【作者】 任军；

【导师】 贾凌云；

【作者基本信息】 大连理工大学 ， 生物化工， 2009， 博士

【摘要】 用于血液净化治疗自身免疫性疾病的蛋白A吸附柱属于三类医疗器械,其内部填充的蛋白A吸附剂能够快速、有效地吸附清除患者血液中的致病抗体和免疫复合物,实现救治病人的目的,其临床效果已得到医生和病人的广泛认可。由于作为吸附剂关键功能基的基因工程蛋白A存在售价昂贵、不易保存和消毒等缺陷,导致蛋白A吸附柱的临床应用受限。本论文的主要研究内容包括以下3个方面:(1)计算机分子模拟固定化配基与血浆中高丰度蛋白的结合,旨在有目的的筛选可能替代蛋白A的小分子功能基;(2)在分子模拟的基础上,实验筛选对IgG具有选择性吸附能力的小分子功能基,优化吸附剂的合成并对其吸附性能进行评价;(3)研究筛选出的小分子功能基吸附剂对血浆中致病因子的清除能力及其安全性,并与商业化的蛋白A吸附剂进行比较,评价其取代蛋白A吸附剂进行血液净化治疗的可行性。论文首先利用分子模拟软件DOCK 5.0模拟了固定化配基与血浆中的两种高丰度蛋白质IgG及HSA的表面作用特征,分析了两种蛋白质表面疏水区域的分布特点。分析结果表明,IgG表面的疏水区域与HSA相比差异很大,IgG表面的疏水位点较多并相对分散,疏水口袋较浅,固定化配基可接触程度较高;而HSA表面的疏水位点相对集中,疏水口袋较深,固定化配基可接触程度较低。根据上述两种蛋白质的表面特点,确认选择固定化疏水配基有可能实现对IgG的选择性吸附。在上述研究基础上,选择了16种带有不同疏水特性的小分子化合物为配基合成吸附剂,利用IgG和HSA为样本评价了其对IgG的选择性吸附能力,发现,兼具疏水性和富电子特性的功能基对IgG的选择性吸附能力远高于HSA。进一步利用血清样本评价其在复杂样本中的选择吸附特性,结果表明4-巯基乙基吡啶(MEP)吸附剂效果最佳。吸附剂采用Sepharose CL-6B为载体,溴丙烯活化、N-溴代丁二酰亚胺溴化的方法偶联MEP,以对血清中IgG具有较高吸附容量同时非特异吸附较低为目标,优化了MEP吸附剂的合成条件,较优化的合成条件为:Sepharose CL-6B在1.2 mol/L NaOH溶液中(凝胶与溶液体积比为1:1)经20%溴丙烯(相对于凝胶的体积比)活化反应4 h;带有烯丙基活性基团的Sepharose CL-6B经N-溴代丁二酰亚胺溴化后,在pH 10的溶液条件下偶联MEP分子。上述反应条件可控制吸附剂的配基密度在100-150μmol/mL gel,对IgG的动态结合容量为36.21 mg/mL,对人血浆中抗体组分的吸附容量约为30 mg/mL,占其血浆蛋白吸附总量的85%以上,其抗体结合能力和选择性能够满足血液净化吸附剂的要求。对MEP吸附剂清除自身免疫性疾病病人血清中致病因子的能力进行了系统研究。对于分子大小差异较大的致病因子,不同固定化密度的MEP吸附剂对其清除能力呈现出不同的规律。对于分子量近900 KDa的类风湿因子,当固定化MEP密度达到60μmol/mL gel时,对其清除能力达到95%以上,而对于系统性红斑狼疮的致病因子抗双链DNA抗体,随着固定化MEP密度的升高,病人血浆中抗双链DNA抗体的去除率升高,当固定化MEP密度达到100μmol/mL gel时,抗双链DNA抗体的去除率达到85%。进一步利用采自不同病人的样本考察了MEP吸附剂对类风湿和系统性红斑狼疮中致病因子清除的个体差异,并与商品化的Protein A吸附剂进行比较。研究结果表明:MEP吸附剂对类风湿因子的清除率达到90%以上,其中阴性率为75%,基本没有个体差异;而商品化的蛋白A吸附剂吸附后的阴性率仅为10%,大部分的清除率在12%至83%之间,个体差异明显。但对于系统性红斑狼疮的致病因子抗核抗体,MEP吸附剂的清除有效率为78%,商品化蛋白A清除有效率为57%,个体差异均较大。在总的致病因子清除效果方面,MEP吸附剂优于商品化的蛋白A吸附剂。论文进一步研究了MEP吸附剂的生物安全性,吸附剂浸出液的小鼠急性毒性实验表明,MEP吸附剂的浸出液未对小鼠产生毒性,说明其中没有有毒物质脱落。对狗进行的MEP吸附剂吸附实验表明,利用该产品的治疗过程安全可靠。上述研究结果证实,MEP吸附剂对病人血浆中的致病因子具有较高的选择性和较大的吸附容量,同时兼具价格低廉、易于消毒等优势,与商品化蛋白A吸附剂的对比研究表明,MEP吸附剂在致病因子的去除上具有更优异的性能,具有替代蛋白A吸附剂应用于临床的潜力。更多还原

【Abstract】 Extracorporeal immunoadsorption(ECI) therapy using Staphylococcal Protein A columns has proven effective for the selective removal of autoantibodies and circulating immune complexes from patients.It currently provides an effective treatment for autoimmune diseases.However,due to the drawbacks of Protein A in terms of cost and stability,the widespread use of Protein A based ECI is limited.The objective of this study was to develop a synthetic compound-based adsorbent with binding selectivity for antibodies,and to investigate its feasibility as an alternative to Protein A for autoantibody removal therapy.The interaction between immobilized ligands and two plasma high-abundant proteins, IgG and HSA,was simulated using DOCK 5.0 software,and the surface hydrophobic distributions of the two proteins were characterized.The results showed that IgG and HSA markedly differed in surface hydrophobicity.IgG had more hydrophobic patches,and these hydrophobic patches were scattered in the form of superficial pockets,making them readily accessible to the immobilized ligands.In contrast,hydrophobic patches on HSA surface were more concentrated and had deeper pockets,making them relatively less accessible to immobilized ligands.The surface characters of the two proteins suggested that immobilized hydrophobic ligands were likely to have selectivity for IgG adsorption.Based on the above study,sixteen compounds with various hydrophobic features were chosen for ligand screening.The compounds were each immobilized onto Sepharose CL-4B and then their adsorptions with IgG and HSA were evaluated.Compounds that had both hydrophobic and electron-rich properties remarkably contributed to IgG selectivity.Further investigation employing plasma as complex samples indicated that 4-mercaptoethylpyridine (MEP)-Sepharose was the best adsorbent for IgG with a binding capacity of more than 30 mg/mL,and a selectivity of more than 80%.Further efforts to optimize MEP adsorbent were focused on the activation conditions and ligand coupling method.A relatively optimal synthesis strategy was obtained:Sepharose CL-6B beads were activated with 20%allyl bromide(2 mL for 10 mL gel) in 1.2 mol/L NaOH solution for 4 h.The activated Sepharose beads were brominated with N-bromosuccinimide,and then coupled with MEP in solution at pH 10.The resulting MEP-Sepharose adsorbent with a ligand density of 100-150μmol/mL gel could achieve a dynamic binding capacity of 36.21 mg/mL for IgG.It had an affinity adsorption constant of 7.8×10~5 L/mol,and more than 85%of the total proteins adsorbed from the plasma were antibodies.The performance of MEP-Sepharose adsorbent for autoantibody removal was further investigated.It was found that ligand density was an adjustable parameter for the synthesis of adsorbents aiming at different pathogenic factors,depending on the class of antibody. MEP-Sepharose with a ligand density of 98.8μmol/mL gel could remove 80%of the anti-double-stranded DNA antibodies from human serum,whereas a ligand density of 64.5μmol/mL gel was enough to remove 96%of the rheumatoid factor(RF) in the serum. Moreover,MEP-based adsorbents showed a lower degree of individual differences compared to Protein A-Sepharose.RF removal of 90%was achieved for all twelve serum samples from different individuals.Among the fourteen serum samples derived from systemic lupus erythematosus patients,eleven samples had markedly reduced antinuclear antibody titers.The safety of the adsorbent as a therapeutic agent for removing autoantibodies from patient’s blood was evaluated by conducting an acute general toxicity test.In addition,a blood purification treatment using MEP-Sepharose was performed on a dog.The results suggested that the adsorbent was safe for clinical use.The above results indicated that MEP adsorbent could remove pathogenic factors from human plasma with marked selectivity and capacity.In addition,MEP adsorbent performed better than commercial Protein A adsorbent in autoantibody removal,having greater stability and being cheaper than Protein A.These results suggested that MEP adsorbent could beeome an alternative to Protein A adsorbents for clinical application.更多还原