中国人RHD合子型研究和Rh蛋白复合体组分CD47功能研究

发布时间：2018-07-07 17:49

本文选题：RHD合子型 + Rhesus盒　；参考：《华东师范大学》2009年硕士论文

【摘要】： Rh血型系统是非常复杂的血型系统,在临床上的重要性仅次于ABO血型系统。D抗原是所有Rh血型抗原中,免疫原性最为强烈的。由D抗原所引起的输血反应及新生儿溶血症也较为常见。D抗原由RHD基因编码,RHD合子型测定是一项新兴技术,其对诊断、监护和防治Rh同种免疫及新生儿溶血病等有重要临床价值,2000年Wagner等建立了限制性片段长度多态性(RFLP)方法检测RHD基因合子型,而在大多数中国人群中,仅使用RFLP一种方法检测中国人RHD合子型,并以此推测RhD血清学表型可能会出现误判,我们从Rhesus盒子及RHD基因自身两个角度出发进行探索。本研究265例标本(93例RhD阳性、72例RhD阴性、84例Del、16例其它D变异型)均取自2006年9月至2007年12月期间上海市血液中心健康献血者及新疆地区兄弟血站的交流样品,其中除5名维吾尔族和1名回族献血者外,大部分为汉族。使用IgM、IgG单克隆抗一D试剂对样本进行RhD抗原和弱D测定后,用D表位分型试剂盒测定不完全D变异型,吸收放散实验检测Del表现型,TIANamp GenomicDNA kit提取试剂盒提取样本基因组DNA。通过3种方法对标本进行RHD合子型检测,包括PCR-RFLP、同时扩增RHD第1外显子和杂交Rhesus盒的双重PCR方法、同时扩增RHD第5和第7外显子的实时荧光定量PCR,结果显示PCR-RFLP和双重PCR的检测结果一致,而有9例标本(3例RhD阴性、2例Del和4例其它D变异型)与实时荧光定量PCR检测结果不一致,对这9例标本进行进一步的多重PCR和测序研究发现,这些标本的基因型分别为DVIⅢ/RHD-,RHD-CE(2-9)-D/RHD-,RHD1227A/RHD711 del C。本实验引入实时定量PCR技术,通过检测RHD基因外显子直接判断RHD合子型,既部分解决了以往各种方法对“无效RHD基因”的误判,又解决了一般PCR技术只能定性检测RHD基因外显子是否存在而无法判断其合子型的问题。结合RFLP检测Rhesus盒及定量PCR检测RHD基因两个不同的外显子这两种方法去判断RHD合子型是可行的。另外随机抽取20例不同RhD表形的标本(7例Rh阳性,7例Del,3例Rh阴性,3例其它D变异型)对其Rhesus盒进行测序分析,结果显示杂交Rhesus盒没有出现多态性,与国内外前期研究一致。而下游Rhesus盒则发现7个变异位点,4951G→A突变,5161T→C突变,5222G→A突变,5442G→A突变,5467 G→C突变,7403G→C突变,7443A→T突变。但由于测序标本数不多,未能涵盖中国人群中所有RhD表型,且7个变异位点分布没有规律,故并没有证据显示RhD表型同这些变异位点之间具有相关性。作为Rh蛋白复合体组分,CD47的功能研究近年来成为一个新的热点,红细胞膜上的CD47可作为红细胞的一个自身标记,通过与吞噬细胞上的SIRPα作用,向吞噬细胞提供了一个抑制吞噬的信号,阻止了吞噬细胞吞噬年轻正常的红细胞。因此推断CD47分子在调控血液循环中血细胞的寿命中具有重要意义。本研究随机抽取上海市血液中心18例献血者新鲜血样,取压积红细胞通过毛细管离心法分成年轻、中间和年老红细胞,采用流式细胞术分别检测不同年龄阶段红细胞上CD47的量。将分好的红细胞4℃保存在红细胞保养液中,实现红细胞体外老化,每周跟踪检测一次,持续五周共取得5组有效数据。对不同年龄段的红细胞采取跟踪监测的方式进行精确定量分析,结果显示虽然不同个体红细胞上CD47的表达量出现差异,但在红细胞逐渐老化的过程中无论是体内老化还是体外老化过程CD47表达量均出现下降的趋势,而下降百分比亦存在个体差异,总红细胞保存28天后其细胞膜表面CD47的量下降51%,年轻红细胞下降44%,年老红细胞下降46%。同次检测的年老红细胞同年轻红细胞相比其膜表面CD47的量平均下降40%。这一现象表明无论红细胞处于体内老化或者体外老化过程,其膜表面CD47的表达量均出现较大的下降趋势,证明在红细胞老化过程中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.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【参考文献】