1.Prevalence of Parvovirus B19 Infection in Taiwan 2.GPI- anchored Proteins Analysis of Patients with Hematopoietic Stem Cell Disorders

• Main Authors: CHANG, TZU-HUI, 張慈惠
• Other Authors: LIANG-IN LIN
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/06322218335940869688

碩士 === 國立臺灣大學 === 醫事技術學系 === 85 === 1. 病毒是引起再生不良性貧血 ( aplastic anemia，AA ) 的因素之一， 其中以 Parvovirus B19 的病例報告最多。Parvovirus B19 是於 1975 年所發現的，為一單股去氧核糖核酸 ( ssDNA ) 的病毒。Parvovirus B19 可以將細胞表面的 P抗原當做受體，進入人類骨髓內分裂旺盛的紅血 球母細胞中繁殖，而且具有細胞毒性 ( cytotoxicity ) 使造血系統受到 傷害，主要經由呼吸道感染，輸血、胎盤感染亦常發生。Parvovirus B19 的流行並無明顯的季節性，但是台灣地區以春末夏初的病例較多，通常感 染 Parvovirus B19 的潛伏期為 4 ~ 20 天，對正常人而言，症狀類似感 冒，後期可能有發疹性的傳染性紅斑症；當患有慢性溶血性貧血 ( chronic hemolytic anemia ) 的病人感染Parvovirus B19 時，通常會造 成暫時性造血不良 ( transient aplasticcrisis，TAC )；而免疫機能不 全的病人感染後則會有持續性貧血( persisent anemia ) 的現象；若是 孕婦感染 Parvovirus B19，會導致胎兒先天性貧血或死亡。通常醫師以 注射免疫球蛋白來改善病患的貧血症狀。本實驗所用的 Parvovirus B19 診斷方法有Parvovirus B19 特異性 IgG、IgM 以及 DNA 的偵測，並將 PCR 產物做 核酸序列的分析。本實驗之目的為：(1) 統計出台灣地區捐 血人曾經感染 Parvovirus B19 的比例，並篩檢出正在感染的病例；(2) 針對一些和 Parvovirus B19 有相關性的疾病，包括乙型地中海貧血 ( b-thalassemia major )、紅斑性狼瘡 ( systemlupus erythematosus， SLE )、再生不良性貧血等病人做 ParvovirusB19 感染率的調查。由實驗 結果發現，捐血人感染率為 24.75%，乙型地中海貧血病人感染率為 26.9 %，再生不良性貧血病人感染率為 12.5 %，紅斑性狼瘡病人感染率為 34.48 %。在捐血人中，偵測出 4 位捐血人有 Parvovirus B19 DNA 的存 在，其 PCR 產物 ( 3002 ~ 3291 nt ) 的核酸序列和 Parvovirus B19- Au 的序列相似。我們由實驗結果推論，Parvovirus B19 的感染率和年齡 及捐血次數有明顯的相關性，而且在台灣地區以春季為 Parvovirus B19 流行的時期。捐血人中，的確發現一些正值感染的病例，所以我們建議對 血液疾病患者欲輸入體內之血液及孕婦做 Parvovirus B19 的篩檢，避免 因為感染 Parvovirus B19引起病人嚴重的併發症以及胎兒的異常。 2. 幹細胞疾病是造成 bone marrow failure 的主要原因，包括驟發性夜 間血紅素尿症 ( Paroxysmal nocturnal hemoglobinuria，PNH ) 、再生 不良性貧血 ( aplastic anemia，AA )、Myelodysplastic syndrome ( MDS ) 等。PNH 是由於glycosylphosphatidylinositol ( GPI ) anchor 合成過程中參與的PIG-A 基因發生突變，無法合成GPI anchor，造成血球 表面的GPI-anchored 蛋白出現缺乏的情形，尤其是 CD55 和 CD59，引發 血管內 complement-mediated hemolysis，使細胞被破壞、溶解。根據臨 床報告發現，部分 AA 病人會轉形為 PNH，亦發生 MDS 轉形為 PNH，或 者 PNH 轉形為 MDS 的病例，但是GPI-anchored 蛋白在 AA 以及MDS 二 種疾病所扮演的角色尚未明確，所以此三種疾病間的關係仍然不清楚。本 實驗的目的，乃藉分析正常人和上述幹細胞疾病病人之周邊血球表面的 GPI-anchored 蛋白表現情形，並加以比較，以探討 GPI anchor 在這些 病人身上所扮演的角色。我們分別將所需的各種血球細胞分離、稀釋，加 入單株抗體反應 ( 紅血球：CD59，顆粒球：CD13 及 CD67，T 型淋巴球 ：CD2 及 CD59，B 型淋巴球：CD19 及 CD48 )，洗淨後，利用流式細胞 計數儀做 GPI-anchored 蛋白的分析。在實驗結果中，PNH 病人周邊血液 之紅血球、顆粒球及 B 型淋巴球皆出現明顯的 GPI-anchor 蛋白缺乏； 而少數正常人以及部分 MDS 病人有小部分 CD67 表現稍弱的顆粒球，其 餘細胞的 GPI-anchored 蛋白則表現正常。經實驗發現，少數正常人有一 群 GPI-anchored 蛋白表現較弱的細胞，由此推論可能在少部分正常人體 內存在著一些 GPI-anchored 蛋白表現較弱的細胞 。 另外，3 位 MDS 病人的顆粒球出現 GPI-anchor 蛋白缺乏，我們懷疑病人是否處於由 MDS 轉變為 PNH 症狀的早期，因此必須長期做追蹤檢查，以了解 GPI anchor 缺失在他們身上所扮演的角色。 1. Human Parvovirus B19, discovered in 1975, is a single- strandedDNA virus. Parvovirus B19 replicates in rapidly dividing bonemarrow cells of the erythroid lineage ( CFU-E, BFU-E ) through theblood group P antigen, and impairs hematopoiesis system with itscytotoxicity. The spreading of Parvovirus B19 is usually via therespiratory route, and sometimes via transfusion or placenta. Innormal individuals, Parvovirus B19 can cause erythema infectiosum( fifth disease ); in patients with underlying hemolytic disorders,infection of Parvovirus B19 is the primary cause of transient aplasticcrisis ( TAC ); whereas in immunocompromised patients, persistentParvovirus B19 infection may develop pure red cell aplasia andchronic anemia. The infection of Parvovirus B19 in utero can resultin fetal death, hydrops fetalis or congenital anemia. Treatment ofpersistent Parvovirus B19 infection with immunoglobulin leads to arapid resolution of the anemia. The purpose of this study is toestablish the proportion of blood dornors who ever had ParvovirusB19 infection in Taiwan, and to investigate the infective proportionof patients with b-thalassemia major, systemic lupus erythematosus( SLE ), aplastic anemia ( AA ), pancytopenia and thrombocytopenia.We detected Parvovirus B19 specific IgG / IgM and DNA by ELISAand PCR, respectively. The PCR products were then sequenced. Ourresults showed that Parvovirus B19 IgG (+) proportion were 24.75 %in blood donors, 26.9 % in b-thalassemia major, 12.5 % in AA, 34.48% in SLE, 30 % in thrombocytopenia, and 33.3 % in pancytopenia.PCR results were positive in four blood donors; the sequence of their PCR products ( 3002 ~ 3291 nt ) were similar to Parvovirus B19-Au. These data suggest that the prevalence rate is higher in olderindividuals and in the blood donated in late spring and early summer.Higher prevalence rate in patients with b- thalassemia major mayresult from repeated transfusion. For the presence of Parvovirus B19DNA (+) in some blood donors, the establishment of Parvovirus B19screening for some patients and pregnant women should beconsidered to avoid serious complications. 2. The major causes of bone marrow failure are stem cell disorders,including paroxysmal nocturnal hemoglobinuria ( PNH ), aplastic anemia( AA ), myelodysplastic syndrome ( MDS ) et al. PNH is an acquiredanemia caused by complement-mediated hemolysis. Patients with PNHhave abnormal blood cells that are sensitive to complement due to thedeficiency of complement regulatory membrane proteins, especially CD55and CD59 which are glycosylphosphatidylinositol ( GPI )-anchoredproteins. Because of mutations on PIG-A gene, GPI anchor cannot besynthesized in PNH cells. According to other reports, we found that some patients with AA or MDS can transform to PNH, some patients withPNH also can transform to MDS. The relationship between these threedisorders is not clear yet. The puropse of this study is to analyze andcompare the presence of GPI-anchored proteins in normal adults andpatients with stem cell disorders. RBC, granulocytes, andlymphocytes were isolated for staining with monoclonal antibodies,and GPI-anchored proteins on the cell membrane were analyzed byflowcytometer. Our results showed that RBC, granulocyte, and B-lymphocyte expressed deficient GPI- anchored proteins in PNHpatients. A small number of normal adults and patients with MDShad a few abnormal granulocytes. From these data, we consider thatthe three MDS patients were at early stage of PNH. The long-termfollow up of flowcytometry data will be essential to understand theimportance of GPI anchor to these patients.