò)毛細(xì)管離心法分成年輕、中間和年老紅細(xì)胞,采用流式細(xì)胞術(shù)分別檢測(cè)不同年齡階段紅細(xì)胞上CD47的量。將分好的紅細(xì)胞4℃保存在紅細(xì)胞保養(yǎng)液中,實(shí)現(xiàn)紅細(xì)胞體外老化,每周跟蹤檢測(cè)一次,持續(xù)五周共取得5組有效數(shù)據(jù)。對(duì)不同年齡段的紅細(xì)胞采取跟蹤監(jiān)測(cè)的方式進(jìn)行精確定量分析,結(jié)果顯示雖然不同個(gè)體紅細(xì)胞上CD47的表達(dá)量出現(xiàn)差異,但在紅細(xì)胞逐漸老化的過(guò)程中無(wú)論是體內(nèi)老化還是體外老化過(guò)程CD47表達(dá)量均出現(xiàn)下降的趨勢(shì),而下降百分比亦存在個(gè)體差異,總紅細(xì)胞保存28天后其細(xì)胞膜表面CD47的量下降51%,年輕紅細(xì)胞下降44%,年老紅細(xì)胞下降46%。同次檢測(cè)的年老紅細(xì)胞同年輕紅細(xì)胞相比其膜表面CD47的量平均下降40%。這一現(xiàn)象表明無(wú)論紅細(xì)胞處于體內(nèi)老化或者體外老化過(guò)程,其膜表面CD47的表達(dá)量均出現(xiàn)較大的下降趨勢(shì),證明在紅細(xì)胞老化過(guò)程中CD47有可能起到重要的抑制吞噬作用。
[Abstract]:The Rh blood group system is a very complicated blood type system. The importance of the clinic is second only to the ABO blood type system.D antigen is all Rh blood group antigen, the immunogenicity is most intense. The blood transfusion reaction caused by the D antigen and the newborn hemolytic disease are also more common.D resistant to the RHD gene coding. The RHD zygote type determination is a new technology, which is a new technique. The diagnosis, monitoring and prevention of Rh homoimmunization and hemolytic disease of the newborn have important clinical value. In 2000, the restrictive fragment length polymorphism (RFLP) method was established to detect the RHD zygote type. In most Chinese people, only a RFLP method was used to detect the Chinese RHD zygote type, and the RhD serological phenotype was presumed to be possible. There will be misjudgement. We will explore from the two perspectives of Rhesus box and RHD gene itself.
In this study, 265 specimens (93 RhD positive, 72 RhD negative, 84 Del, and 16 other D variant) were collected from September 2006 to December 2007 in the blood center of the Shanghai blood center and the brotherhood blood stations in the Xinjiang region. Among them, the majority of the Han people except 5 Uygur and 1 Hui blood donors. The use of IgM, IgG monoclonal anti After a D reagent was used to determine the RhD antigen and weak D of the sample, the D epitope was used to determine the incomplete D variant, and the absorption and dispersion test was used to detect the Del phenotype. The TIANamp GenomicDNA kit extraction kit was used to extract the sample genomic DNA..
3 methods were used to detect RHD zygote type, including PCR-RFLP, double PCR method of RHD first exon and hybrid Rhesus box, and real-time quantitative PCR of RHD fifth and exon seventh. The results showed that the results of PCR-RFLP and double PCR were the same, and 9 specimens (3 RhD negative, 2 Del and 4 other D changes). It was not consistent with the results of real-time fluorescence quantitative PCR detection. Further multiple PCR and sequencing studies on these 9 specimens showed that the genotypes of these specimens were DVI III /RHD-, RHD-CE (2-9) -D/RHD-, RHD1227A/RHD711 del C. experiment introduced real-time quantitative PCR technology, and the RHD zygote type was directly judged by the detection of the RHD gene exons. Some of the previous methods have solved the misjudgment of the "invalid RHD gene" in the past. It also solves the problem that the general PCR technique can only determine whether the RHD exons exist and can not judge the zygote. The two methods of detecting the RHD zygote with the RFLP detection Rhesus box and the quantitative PCR for the two different exons of the RHD gene can be used to determine the RHD zygote type. Yes.
In addition, 20 specimens of different RhD forms (7 cases of Rh positive, 7 Del, 3 Rh negative, 3 other D variant) were sequenced and analyzed. The results showed that the hybrid Rhesus box did not appear polymorphism, which was consistent with the previous studies at home and abroad. The downstream Rhesus box found 7 variation sites, 4951G to A mutation, 5161T C mutation. A mutation, 5442G to A mutation, 5467 G to C mutation, 7403G to C mutation, 7443A to T mutation. But because the number of sequencing specimens is not much, it can not cover all the RhD phenotypes of the Chinese population, and the distribution of the 7 Variation sites is not regular, so there is no evidence that the RhD phenotype is associated with these mutational sites.
As a component of Rh protein complex, the functional study of CD47 has become a new hot spot in recent years. The CD47 on the erythrocyte membrane can be used as a marker of red cells. By the effect of SIRP alpha on the phagocyte, it provides a signal to inhibit phagocytosis and prevents phagocytosis from phagocytosis of young normal red cells. Breaking CD47 molecules is of great importance in regulating the lifespan of blood cells in blood circulation.
In this study, the blood samples of 18 blood donors in Shanghai blood center were randomly selected to be divided into young, intermediate and old red cells by capillary centrifugation. Flow cytometry was used to detect the amount of CD47 on red blood cells of different ages. The red blood cells were preserved in the red cell maintenance fluid to achieve the erythrocyte body at 4 degrees centigrade. After five weeks of five weeks, 5 groups of effective data were obtained. The results showed that there was a difference in the expression of red cells in different ages, but in the process of aging red blood cells in vivo and in vitro In the aging process, the expression of CD47 decreased, and the percentage of the decrease was also different. The amount of CD47 in the membrane surface of the cells decreased by 51% after 28 days of the total erythrocyte preservation, and the young red blood cells decreased by 44%. The average red blood cells of the old red blood cells decreased by 46%. in the same time compared with the young red blood cells on the average of 40%. on the surface of the membrane surface of the red blood cells. One phenomenon shows that the expression of CD47 in the membrane surface of the erythrocytes in the aging process in vivo and in vitro is greatly decreased, which shows that CD47 may play an important inhibition of phagocytosis in the process of red cell aging.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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相關(guān)期刊論文 前3條

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本文編號(hào)：2105755