【作者】 谷亚伟；

【导师】 杨丽；

【作者基本信息】 天津医科大学 ， 神经病学， 2008， 硕士

【摘要】 目的:探讨全血灌流免疫吸附治疗实验性变态反应性神经炎的效果和作用机制。方法:分别应用髓鞘碱性蛋白(MBP)和牛坐骨神经免疫新西兰兔,从中选择适合全血灌流的动物模型。健康兔随机分为4组,分别为实验组1组(接受牛坐骨神经免疫,在免疫后第14天给予1次全血灌流免疫吸附治疗),实验组2组(接受牛坐骨神经免疫,在免疫后第14天和第16天分别给予1次全血灌流免疫吸附治疗),阳性对照组(接受牛坐骨神经免疫,但不给予任何治疗处理)和阴性对照组(给予生理盐水5 ml多点皮内注射,在免疫后14天接受1次全血灌流)。应用Graham K临床评分对实验兔进行临床评定。阴性对照组实验兔在免疫后第14天进行全血灌流,通过检测灌流前后血细胞的变化进行血液相容性的评价,在免疫后第18天同阳性对照组实验兔一同进行运动神经传导速度、波幅和F波测定,两组还需在免疫后第35天取坐骨神经进行髓鞘染色,切片进行光镜检查。实验组1组和实验组2组在免疫后第14天进行灌流治疗,两天后实验组2组进行第2次灌流治疗,灌流前后取血进行血清成分分析,在免疫后第35天两组兔进行神经电生理检测和病理观察。定期留取4组实验兔血清并测量其血清P2抗体的滴度变化。应用木瓜蛋白酶和胃蛋白酶水解健康兔IgG,水解片段由吸附剂进行吸附,并比较各水解片段的吸附率。结果:用MBP和坐骨神经均可制备新西兰兔EAN,后者更适合全血灌流操作。阳性对照组兔自免疫后14.13±0.64天出现临床症状,在免疫后第28～32天症状达到高峰,自免疫后35.00±0.63天存活兔开始恢复,总病程约60.80±0.84天。实验组1组实验兔在免疫后第32.13±0.64天开始恢复,总病程为42.38±0.50天。实验组2组实验兔在免疫后第30.25±0.46天开始恢复,总病程为38.38±0.52天。坐骨神经病理变化在阳性对照组实验兔病情高峰期表现为髓鞘脱失严重,轴索有损伤。经灌流治疗后,神经脱髓鞘现象明显减轻,这种改善在实验组2组表现的最为显著。阳性对照组在免疫后第18天电生理检查示神经传导速度轻度降低,在免疫后第35天进展到重度减慢;波幅和F波出现率为中度减低。经灌流治疗后,神经传导速度改善到中度减低,波幅和F波出现率转变为正常。坐骨神经免疫组实验兔在免疫后第5天即可检测到血清P2抗体,其血清浓度在阳性对照组总体上经历了一个先是急剧上升然后逐渐下降的曲线变化。在接受灌流治疗后,曲线变得较为平缓,曲线的这种变化在实验组2组体现地更为明显。阴性对照组实验兔灌流前后的血常规、血脂及电解质无显著性变化(p＞0.05)。灌流治疗对血清白蛋白无明显影响,但可降低血清球蛋白浓度。酶水解IgG可得F(ab’)2、Fc和Fab,吸附率分别为17.94±4.10%、2.48±2.63%和0.02±0.05%。结论:球形纤维素为载体色氨酸为配基的吸附剂血液相容性良好,对IgG具有非特异性的吸附作用,作用靶点可能为IgG的铰链区。同阳性对照组相比,全血灌流免疫吸附治疗可使免疫兔在病理、电生理和血清P2抗体浓度的检测上呈现出改善的趋势。基于此,可认为全血灌流免疫吸附治疗对EAN兔具有积极的治疗作用,且治疗效果同灌流次数有一定关系,这或许可为GBS提供新的治疗策略。更多还原

【Abstract】 Objective:Evaluate the therapy of extracorporeal whole blood immunoadsorption on experimental allergic neuritis and access the mechanism of cellulose-tryptophan on the adsorption.Methods:For optimize the immunogen of EAN rabbits,myelin basic protein (MBP)was extracted from bovine sciatic nerve.The New Zealand rabbit EAN model was induced by intradermic injection of MBP or bovine sciatic nerve.The healthy rabbits were divided into four groups,i.e.experimental group one(treated with immunoadsorption for one time at the day 14 after immunization),experimental group two(underwent immunoadsorption twice,at the day 14 and the day 16 after immunization),positive control group(EAN rabbits without any treatment)and negative control group(physiologic saline solution instead of MBP and underwent immunoadsorption at the same time with group two).According to Graham K,the clinical manifestation of the EAN rabbits was graded in 7 levels.The blood samples were taken from the rabbits before and after therapy for the assessment of the biocompatibility.To the negative and positive control groups,the electrophysiological functions,including motor nerve conduction velocity(MNCV),motor evoked potential amplitude,F wave were evaluated at the day 18 after immunization.All the rabbits were comprehensively examined,e.g.electrophysiological function and pathological changes,at the day 35 after immunonization.ELISA was introduced for the measurement of serum P2 protein antibody.The blood samples for the detection of P2 protein antibody were regularly drawn from all the rabbits,from the day 0 to the day 60 after immunization.F(ab’)~2,Fab,and Fc fragments of IgG from the health rabbits were achieved by pepsin and papain,for the further investigation on the mechanism of cellulose-tryptophan.Results:EAN was successfully induced either by MBP or sciatic nerve.EAN rabbits set up by the sciatic nerve undertook the extracorporeal whole blood immunoadsorption.In positive control group,symptoms climbed to the climax from the day 28 to the day 32,remitted from the day 35.00±0.63,and the mean duration was 60.80±0.84 days.The rabbits in the experimental group one developed the neurological signs of EAN at the day 14.25±0.46,ameliorated since the day 32.13± 0.64,the duration is 42.38±0.50.The experimental group two,rabbits recovered at the day 30.25±0.46,the duration is 38.38±0.52.The perivascular infiltration of macrophages and demyelination in the sciatic nerve were found in positive control group.Experimental group showed lighter pathological changes,especially in experimental group two.The positive control group,at the day 18,MNCV retarded obviously,amplitude fell down,the detection rate of F wave reduced and the electrophysiological function continued to deteriorate until the day 35.Compared with positive control group,the electrophysiological function in the experiment groups significant improved(p＜0.05).Anti-P2 IgG could be detected at the day 5 post-inoculation and plasma levels showed a sharply rise and gradual fell down throughout the experimental period in positive control group.No significant changes of blood cells,electrolytes and blood lipids were found.Serum albumin was not altered by adsorbent,but the concentration of globulin dropped down.The adsorption rate of F(ab’)~2 and Fc was 17.94±4.10%and 2.48±2.63%,no adsorption was observed with Fab.Conclusion:Imunoadsorbent with cellulose as carrier tryptophan as ligand was biocompatible with the blood system and had specific affinity with Globulin.It was inferred that cellulose-tryptophan most likely bond with the hinge region of IgG. Compared with positive control,experimental group displayed less severe clinical sign,faster recovery of electrophysiological and pathological characteristics of sciatic nerves.Based on these results,it can be concluded that extracorporeal whole blood immunoadsorption has therapeutical effect on EAN rabbits.更多还原