含种子细胞的丝素组织工程神经移植物修复大鼠脊髓损伤

Repair of spinal cord injury by using the tissue engineered nerve grafts constructed by silk fibroin / filament including seed cells

目的 探讨体外构建含种子细胞的蚕丝丝素组织工程神经移植物（TENGs）的方法,评价其对大鼠脊髓损伤修复的影响。方法 分离大鼠皮肤前体细胞并向施万细胞诱导分化,S-100免疫荧光染色鉴定。将皮肤前体细胞诱导分化的施万细胞（SKP-SCs）作为种子细胞,联合蚕丝丝素神经导管和纤维支架共培养。共培养7d后将蚕丝丝素TENGs置入大鼠背侧T8-T10半横断损伤的脊髓中,于术后不同时间点利用BBB评分观察行为学的变化,术后8周取材,切片,免疫荧光染色观察脊髓损伤修复情况以及种子细胞的存活情况。结果 相差显微镜下体外培养的SKP-SCs大部分细胞形态呈双极或3极,免疫荧光染色显示,SKP-SCs呈S-100阳性,将SKP-SCs与蚕丝丝素支架材料共培养,蚕丝丝素支架表面均匀贴附大量的细胞,生长状态良好。将该移植物移植入大鼠T8-T10半横断损伤脊髓处,术后BBB评分显示,从4周起至8周均优于对照组,且结果具有统计学差异;术后8周时取材切片仍能观察到大鼠体内有大量种子细胞存活。结论 含种子细胞的蚕丝丝素组织工程神经移植物对于修复大鼠脊髓损伤具有一定的促进作用。

Objective To explore a method for construction of tissue engineered nerve grafts （tissue-engineered nerve grafts, TENGs ） using silk fibruin/filament in vitro and to evaluate their roles fur repairing spinal curd injury. Methods Isolation and induction of rat skin derived precursors into Schwann cells （SCs）. S-100 immunofluorescence histochemical staining was applied for the SCs identification. As seed cells, SCs differentiated from skin derived precursors （SKPs） cultured with silk fibroin conduit and silk filament scaffold in vitro. After culturing for 7 days, the TENGs were transplanted into the injury site of rat dorsal spinal cord （T8-T10） hemisection model. BBB score was evaluated at different time points after the operation. The spinal cord samples were obtained 8 weeks after the operation and immunofluorescence histochemical staining was applied to evaluate the regeneration of the injured spinal cord and the survival of seed cells. Results Under a phase contrast microscope, most of the seed cells were bipolar or tripolar. The immunofluorescence staining showed that SKP-SCs were S-100 positive. After the transplantation of the TENGs into spinal cord injury site, BBB scores were significantly higher in TENGs group than in control group from 4 weeks tu 8 weeks. The GFP-positive seed cells were also investigated after 8 weeks after the operation. Conclusion The TENGs constructed by silk fibroin/filament including seed cells may promote repairing of rat spinal cord injury.