Immunological study on human serum PAF(platelet-activating factor) acetylhydrolase deficiency

血小板活化因子（PAF）是由参与炎症和过敏反应的许多细胞类型合成的磷脂，并且被认为是参与许多不同临床病症（包括哮喘、血栓形成、循环障碍和移植物排斥）的重要生物介质。PAF从生产细胞释放到血液中，被血浆/血清PAF乙酰水解酶（PAF-AH）迅速水解为无活性的lysoPAF。我们发现一些日本家庭与血清PAF-AH缺乏症，这似乎是通过常染色体隐性遗传。哮喘患儿中、重度症状组PAF乙酰水解酶缺乏的发生率明显高于对照组（P<0.01）。提示PAF乙酰水解酶缺乏可能是哮喘患儿严重呼吸道症状的遗传因素之一，为进一步阐明PAF乙酰水解酶缺乏是否由哮喘患儿呼吸道功能异常引起， 关于我们 血清PAF-AH酶蛋白的表达水平及血清PAF-AH的产生细胞类型，建立PAF-AH酶蛋白的免疫分析方法。首先，我们从人混合血清中纯化PAF-AH，通过磷钨酸沉淀，吐温20增溶，DEAECellufineAM离子交换柱，Q-Sepharose柱，TSK-GelHW-60 HPLC柱，羟基磷灰石HPLC柱，AsahipakGS-510 HPLC柱和MonoQFPLC柱进行层析，使PAF-AH在电泳上为单一条带。接下来，我们从注射了上述纯化PAF-AH的兔中制备PAF-AH抗血清。该抗血清与纯化的PAF-AH及不含PAF-AH活性的脂蛋白均有交叉反应。因此，为了获得对PAF-AH具有高度特异性的抗体，我们正在利用由PAF-AH免疫的BALB/C小鼠的脾细胞与骨髓瘤细胞融合得到的杂交瘤制备单克隆抗体。另一方面，我们阐明了（1）川崎病患者的血清PAF-AH活性显著高于健康儿童，（2）系统性红斑狼疮（SLE）患者的血清PAF-AH活性随病程而改变;（3）水浸应激引起大鼠胃粘膜病变，血清PAF-AH活性显著升高。此外，尽管已经报道了中性粒细胞显然保留了它们合成的PAF，但我们鉴定了人PAF-AH缺陷血清中的载体蛋白，其提取了人中性粒细胞中产生的PAF。少

Platelet-activating factor(PAF) is a phospholipid synthesized from many cell types involved in inflammatory and allergic reactions, and is thought to be a significant biological mediator involved in many diverse clinical conditions including asthma, thrombosis, circulatory disorders and graft rejection. PAF, which is released from producing cells into blood, is hydrolyzed rapidly by plasma/serum PAF acetylhydrolase PAF-AH) to be inactive lysoPAF. We found some Japanese families with serum PAF-AH deficiency, which appeared to be transmitted by autosomal recessive heredity. The probability of PAF acetylhydrolase deficiency among asthmatic children was significantly higher in groups with severe symptoms (moderate and severe)(P<0.01). From these observations, we proposed that deficiency of serum PAF acetylhydrolase might be one hereditary factor leading to severe respiratory symptoms in as thmatic children.Furthermore, in order to elucidate whether PAF-AH deficiency results from abnormalit … More y of serum enzyme protein and what kind of cells produces serum PAF-AH, we aimed to develop the immunoassay for PAF-AH enzyme protein. First, we purified PAF-AH from human pooled serum to be a single band on electrophoresis through Phosphotungstic acid precipitation, solubilization with Tween 20, chromatographies using DEAE Cellulofine AM ion exchange column, Q-Sepharose column, TSK-Gel HW-60 HPLC column, Hydroxyapatite HPLC column, Asahipak GS-510 HPLC column and Mono Q FPLC column. Next, we prepared PAF-AH antiserum from rabbits into which above described purified PAF-AH was injected. This PAF-AH antiserum had cross-reactivity with not only purified PAF-AH but also lipoprotein containing no PAF-AH activity on immunoelectrophoresis. Hence, in order to obtain antibody highly specific for PAF-AH, we are in progress to prepare monoclonal antibody using the resulting hybridomas that spleen cells from BALB/C mouse immunized with PAF-AH were fused with myeloma cells.On the other hand, we elucidated that (1) serum PAF-AH activity in subjects with Kawasakis disease was significantly higher than that in healthy children, (2) that in subjects with systemic lupus erythematosus(SLE ) changed during the course of the sickness, and (3) the onset of water-immersion stress toward rat caused the development of gastric lesions associated with a significant increase in serum PAF-AH activity. Furthermore, although it has been reported that neutrophils apparently retain the PAF they synthesize, we identified the carrier protein in human PAF-AH deficient serum which drew out PAF produced in human neutrophils. Less