海藻酸钠/壳聚糖微胶囊小鼠腹腔移植的生物学评价

Biological evaluation of transplantation of sodium alginate/chitosan microcapsule into abdominal cavity of rats

目的:实验证明海藻酸钠/壳聚糖微胶囊具有一定的通透性和免疫隔离特性,用于体外细胞培养取得了较好的效果,但植入体内出现了不同程度的细胞黏附和纤维化现象,因此对海藻酸钠/壳聚糖微胶囊作为细胞移植用的免疫隔离载体在体内的移植效应进行评价。方法:实验于2004-03/07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实验室完成。采用自制静电液滴发生器制备海藻酸钙凝胶微球,以一定浓度海藻酸钠溶液中和成膜后的海藻酸钙微球即得到海藻酸钠/壳聚糖微囊。将对数生长的L929细胞消化后与海藻酸钠溶液混匀,转移至静电液滴发生器,制备包封细胞微囊。取小鼠84只,雌雄各半,随机分为7组,每组12只。①对比观察材料对移植的影响:取3组小鼠,每组小鼠分别腹腔注射移植单纯的海藻酸钙胶珠、壳聚糖成膜的海藻酸钠微囊和最外层用海藻酸钠中和的壳聚糖/海藻酸钠微囊,在移植后的第7天和14天观察微囊回收率。②对比观察粒径对移植的影响:另取两组小鼠,分别移植粒径为200μm左右和粒径为500μm左右的海藻酸钠/壳聚糖微囊,于移植后1,4,14d观察微囊回收率。③对比观察细胞的存在对移植的影响:取两组小鼠分别移植空的微胶囊和包封有L929细胞的微胶囊。于移植后4,14d观察微囊回收率。回收率=(回收微囊体积/移植微囊体积)×100%。结果:84只小鼠均进入结果分析。①不同材料微囊回收率:壳聚糖成膜的胶珠引发的黏附最重,外层以海藻酸钠中和后的微囊其次,而单纯的海藻酸钠胶珠在所考察的时间内基本没有引起黏附。②不同粒径微囊回收率:粒径为200μm左右的微囊相对于粒径为500μm的微囊引发的黏附略有增加,但差别不明显,粒径200μm左右的微囊彼此聚集的特点导致回收率低于粒径为500μm左右的微囊。③在细胞微囊与空微囊回收率:与空胶囊比较,包封细胞的微囊黏附程度明显增加。结论:黏附程度随着粒径的增加而略有增加,壳聚糖是引发黏附的主要原因,细胞的存在提高了黏附程度。

AIM：The study certificates that the sodium alginate/chitosan microcapsule, which has certain permeability and immunity insulation peculiarit, gains better effects on the cell culture in vitro, but the phenomena of cell sticky and fibrosis emerge in different degree when implanted in vivo. Therefore, the transplantation effect of sodium alginate/ chitosan microcapsule as immunity insulation carrier used for cell transplantation was evaluated primarily. METHODS：The experiment was done in the Lab of Biomedical Material Engineering, Department of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences between March and July 2004. The self-restraint static electricity drip generator made the calcium alginate gelatum micro-beads. The sodium alginate solution of certain concentration neutralization, after making into membrane, with calcium alginate microbeads, the sodium alginate/chitosan microcapsule was gained. After the digestion of the logarithm growth L929 cells, they were mixed with the sodium alginate solutions, and then moved into the static electricity drip generator to make the close packing cell microcapsule. Eighty-four male and female （half and half） mice were selected and assigned randomly into 7 groups with 12 mice in each group.① To observe the effects of materials on the transplantation comparatively： The mice in three groups were obtained, and the mice in each group were treated with pure calcium algihate gelatum beads, sodium alginate of chitosan membrane and chitosan membrane/sodium alginate microcapsule that the extra-layer was neutralized with sodium alginate. The microcapsule retrievability rate was observed at the 7^th day and the 14^th day after transplantation. ②To observe the effects of granulation diameter on the transplantation comparatively： The other two group of mice were selected, and were treated with the sodium alginatc/chitosan microcapsule with around 200μm and around 500μm granulation diameter. The microcapsule retrievability rate was observed at the 1^st, 4^th, and 14^th days after transplatation.③ To observe the effects of the existence of the cells on the transplantation： Two groups of mice were gained and treated with empty microcapsule and packing microcapsule with L 929 cells. The microcapsule retrievability rate was observed at the 4^th and 14^th days after transplantation. The retrievability rate=（Retrieving microcapsule volume/transplantation microcapsule volume）×100%. RESULTS：Totally 84 mice were involved in the result analysis.①The microcapsule retrievability rate in different materials： The adherence induced by gelatum beads forming membrane of chitosan was the most serious.Followed by the microcapsule after neutralizing of the outer layer sodium alginate, while there was no adherence of the pure sodium alginate gelatum beads within the inspection time.② The microcapsule retrievability rate in different granulation diameter： The adherence induced by mi-crocapsule with around 200μm granulation diameter increased thanthat in-duced by microcapsule with around 500μm granulation diameter, but the difference was insignificant. The collective character of the microcap-sule with around 200μm granulation diameter led to the retrievability rate was lower than that of the microcapsule with around 500μm granulation diame-ter.③The retrievability rate of the cell microcapsule and the empty mi-crocapsule： Compared with the empty microcapsule, the adherence degree of the close packing microcapstde increased significantly. CONCLUSION： Adherence degree increases slightly with the diameter, The chitosan is the main component induce to adherence, and the existence of cell enhances the adherence degree.