免疫相关血细胞减少症患者外周血淋巴细胞线粒体DNA D-环区多态性研究
发布时间：2018-09-11 09:45
【摘要】：目的:第一部分:在血液系统的疾病中,我们常常最需要诊断的一类为全血细胞减少症,而其中免疫相关血细胞减少症(IRP)作为以骨髓衰竭为表现的疾病体系被细分出来,近年来研究表明其发病机制是由于机体受到某种未知病原的刺激后,树突细胞亚群作为主要的呈递抗原率先出现异常,随后传递至T淋巴细胞,再导致下游的B淋巴细胞在功能、数量及亚群等各方面出现不同程度的异常,从而形成自身抗体,而这类自身抗体可以专门针对骨髓的造血细胞,它们可以通过封闭造血细胞膜上的功能蛋白、或通过激活补体原位溶血、亦或可通过介导巨噬细胞吞噬等多种途径导致骨髓的造血细胞增殖分化被抑制,最终导致机体无效造血,骨髓出现衰竭[35]。所以尽快的正确诊断,将IRP与其他不同种类、不同治疗方案的全血细胞减少症鉴别出来至关重要。近年来大量的实验研究都集中在细胞核内的基因组遗传物质与血液疾病的是否存在相关性,本试验检测的线粒体DNA中的D-环区着眼于细胞核外DNA的突变与免疫性血液系统疾病是否同样存在相关性,立志于为疾病的临床诊断与发病机制提供新的思路。线粒体作为高等动物细胞内除细胞核外的唯一含有DNA的细胞器,且不依赖基因组进行复制、转录及翻译。尽管如此,但是当线粒体DNA长期在氧化磷酸化中裸露时却极其容易被周围的氧自由基等物质所损伤,这主要是由于其缺乏有效的抗氧化修复机制,大量研究表明其同样缺乏与组蛋白的有效结合,因此无法获得组蛋白保护,导致线粒体DNA的与细胞核DNA相比,远高于10倍以上。目前大量研究也表明,线粒体DNA的损伤氧化与肿瘤的发病风险、疾病的预后、人类糖尿病等慢性疾病以及人类衰老等多方面存在相关性。此外,线粒体DNA的变异还会影响线粒体呼吸链功能的障碍从而导致机体多种免疫相关性疾病发生。其中的D-环区位于线粒体DNA的t RNApro与t RNAphe之间(nt16024-nt575)总长度约为1120bp左右,为线粒体DNA主要的非编码区域,是线粒体DNA内最大的遗传变异区域,D-环区还是线粒体DNA复制与转录的调控区,以及线粒体DNA的轻链与重链的开始位点也均在D-环区,因此近年来对该区域的探索成为对线粒体DNA研究的重点区域。单核苷酸多态性(SNP),研究认为其在DNA序列的变异中是极为常见的,在高等动物的基因组内变异频率通常可㧐1%,它主要是由于DNA序列上单个核甘酸的变化导致的基因组序列的多态性,比例在已知的所有多态性中占有90%以上。因此SNP被认为与物种的多样性、与药物治疗个体差异、与某些疾病的易感性均存在相关因素。由于人类的基因组是存在连锁不平衡的,所以,尽管大多数的SNP不会直接的影响到对基因产物的表达,但是其依旧会被大多数学者当成遗传标记的一个类别来深入研究,当某些可以导致疾病的基因其在功能上或在结构上产生变化时,我能仍可以将SNP用于定位这些临近的基因或基因组。如果SNP恰好位于某些基因的启动子中,其功能起着对该基因进行编码及调控时,那么此时的SNP就有可能会对此基因造成影响。而本次将要研究的D-环区域线粒体DNA复制的控制区、线粒体DNA的轻链及重链的复制起始点以及承担调控线粒体DNA转录和复制的重要区域,其遗传上的高度多态性势必会对线粒体产生一定的影响。因此我们进行了对免疫相关性血细胞减少症(IRP)患者外周血淋巴细胞线粒体DNA(mtDNA)D-环区多态性的意义、与免疫指标相关性以及与患者骨髓及外周血细胞形态学特征相关性的研究。第二部分:幼淋巴细胞白血病(prolymphocytic leukemia,PLL)为一种特殊类型的淋巴细胞白血病,一般认为属罕见的慢性淋巴细胞白血病的变异型,但也有人认为是急淋的一种(Ⅱ型)。Auer小体是急性髓系白血病一个具有诊断意义的形态学特征,其在急性髓系白血病的检出、分布情况及与疗效的关系有诊断性的临床意义。而我们此次探讨的是在幼淋巴细胞白血病细胞的胞浆中出现了形态学类似Auer小体的Auer小体样内含物的特征。方法:第一部分:1.提取43例初治IRP患者及41例正常对照者外周血淋巴细胞的线粒体DNA,对所有样本的D-环区域采用PCR扩增的方法,并通过正向及反向直接测序对其D-环区域的PCR扩增产物基因序列进行检测,将检测出的结果与人线粒体DNA文库记录的剑桥序列以及mt DB数据库的Polymorphic Sites子数据库相关数据库进行对比。2.利用流式细胞术(FACS)检测43例初治IRP患者的骨髓造血细胞CD15+、Gly Co A+细胞以及CD34+细胞膜表面抗体的阳性率(Ig G与Ig M)3.利用流式细胞术(FACS)检测43例初治IRP患者的外周血B淋巴细胞及T淋巴细胞亚群阳性率4.利用瑞氏染色法对43例初治IRP患者的骨髓涂片,计数200个骨髓细胞,分别记录其骨髓的小粒、油滴、增生程度、粒红系比例及病态造血情况、淋巴细胞比例、巨核细胞数量以及血小板等5.利用利用瑞氏染色法对43例初治IRP患者的外周血涂片,计数100个白细胞,分别记录其粒细胞比例、成熟红细胞、淋巴细胞比例、单核细胞比例以及血小板等第二部分:用显微镜观察骨髓细胞形态学、细胞化学染色的特点,用流式细胞术检测骨髓细胞的免疫表型,用电镜扫描分析其成份。结果:1.43例IRP患者的外周血淋巴细胞线粒体DNA D-环区中共有110个变异位点,其中62个为多态位点(SNP),48个为突变位点,其中14个位点为数据库中未提及的新突变位点。2.110个变异位点中共发现516个碱基改变,最主要改变是410个碱基置换,常见的碱基置换为T/C(184/410)和A/G(113/410)3.110个变异位点中高频变异位点73和263,其变异率为43/43,位点311的变异率为32/43,位点310和16224的变异率为27/43,16519的变异率为25/43,位点489和16362为24/43。4.IRP患者外周血淋巴细胞线粒体DNA D-环区多态性与该类患者骨髓单个核细胞的相关性统计中发现,以年龄18岁为界的35例成年组中其淋巴细胞线粒体DNA D-环区多态性与患者骨髓单个核细胞的CD15Ig M、Gly Co A+细胞Ig M、CD34+细胞Ig G、CD34+细胞Ig M存在显著正相关性。5.骨髓细胞形态学提示为幼淋巴细胞白血病,部分胞浆中见有Auer小体样内含物,细胞化学染色及流式细胞术分析亦为B淋巴细胞,电镜扫描其为基质密度增高,内部结构消失的变性线粒体。结论:1.IRP患者外周血淋巴细胞的线粒体DNA D-环区存在高频突变。2.最主要的碱基改变类型为T/C以及A/G3.高频突变位点分别为73、263、311、310、16224、16519、489以及163624.且在成年组(年龄≥18岁)中与骨髓单个核细胞膜结合自身抗体显著正相关,可能是免疫相关性血细胞减少症发病机制中的重要环节。5.在淋巴系统的肿瘤细胞胞浆中变性的线粒体经瑞氏染色后可呈现Auer小体样内含物。Auer小体在形态学上是非常有价值的发现,但不能仅通过形态学观察,还应结合其他相关检查进一步对白血病细胞的类别分型。
[Abstract]:OBJECTIVE: PART I: In hematological diseases, pancytopenia is the most frequently diagnosed type, in which immune-associated hematocytopenia (IRP) is subdivided as a disease system characterized by bone marrow failure. Recent studies have shown that the pathogenesis of IRP is stimulated by an unknown pathogen. After that, dendritic cell subsets as the main presenting antigens first appear abnormal, then transmit to T lymphocytes, and then lead to the downstream B lymphocytes in the function, number and subsets of abnormalities in varying degrees, thus forming autoantibodies, which can be specifically targeted at bone marrow hematopoietic cells, they can pass through Blockade of functional proteins on hematopoietic cell membranes, activation of complement in situ hemolysis, or mediation of macrophage phagocytosis may lead to inhibition of bone marrow hematopoietic cell proliferation and differentiation, resulting in invalid hematopoiesis and bone marrow failure [35]. In recent years, a large number of experimental studies have focused on whether there is a correlation between genomic genetic material in the nucleus and blood diseases. The D-loop region of mitochondrial DNA detected in this study focuses on whether mutations in extranuclear DNA and immune hematological diseases exist as well. Mitochondria are the only DNA-containing organelles in the cells of higher animals other than the nucleus and are not dependent on the genome for replication, transcription and translation. Oxygen free radicals and other substances around the damage, mainly due to its lack of effective antioxidant repair mechanism, a large number of studies have shown that it is also lack of effective binding with histone, so it can not obtain histone protection, resulting in mitochondrial DNA and nuclear DNA compared to much more than 10 times. In addition, the variation of mitochondrial DNA also affects the function of mitochondrial respiratory chain and leads to the occurrence of many immune-related diseases. The D-ring region of mitochondrial DNA is located in the t RN of mitochondrial DNA. The total length between Apro and t RNAphe (nt16024-nt575) is about 1120 bp, which is the main non-coding region of mitochondrial DNA, the largest genetic variation region in mitochondrial DNA, the D-loop region or the regulatory region of mitochondrial DNA replication and transcription, as well as the starting sites of light and heavy strands of mitochondrial DNA in recent years. Single nucleotide polymorphism (SNP) is considered to be very common in the variation of DNA sequences. The frequency of variation in the genome of higher animals is usually? 1%. It is mainly due to the polymorphism of the genome sequence caused by the change of single nucleotide glycine in the DNA sequence. SNP is considered to be associated with species diversity, individual differences in drug treatment, and susceptibility to certain diseases. Since the human genome is linked to disequilibrium, most SNPs do not directly affect the expression of gene products, but their dependence. I can still use SNPs to locate adjacent genes or genomes when certain genes that cause disease change functionally or structurally. If SNPs happen to be located in the promoters of certain genes, their function plays a role in that gene. The high genetic polymorphisms in the D-loop region, the starting point for the replication of light and heavy strands of mitochondrial DNA, and the important regions responsible for the regulation of mitochondrial DNA transcription and replication are bound to occur. Therefore, we have studied the significance of D-loop polymorphism of peripheral blood lymphocyte mitochondrial DNA (mtDNA) in patients with immune-associated hematopenia (IRP), the correlation between D-loop polymorphism and immune indices, and the correlation between D-loop polymorphism and morphological characteristics of bone marrow and peripheral blood cells. Prolymphocytic leukemia (PLL) is a special type of lymphocytic leukemia. It is generally considered to be a rare variant of chronic lymphocytic leukemia, but it is also considered to be a type (type II) of acute lymphocytic leukemia. Auer body is a diagnostic morphological feature of acute myeloid leukemia, which is found in acute myeloid leukemia. Detection, distribution and relationship with therapeutic effect have diagnostic clinical significance. We are exploring the characteristics of Auer body-like inclusions in the cytoplasm of immature lymphoblastic leukemia cells. The mitochondrial DNA of the cells was amplified by PCR in the D-loop region of all samples. The PCR product gene sequences of the D-loop region were detected by direct forward and reverse sequencing. The results were related to the Cambridge sequence recorded by the human mitochondrial DNA library and the Polymorphic Sites subdatabase of the MT DB database. Flow cytometry (FACS) was used to detect the positive rates of CD15 +, Gly Co A + cells and CD34 + cell membrane surface antibodies (Ig G and Ig M) of bone marrow hematopoietic cells in 43 patients with newly diagnosed IRP. Flow cytometry (FACS) was used to detect the positive rates of B lymphocytes and T lymphocyte subsets in 43 patients with newly diagnosed IRP. Bone marrow smears of 43 patients with primary IRP were counted by color method. 200 bone marrow cells were counted. The granules, oil droplets, degree of proliferation, ratio of granulogenous erythroid system and pathological hematopoiesis, ratio of lymphocyte, number of megakaryocyte and platelet were recorded. Peripheral blood smears of 43 patients with primary IRP were counted by Rayleigh staining and 100 white blood cells were counted. The proportion of granulocytes, mature red blood cells, lymphocytes, monocytes and platelets were recorded. Part 2: Morphology and cytochemical staining of bone marrow cells were observed under microscope. Immunophenotype of bone marrow cells was detected by flow cytometry, and their components were analyzed by electron microscopy. Results: 1.43 patients with IRP There were altogether 110 mutation sites in the mitochondrial DNA D-loop region of peripheral blood lymphocytes, of which 62 were polymorphic sites (SNP) and 48 were mutation sites. Of these sites, 14 were new mutation sites not mentioned in the database. A total of 516 base changes were found in the 2.110 mutation sites. The main change was 410 base substitution, and the common base substitution was T/C (T/C). The mutation rates of 73 and 263 of 184/410 and A/G (113/410) 3.110 loci were 43/43, 32/43 for locus 311, 27/43 for loci 310 and 16224, 25/43 for loci 16519, 24/43.4 for loci 489 and 1616622. Mitochondrial DNA D-loop polymorphism in peripheral blood lymphocytes of patients with IRP and bone marrow of these patients According to the correlation statistics of mononuclear cells, there was a significant positive correlation between lymphocyte mitochondrial DNA D-loop polymorphism and CD15Ig M, Gly Co A + cell Ig M, CD34 + cell Ig G, CD34 + cell Ig M of bone marrow mononuclear cells in 35 adult patients aged 18 years. There were some Auer body-like inclusions in the cytoplasm. Cytochemical staining and flow cytometry analysis also showed that B lymphocytes were denatured mitochondria with increased matrix density and disappeared internal structure. The high-frequency mutations were 73,263,311,310,16224,16519,489 and 163624 respectively. In the adult group (age < 18 years old), the high-frequency mutations were positively correlated with autoantibodies binding to bone marrow mononuclear cell membrane, which may be an important link in the pathogenesis of immune-associated hematocytopenia. 5. Auer bodies are very valuable morphological findings, but not only through morphological observation, but also should be combined with other related examinations to further classify leukemia cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R558.2
本文编号：2236337
[Abstract]:OBJECTIVE: PART I: In hematological diseases, pancytopenia is the most frequently diagnosed type, in which immune-associated hematocytopenia (IRP) is subdivided as a disease system characterized by bone marrow failure. Recent studies have shown that the pathogenesis of IRP is stimulated by an unknown pathogen. After that, dendritic cell subsets as the main presenting antigens first appear abnormal, then transmit to T lymphocytes, and then lead to the downstream B lymphocytes in the function, number and subsets of abnormalities in varying degrees, thus forming autoantibodies, which can be specifically targeted at bone marrow hematopoietic cells, they can pass through Blockade of functional proteins on hematopoietic cell membranes, activation of complement in situ hemolysis, or mediation of macrophage phagocytosis may lead to inhibition of bone marrow hematopoietic cell proliferation and differentiation, resulting in invalid hematopoiesis and bone marrow failure [35]. In recent years, a large number of experimental studies have focused on whether there is a correlation between genomic genetic material in the nucleus and blood diseases. The D-loop region of mitochondrial DNA detected in this study focuses on whether mutations in extranuclear DNA and immune hematological diseases exist as well. Mitochondria are the only DNA-containing organelles in the cells of higher animals other than the nucleus and are not dependent on the genome for replication, transcription and translation. Oxygen free radicals and other substances around the damage, mainly due to its lack of effective antioxidant repair mechanism, a large number of studies have shown that it is also lack of effective binding with histone, so it can not obtain histone protection, resulting in mitochondrial DNA and nuclear DNA compared to much more than 10 times. In addition, the variation of mitochondrial DNA also affects the function of mitochondrial respiratory chain and leads to the occurrence of many immune-related diseases. The D-ring region of mitochondrial DNA is located in the t RN of mitochondrial DNA. The total length between Apro and t RNAphe (nt16024-nt575) is about 1120 bp, which is the main non-coding region of mitochondrial DNA, the largest genetic variation region in mitochondrial DNA, the D-loop region or the regulatory region of mitochondrial DNA replication and transcription, as well as the starting sites of light and heavy strands of mitochondrial DNA in recent years. Single nucleotide polymorphism (SNP) is considered to be very common in the variation of DNA sequences. The frequency of variation in the genome of higher animals is usually? 1%. It is mainly due to the polymorphism of the genome sequence caused by the change of single nucleotide glycine in the DNA sequence. SNP is considered to be associated with species diversity, individual differences in drug treatment, and susceptibility to certain diseases. Since the human genome is linked to disequilibrium, most SNPs do not directly affect the expression of gene products, but their dependence. I can still use SNPs to locate adjacent genes or genomes when certain genes that cause disease change functionally or structurally. If SNPs happen to be located in the promoters of certain genes, their function plays a role in that gene. The high genetic polymorphisms in the D-loop region, the starting point for the replication of light and heavy strands of mitochondrial DNA, and the important regions responsible for the regulation of mitochondrial DNA transcription and replication are bound to occur. Therefore, we have studied the significance of D-loop polymorphism of peripheral blood lymphocyte mitochondrial DNA (mtDNA) in patients with immune-associated hematopenia (IRP), the correlation between D-loop polymorphism and immune indices, and the correlation between D-loop polymorphism and morphological characteristics of bone marrow and peripheral blood cells. Prolymphocytic leukemia (PLL) is a special type of lymphocytic leukemia. It is generally considered to be a rare variant of chronic lymphocytic leukemia, but it is also considered to be a type (type II) of acute lymphocytic leukemia. Auer body is a diagnostic morphological feature of acute myeloid leukemia, which is found in acute myeloid leukemia. Detection, distribution and relationship with therapeutic effect have diagnostic clinical significance. We are exploring the characteristics of Auer body-like inclusions in the cytoplasm of immature lymphoblastic leukemia cells. The mitochondrial DNA of the cells was amplified by PCR in the D-loop region of all samples. The PCR product gene sequences of the D-loop region were detected by direct forward and reverse sequencing. The results were related to the Cambridge sequence recorded by the human mitochondrial DNA library and the Polymorphic Sites subdatabase of the MT DB database. Flow cytometry (FACS) was used to detect the positive rates of CD15 +, Gly Co A + cells and CD34 + cell membrane surface antibodies (Ig G and Ig M) of bone marrow hematopoietic cells in 43 patients with newly diagnosed IRP. Flow cytometry (FACS) was used to detect the positive rates of B lymphocytes and T lymphocyte subsets in 43 patients with newly diagnosed IRP. Bone marrow smears of 43 patients with primary IRP were counted by color method. 200 bone marrow cells were counted. The granules, oil droplets, degree of proliferation, ratio of granulogenous erythroid system and pathological hematopoiesis, ratio of lymphocyte, number of megakaryocyte and platelet were recorded. Peripheral blood smears of 43 patients with primary IRP were counted by Rayleigh staining and 100 white blood cells were counted. The proportion of granulocytes, mature red blood cells, lymphocytes, monocytes and platelets were recorded. Part 2: Morphology and cytochemical staining of bone marrow cells were observed under microscope. Immunophenotype of bone marrow cells was detected by flow cytometry, and their components were analyzed by electron microscopy. Results: 1.43 patients with IRP There were altogether 110 mutation sites in the mitochondrial DNA D-loop region of peripheral blood lymphocytes, of which 62 were polymorphic sites (SNP) and 48 were mutation sites. Of these sites, 14 were new mutation sites not mentioned in the database. A total of 516 base changes were found in the 2.110 mutation sites. The main change was 410 base substitution, and the common base substitution was T/C (T/C). The mutation rates of 73 and 263 of 184/410 and A/G (113/410) 3.110 loci were 43/43, 32/43 for locus 311, 27/43 for loci 310 and 16224, 25/43 for loci 16519, 24/43.4 for loci 489 and 1616622. Mitochondrial DNA D-loop polymorphism in peripheral blood lymphocytes of patients with IRP and bone marrow of these patients According to the correlation statistics of mononuclear cells, there was a significant positive correlation between lymphocyte mitochondrial DNA D-loop polymorphism and CD15Ig M, Gly Co A + cell Ig M, CD34 + cell Ig G, CD34 + cell Ig M of bone marrow mononuclear cells in 35 adult patients aged 18 years. There were some Auer body-like inclusions in the cytoplasm. Cytochemical staining and flow cytometry analysis also showed that B lymphocytes were denatured mitochondria with increased matrix density and disappeared internal structure. The high-frequency mutations were 73,263,311,310,16224,16519,489 and 163624 respectively. In the adult group (age < 18 years old), the high-frequency mutations were positively correlated with autoantibodies binding to bone marrow mononuclear cell membrane, which may be an important link in the pathogenesis of immune-associated hematocytopenia. 5. Auer bodies are very valuable morphological findings, but not only through morphological observation, but also should be combined with other related examinations to further classify leukemia cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R558.2
【参考文献】
相关期刊论文 前10条
1 陈洁;赵明峰;;活性氧在免疫应答中的作用[J];中国免疫学杂志;2015年06期
2 白亚玲;徐金升;艾晓璐;张胜雷;张俊霞;崔立文;张慧然;;线粒体DNA D环基因多态性与慢性肾脏病发病风险关系的研究[J];中国现代医学杂志;2014年29期
3 刘惠;付蓉;邵宗鸿;;免疫相关性血细胞减少症的诊断及鉴别诊断[J];临床血液学杂志;2013年04期
4 冯延;徐瑜;胡义德;;恶性肿瘤细胞能量代谢特点及研究进展[J];中华肺部疾病杂志(电子版);2013年03期
5 韩卉;李大启;陈萍;邵建华;赵红玉;董学斌;顾琳萍;;急性白血病骨髓细胞线粒体DNA D-loop区的突变检测[J];中国实验血液学杂志;2013年01期
6 Gazi Nurun Nahar Sultana;Atiqur Rahman;Abu Din Ahmed Shahinuzzaman;Rowshan Ara Begum;Chowdhury Faiz Hossain;;Mitochondrial DNA mutations-candidate biomarkers for breast cancer diagnosis in Bangladesh[J];癌症;2012年09期
7 王勉;张旭;;线粒体与肿瘤关系的研究进展[J];承德医学院学报;2009年03期
8 严庆丰;管敏鑫;;线粒体疾病与核基因-线粒体基因的表达调控[J];生命科学;2008年04期
9 张倩乔;李晓;;线粒体DNA与骨髓增生异常综合征[J];中国实验血液学杂志;2008年03期
10 ;ROLE OF B LYMPHOCYTE AND ITS SUBPOPULATIONS IN PATHOGENESIS OF IMMUNORELATED PANCYTOPENIA[J];Chinese Medical Sciences Journal;2007年03期
，本文编号：2236337
本文链接：https://www.wllwen.com/yixuelunwen/xxg/2236337.html
最近更新
教材专著
