【作者】 朱玲；

【导师】 陆惠民；

【作者基本信息】 苏州大学 ， 病原生物学， 2003， 博士

【摘要】 目的：1) 从细胞分子水平研究Ⅰ型超敏反应的免疫病理发生机制。2) 通过基因克隆策略和分子生物学技术从人的Daudi细胞中获得含人磷脂酶D2的功能保守基序的基因，并使其在大肠杆菌中表达，最终拿到具有生物学活性的蛋白质产品：rhPLD2。3) 研究该蛋白在哮喘等Ⅰ型超敏反应的炎症过程中所具有的一系列免疫学生物活性。方法：1) 采用微量免疫荧光、免疫酶标、细胞培养、电镜等技术观察当肥大细胞受到相同过敏原再次刺激时释放炎症介质的过程。并将抗原、抗体结合的高特异性与DAB、中性红染色的高灵敏性相结合，测定肥大细胞被IgE致敏的百分率。2) Daudi细胞经5％CO2，37℃条件下，以20％小牛血清、100μg·ml^-1链霉素、100U·ml^-1青霉素的DMEM培养基培养，调整细胞密度达10¹²个·ml^-1。经1,500rpm，5min离心，收集沉淀细胞。3) 提取mRNA，并根据所设计5′和3′端寡核苷酸引物及突变引物进行RT-PCR。I型超敏反应的细胞分子机制及其抗炎蛋自质的研究 4)PCR产物采用基因内引物PCR扩增和基因内限制性内切酶酶切方 法进行分析鉴定正确后，克隆到载体puCm一Tvector/psK上。酶切， PCR及测序鉴定其正确性。将rhPLDZ的DNA全长片段克隆至原 核表达载体pET30a（+）中，构建出能表达出带有6 X HIS纯化标记的 pET30a一rhPLDZ的融合蛋白的原核表达载体。所获重组蛋白经分 离、纯化，SDS一队GE，Western Blot鉴定分子量和纯度。并采用 BCA Protein ASSay Reagent Kit测定其蛋白含量。5)参照Amplex Red Phospholipase D Assay Kit提供的protoeol进行人重 组磷脂酶DZ蛋白质的活性测定。6)蛋白质产品rhPLDZ进行动物过敏实验和毒性实验。利用速发 型超敏反应的动物模型，在体外抗炎实验的基础上进一步研 究用药前后，实验动物临床症状的表现及动物循环血液中嗜 酸性粒细胞（Eos）的水平。6)观察rhPLDZ对LPS诱导小胶质细胞释放炎症因子的干预作用。结果:l)观察到了MC特异性脱颗粒的过程，并实拍到了其动态变化。 2)建立了哮喘监测、诊疗的新指标。3)从人Daudi细胞中提取 mRNA，经剪切、拼接变构，克隆获得了不含膜结合部位及信号肤 的，可编码631个氨基酸的PLDZ cDNA序列（GenBank登录 号:AY178289）。4)构建了不含信号肤及氨基端的:hPLDZ的E.Coli 工程菌株的表达载体，在细菌细胞的包涵体中成功获得了 11l型超敏反应的细胞分子机制及其杭炎蛋白质的研究膜蛋白的表达产物:rhPLDZ的变构蛋白质，且具有较好的生物活性;与提取的天然膜蛋白及纯化的PLD标准（Sigma）比较，rhPLDZ具有较好的生物活性:达到47.982 mu/mL（o.gO75mg/mL）（rhPLDZ第一批）;53.967mu/mL（0 .93493mg/mL）（r hPLDZ第二批）。5)己成功建立了稳定的哮喘动物模型。并初步进行了:hPLDZ的动物过敏试验，表明:rhPLDZ经腹腔注射0.4537 mg/mL及1 .815mg/mL，对动物无致敏及任何毒性作用。6)thPLDZ对动物模型的哮喘状态有一定的拮抗作用。用药前后其E 05的水平经t检验比较P<0.01，差异具有显著性。7)初步证实了rhPLDZ对LPS诱导小胶质细胞释放炎症因子有抑制性的干预作用。结论:1)特异性释放颗粒时，肥大细胞（MC）内有曲折的膜被迷路 或脱颗粒管道形成，并与细胞膜合并形成开口，胞浆颗粒由此释放 到细胞外。颗粒被释放后，膜性开口重新闭合。脱颗粒后的MC 胞浆内空泡化明显，电子密度明显降低2)肥大细胞特异性脱颗 粒可被DAB加中性红双染色法染色。其优点是:不受肥大细胞自 然脱颗粒（非特异性脱颗粒）的影响。即特异性高，灵敏度好。3) rhPLDZ的变构蛋白质，是一个具有生物学活性及一定免疫学活性的 功能蛋白质。4) rhPLDZ可在大肠杆菌中得到较好的活性表达。5) rhPLDZ在炎症过程中发挥着重要的抗感染作用。更多还原

【Abstract】 Objective: To research the mechanism of immune pathology of type I allergy at the level of cell and molecule. To obtain the cDNA encoding human recombinant phospholipase D2 (rhPLD2) from human daudi cell , which was an NH2 -terminal PLD2 deletion mutant expressed in E.coli; and to investigate the activities of the protein in vive or vitro.Methods: 1) Used transmission electron microscope observing the process of mast cell degranulation induced by anti-IgE-HRP, and the mast cells have stained by ELISA and immunofluorescence. 2) Daudi cells was cultured in Hepes-buffered DMEM that was supplemented with 10% (v/v) fetal bovine serum, 100 units/mL of penicillin and 100 mg/mL of streptomycin, at 37℃ in a humidified, CO2 controlled (5%) incubator. At culture dishes at l*1012 cells/dish., the Daudi cells were deprived of serum by centrifuged at 500 x g for 5 min. 3) we performed reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers that were designed according to the Genbank database. 4) A cDNA encoding hPLD2 was engineered by PCRand introduced into a pET30a(+) vector for expression with a 5’ 6X His tag. The recombinant protein was purified and electrophoresed in SDS-polyacrylamide gelelectrophoresis gels in order to identify its molecule weight and purity. The guantity of rhPLD2 was detected with BCA Protein Assay Reagent Kit. The supernatant was subjected to Western blot with 6X His monoclonal antibody albumin free and PLD ( carboxy terminus ) goat polyclonal IgG; respectively. 5) On the other hand, the supernatant was used for assay of PLD activity adopted Amplex Red Phospholipase D Assay Kit. 6) We researched the hypersusceptibility and toxicity of rhPLD2 with guinea pigs, and studied the behave of the model of immediate hypersensitivity animals and the change of the level of eosinophil comparing between the before and after used drugs. 7)To investigate the effect of rhPLD2 meddled in the expression of the genes encoding IL-1B in the murine microglial cell line N9 which was stimulated with LPS.Results: 1) Observed and photographed the kinetic process of mast cell specific degranulation . 2) Set up the new criterior to supervise and diagnosis and treat the asthma. 3) Obtained a recombinant protein which without membrane band site and signal peptide from human Daudi cells .The cDNA sequence can encode a protein with 631 aa of rhPLD2 (accession numbers: AY 178289) . 4) constructed the recombinant expression plasmid pET30a-rhPLD2,and obtained successfully the production of membrane protein from the precipitate of inclusion bodies in E.coli. Expression of this cDNA clone in E. coli shows a functional PLD2 activity similar to that of the natural PLD2. As we predicted that the activity of expressed rhPLD2 was estimated to be 50.9745 raU/mL ( 0.9212mg/mL ) . 5) Founded successfully a animal model of asthma. The principium results showed injected 0.4537 mg/mL or 1.815mg/mL rhPLD2 to animals abdomen which have no hypersusceptibility and toxicity. 6) The protein of rhPLD2 can resist the asthma outbreak. 7) To approve primarily rhPLD2 can inhibit the expression of inflammatory cytokines such as IL-1B in the murine microglial cell line N9 which was stimulated with LPS.Conclusion: 1) The track of mast cell specific degranulation is the membranous aisle in which granule was released . After that, the membranous hole closed and cytoplasma became cellular clearly . 2) We found that only specific sensitized mast cells was stained orange-brown colour. ConclusionThis method can be used to forecast the development of type I allergy, such as: asthma etc. and to conduct some tests related to the degranulated mast cells. 3) The mutation protein of rhPLD2 is a functional protein which possess some biologic and immune activities. 4) A preferable activity of the mutation protein of rhPLD2 can be expressed in E.coli. 5) Themutation protein of rhPLD2 plays an important role which is against to inflammation.更多还原