微囊化PC12细胞移植治疗帕金森病 微胶囊材料的生物学特征(英文)

Biological property of microencapsulating material in treatment of Parkinson disease with encapsulated PC12 cell 

背景:帕金森病系中脑黑质多巴胺能神经元变性,使纹状体内多巴胺减少而出现一系列临床症状。将分泌多巴胺的细胞或转染多巴胺合成限速酶基因的细胞进行脑内移植,能一定程度地逆转或改善症状,但存在免疫排斥的问题。目的:探讨在不使用免疫抑制剂的情况下,应用免疫隔离的方法进行细胞移植治疗帕金森病大鼠,观察微胶囊的生物相容性及其机械强度。设计:随机对照实验。单位:中国科学院大连化学物理研究所生物技术部生物医用材料工程组,吉林大学第二医院神经内科。材料:实验于2003-08/2004-02在吉林大学第二临床学院动物实验中心完成。选择雄性Wistar大鼠40只。PC12细胞由中国科学院上海细胞生物学研究所提供。方法:在纹状体内注射6-羟多巴胺盐溶液制备帕金森病动物模型。其中制备模型成功大鼠25只,随机分为微囊化细胞移植组12只:将25μL的微囊化细胞悬液(相当于2.5×104个细胞)分两点定位注射到动物模型右侧(损毁侧)纹状体内。非微囊化细胞移植组7只:植入25μL裸PC12细胞悬液(相当于5×104个细胞)。空微胶囊移植组6只:植入25μL的空微胶囊悬液。各组大鼠在移植后第7天注射阿朴吗啡(0.05mg/kg),然后记录每只大鼠的旋转行为,1次/周,共12周。术后12周处死大鼠,取脑组织进行病理学观察,回收微胶囊观察生物相容性和免疫隔离作用。主要观察指标:①微胶囊在大鼠纹状体内的生物相容性。②微胶囊的免疫隔离作用。③各组大鼠移植前后平均旋转圈数。结果:进入结果分析25只,其余均为模型制备不成功而脱落。①微胶囊的生物相容性:回收的海藻酸钠-壳聚糖-海藻酸钠微胶囊,形态完整,表面光滑,无炎细胞浸润。②微胶囊的免疫隔离作用:微胶囊内的PC12细胞继续增殖,部分形成了细胞团。③各组大鼠移植前后平均旋转圈数:移植前各组大鼠的单侧旋转记录无明显不同(P>0.05)。微囊化细胞移植组移植2周后平均旋转圈数明显低于移植前,甚至停止旋转,症状的改善保持至移植后12周。非微囊化细胞移植组的平均旋转圈数也明显低于移植前,但8,12周又有上升趋势,与移植前无明显变化(P>0.05)。空微胶囊移植组移植前后的旋转圈数基本一致。结论:①海藻酸钠-壳聚糖-海藻酸钠微胶囊有良好的生物相容性和机械强度,在移植部位不引发炎症反应,并保持形态的完整性。②海藻酸钠-壳聚糖-海藻酸钠微胶囊有免疫保护作用,包封的PC12细胞移植到帕金森病大鼠的脑内可以长期存活,继续保持正常的生理功能,通过合成和释放多巴胺,改善帕金森病大鼠的症状。

BACKGROUND： Parkinson disease（PD） is a series of clinical symptom induced by decreased dopamine （DA） in the striatum due to nigral dopaminergic neuronal degeneration. The intracerebral transplantation of secretory DA can reverse or improve the symptoms to a certain extent, but immunologic rejection is still existed. OBJECTIVE： To probe into cell transplantation with immunoisolation in treatment of in rats without application of immunosuppress and observe its mechanical intensity and the biocompatibility of microcapsule . DESIGN： Randomized controlled experiment was designed. SETTING： Biomedical Material Engineering Group, Dalian Institute of ChemicalPhysics , Chinese Academy of Sciences, and Department of Neurology, Second Hospital of Jilin University. MATERIALS： The experiment was performed in Animal Experimental Center of Second Hospital of Jilin University from August 2003 to February 2004, in which, 40 male Wistar rats were employed. PC12 cell was provided from Shanghai Institute of Cellular Biology of Chinese Academy of Sciences. METHODS： 6-hydroxydopamine solution was infused in the striatum to prepare animal model of Parkinson disease. Twenty-five rats of those had been prepared successfully and were randomized into microencapsulated cell transplantation group （12 rats）, in which, 25μL cell-loading sodium alginate-chitosan-solium alginate（ACA）microencapsul suspension （equal to 2.5×10^4 cells） was injected stereotaxically on two points of the fight （affected side） striatum of animal model; non-microencapsulated cell transplantation group （7 rats）, in which, 25μL PC12 cell suspension （equal to 5×10^4 ceils） was injected ; and empty microcapsul transplantation group （6 rats）, in which, 25μL empty microcapsules suspension was injected . On the Th day after transplantation, in every group, apomorphine （APO） prepared with saline solution was injected （0.05 mg/kg） subcutaneously in the neck; afterwards, the revolving behavior was recorded for each rat, once per week, totally for 12 weeks. In the 12^th week after operation, the rats were sacrificed with anesthesia. The brain tissue was collected for pathological observation and microcapsule were retrieved to evaluation of biocompatibility and immunoisolation. MAIN OUTCOME MEASURES：①Biocompatibility of microcapsule in striatum of rats. ② Immunoisolation of. mierocapsule,③Average revolving numbers before and after transplantation of each .group. RESULTS：Twenty-five rats entered result analysis and the rest was dropped out due to unsuccessful modeling.①Biocompatibility of microcapsule： the retrieved ACA microcapsule was integrative in morphology, smooth on surface and absence of inflammatory cellular infiltration.② Immunoisolation of microcapsule： mieroencapsuled PC12 cells were proliferated continuously and formed partially into cell mass.③Average revolving cycles before and after transplantation of each group： the records of lateral revolving of rats in every group before transplantation were not significantly different （P 〉 0.05）. In microencapsuled cell transplantation group, 2 weeks later, the average number of revolving was significantly Iower thanthat before the transplantation, or even the revolving stopped; the improved symptoms were maintained till the 12^th week after transplantation. In nonmieroeneapsulated cell transplantation group, the average revolving number was also significantly lower than that before the transplantation, but that on the 8^th and 12^th weeks was in tendency of increase, without obvious change compared with that before the transplantation （P 〉 0.05）. The revolving number before and after transplantation in non-mieroeneapsulated transplantation group was similar[（10.5±1.4）, （10.5±1.3） eyelos/min, P 〉 0.05]. CONCLUSION：① ACA microeapsule provides good bioeompatibility without inducing inflammatory reaction and mechanical intensity. ②ACA mieroeapsule provides immune protection. The grafted encapsulated PC12 cells survive for along term in the brain of rats with PD, maintain continuously the normal physiological function and improve the symptoms of PD by synthesizing and releasing DA.