【作者】 王勤；

【导师】 金锡御； 吴雄飞；

【作者基本信息】 第三军医大学 ， 外科学， 2002， 博士

【摘要】 多脏器功能障碍综合征(MODS)死亡率极高，对其进行有效治疗，是临床医学的一项重要任务。大量研究证明，系统性炎症反应综合征(SIRS)是MODS动态发展的基础，肿瘤坏死因子-α(TNF-α)是一个在SIRS发病机制中起十分重要作用的关键性前炎症细胞因子。及时有效地清除过度产生的TNF-α，可以抑制SIRS早期细胞因子网络的变化，有助于改善MODS预后。连续性肾脏替代治疗(CRRT)在国内外被广泛地用于救治MODS，因其仅能部分而不是彻底地清除TNF-α，故不能降低MODS的死亡率。吸附疗法具有显著清除TNF-α的功能，但临床应用受到限制。为此，我们从以下两方面进行了研究。第一，应用杂交瘤单克隆抗体技术制备了抗重组人肿瘤坏死因子单克隆抗体，将其包被在载体微球上，制作成功特异性免疫吸附柱，并进行吸附及相应试验。第二，应用CRRT技术治疗14例大器官移植术后MODS患者，ELISA法检测治疗前后血浆TNF-α、IL-1β、IL-6、IL-8浓度，观察清除TNF-α的效果，并将其与特异性吸附柱的吸附效果及优缺点进行综合对比分析。为今后创造出一种将二者有机结合起来治疗MODS的新方法，提供临床及实验依据。 主要结果和结论如下： 1、应用杂交瘤单克隆抗体技术，共建立了9株分泌抗γHTNF-αMcAb的杂交瘤细胞系，继而制备了含McAb的腹水，经免疫亲和层析法得到了纯化的抗rHTNF-αMcAb。 2、特异性交叉反应测定及Western blotting结果显示，这组抗体只与rHTNF具有特异性反应，还具有不同程度的细胞毒中和能力及亲和力。选 取其中特性及功能最佳的单抗，为研制特异性吸附柱奠定了基础。 3、自行设计制作了过滤柱，行硅烷化处理，经XPS检测天N、S、 CI污染，改善了生物相容性。该柱的通透性及实用性能良好，为相关研究 提供了一个新模型。 －4、选用 PSM作为载体微球，依次包被 PLL及抗 rHmF咀 McAb，经 检测包被满意而且牢固。对二次包被的条件进行了探讨。为相关研究提供 了一种新颖的方法。 5、成功制备了特异性兔疫吸附柱，最大吸附率达90％以上。实验发 现吸附存在饱和现象、酸性溶液可洗脱结合抗原，经碱性液缓冲后，仍有 良好的吸附率，证实该柱具有良好的稳定性和重复使用性。创造了一种吸 附疗法中特异性清除TNF－a的新型模式，具有较大的应用价值。 6、常规应用CRRT技术治疗14例大器官移植术后MODS患者，ELISA 法检测治疗前及治疗后不同时间患者血浆TNFo、IL1、IL6及IL毛浓 度。证实了TNFa是大器官移植术后MODS发生发展的关键细胞因子。 提出动态观察TNF－a变化情况，有助于指导临床工作。 7、CRRT能够部分清除此类患者体内TNFa，以吸附机制为主，最大 清除量达44％左右。可以在一定程度上暂时维持患者内环境稳定，但并未e 明显提高生存率，降低死亡率。 8、TNFo的浓度变化影响了 IL刁 p、IL七及 IL｛的变化，存在正相关 关系。认为早期高效地清除y卜a，将有助于纠正全身性炎症反应，改善 MODS预后。 9、CRRT与特异性吸附柱相比，在治疗MODS中各有优缺点。如将 二者有机地加以结合，将能更加有效地救治MODS患者，改善预后。本研 究为此方面提供了理论及实验依据。更多还原

【Abstract】 It has been an important task that effective treatment to multiple organ dysfunction syndrome (MODS) for clinical medicine, because of the MODS has extremely high mortality. Many researches have approved that the systemic inflammatory response syndrome (SIRS) was the foundation of the dynamic process of the MODS. Tumor necrosis factor-alpha (TNF- a )is a key pro-inflammatory cytokine which play a very important role in SIRS pathogenesis. Timely and effectively removal tumor necrosis factor-alpha when it was excessive product the changes of cytokine net in the early stage of the systemic inflammatory response syndrome could be suppressed and which is beneficial for improving the prognosis of the multiple organ dysfunction syndrome. Continuous renal replacement therapy (CRRT) had been extensively used for the rescue for the MODS in the world for many years. However, it only partly not totally eliminate the TNF- a and could not depress the mortality of the MODS. Adsorption therapy has obvious function for removal the tumor necrosis factor-alpha, but it was restricted in the clinical application .Fof this reason, we performed the reseach from the two undermentioned aspects.Firstly, we prepared for the monoclonal antibodies against recombinant human tumor necrosis factor alpha by hybridoma techniques. It was wraped on the polystyrene microsphere carrier connecting poly-L-lysine. After that, we made successfully up the specific immunoadsorption column for the first time,and took some tests about adsorption. Secondly, we treated to 14 patients with MODS after the big apparatus transplantations by the continous renal replacement therapy techniques. They were detected by ELISA that the concentration of the TNF- a , IL-1 P, IL-6, IL-8 in plasmam before and during the CRRT 1,2,3,6,12 hours respectively.The main results and conclusions were as follows:1. Nine groups of hybridoma cell line which can secrete anti-recombinant human tumor necrosis facter alpha were established, and we got some purificative monoclonal antibodies against recombinant human tumor necrosis facter alpha. It grounded for us in whole research.2. The specific across reaction test and the Western blotting showed that these monoclonal antibodies only reacted specificly to the recombinant human tumor necrosis facter alpha that has 17KDa molecular weight. These McAb possess the neutralization ability for the rHTNF- a cytotoxicity and relative affinity in some degree.3. The filtration column has favourable capability and practicality, the most penetrating volume was about 2500 milliliters per hour. It provided a new model for some correlative researches.4.The polystyrene microsphere was chosen for the carrier and coated by poly-L-lysine and anti-recombinant human tumor necrosis facter alpha in turn. Though examining, the two times coating was firm and satisfying. It offered a novel method for this field.5.We produced triumphantly the specific immunoadsoiption column which the most adsorptive rate was more than 90 percent. This column has good stability and repetition. It created a new pattern of specific eliminate the TNF- a in the adsorption therapy and had biggish application value.6.There was remarkable running up of the concentration of the TNF- a , IL-1 P , IL-6, IL-8 in plasmm of those patients with MODS after the bigapparatus transplantations.lt was proved that the TNF- a is the key pro-inflammatory cytokine in the pathogenesis of the MODS after translantation.Observing the variety of TNF- a with dynamic mehtod, will help to guidance the clinical work.7. Partly the TNF- a in these patients could be removaled by CRRT.The main mechanism was the adsorption potentially. It was found that the maximal clearance was gotten for the removal TNF- a by CRRT at 6 hour, and at the same time , the maximal adsorption efficiency was 44%.Although maintaining the circumstance stabilization of the patients temporarily, CRRT could not increase the survival and decrease the mortality in our observation.8. We substan更多还原