人羊膜缓释支架联合神经干细胞治疗脊髓损伤

Drug delivery human amniotic membrane scaffold combined with neural stem cells transplantation in the treatment of spinal cord injury

目的探讨去上皮细胞羊膜（DMAM）构建药物缓释组织工程支架联合神经干细胞（NSCs）在大鼠脊髓全横断损伤模型中的治疗效果。方法取出生24h内的SD大鼠8只，剥离双侧海马分离、培养、纯化、鉴定NSCs备用。获取健康产妇羊膜，制备成DHAM。裁取两块DHAM4cm×4cm大小，吸取含5×10^9／LNSCs的细胞悬液分别种植其上。一块用2．5ml纤维蛋白黏合剂与18μg鼠神经生长因子的混合剂喷洒于其无细胞面，另一块单纯用18μg鼠神经生长因子喷洒于其无细胞面，卷成圆柱形支架后，分别制成DHAM药物缓释组织工程支架和无缓释功能的DHAM药物组织工程支架。将36只实验大鼠制作脊髓完全横断损伤模型，随机分为3组。A组为DHAM药物缓释组织工程支架移植组；B组为无缓释功能的DHAM药物组织工程支架移植组；C组为旷置缺损组。术后分别在1、2、4、8周对各组运动功能用脊髓损伤后后肢运动功能（BBB）评分系统进行评价。在第8周处死各组大鼠取材，行苏木素-伊红（HE）染色和镀银染色以及免疫组织化学染色观察形态学结果。结果术后8周，A组BBB评分达（11．63±0．58）分，明显高于其余两组（P〈0．05）。苏木素-伊红（HE）染色见A组大部分DHAM支架呈规则平行排列，DHAM支架内可见较多细胞存活生长。部分细胞发出丝状纤维沿DHAM支架间隙生长。B组DHAM支架不能保持平行排列的状态，呈波浪状或网状，细胞发出的丝状纤维混乱生长，缺乏方向。C组脊髓断端可见空洞形成。新长出的轴突杂乱交织在一起。神经元核抗原（Neun）抗体免疫组织化学染色显示部分存活的神经干细胞分化为神经元。镀银染色和神经丝蛋白-200（NF-200）抗体免疫组织化学染色证实支架内存活的细胞发出的丝状纤维为神经轴突。结论DHAM药物缓释支架联合NSCs组建的组织工程支架对大鼠脊髓缺损有良好的治疗潜力。

Objective To research the curative effect of a new medical biomaterial drug delivery denuded human amniotic membrane （DHAM） scaffold combined with neural stem cells transplantation in rat spinal cord injury model. Methods The NSCs were separated, cultured, purified and identified from bilateral hippoeampus of 24 - hour - old SD rats （ n = 8 ）. The human amniotic membrane was obtained from the healthy pregnant women and was prepared to DHAM. Two DHAM was cut into 4 by 4 cm in size. The NSCs （5 × 109 cells/L） were seeded on two DHAM. The mixture of fibrin - glue （2. 5 ml） and nerve growth factor （ 18μg） were aspersed on one no cell side of DHAM to construct drug delivery scaffold. The nerve growth factor （ 18μg） were purely aspersed on one no cell side of another DHAM to construct no drug delivery scaffold. Thirty - six rats were made into spinal cord injury models through spinal cord complete section, and were randomly divided into three groups. In group A, the drug delivery scaffold was transplanted in rat spinal cord injury model. In group B, the no drug delivery scaffold was transplanted. In group C, rats received no treatment. After 1, 2, 4, and 8 weeks of surgery, the hindlimbs functional recovery was evaluated by the Basso - Beattie - Bresnahan （ BBB ） locomotor rating score. Morphological changes were tested by Hematoxylin - Eosin （HE） staining, silver staining and immunohistoehemistry staining after 8 weeks of surgery. Results The BBB score was higher in group A （ 11.63 ± 0. 58 ） than in both other groups（ P 〈 0. 05 ）. HE staining showed different appearance in each group. In group A, most of DHAM arranged parallel. In the scaffold, there were many living cells. Some cells erupted silkiness fibers. And the silkiness fibers were growing in a directional manner. In group B, the DHAM did not keep the parallel arrangement. The DHAM arranged like waves or net. The silkiness fibers erupted by cells tended to tangle and did not have a directional manner. In group C, there were cavities in broken ends of spinal cords. The new born silkiness fibers tangled together. The results of Neun antibody immunohistochemical staining confirmed some living neural stem cells differentiated to neurons. The result of silver staining and NF- 200 antibody immunohistochemical staining showed the silkiness fibers erupted by cells were axons. Conclusion Drug delivery denuded human amniotic membrane scaffold combined with neural stem cells transplantation had therapeutic latent capacity in the treatment of spinal cord injury.