移植自聚合肽纳米纤维材料与RhoA-siRNA复合物修复脊髓损伤

Self-assembling peptide nanofiber scaffold combined with RhoA-siRNA for repair of spinal cord injury

背景:脊髓损伤后RhoA表达增高是神经再生困难的主要原因之一,本课题在前期研究证明自聚合肽纳米纤维材料能较好地促进脊髓损伤后结构与功能修复的基础上,构建了自聚合肽纳米纤维材料与RhoA-siRNA复合材料用于修复脊髓损伤。目的:探讨自聚合肽纳米纤维材料介导siRNA干扰RhoA表达促进脊髓损伤修复。方法:54只昆明小鼠随机数字表法分为4组。假手术组仅作脊髓暴露,其他3组在切除1mm脊髓组织制备全横断脊髓损伤模型后,分别于损伤腔内填充生理盐水、自聚合肽纳米纤维支架或含有RhoA特异性siRNA复合物。通过FAM标记检测siRNA转染效率;免疫组化检测RhoA表达;免疫组化和定量分析检测再生神经纤维;行为学检测评定后肢功能恢复。结果与结论:移植FAM标记的含有RhoA特异性siRNA复合物后,在脊髓内的神经纤维及大脑皮质运动区神经元胞体内均可检测到FAM荧光信号,提示siRNA可从复合材料中释放到组织中并成功导入靶细胞。与SAPNS组和生理盐水组相比,含有RhoA特异性siRNA复合物和自聚合肽纳米纤维支架组可显著降低RhoA在神经元的表达,提高脊髓损伤区内NF阳性神经纤维的密度,促进后肢功能的恢复。结果提示通过自聚合肽纳米纤维支架的介导,含有RhoA特异性siRNA复合物能有效干扰RhoA表达,从而促进脊髓损伤的修复。

BACKGROUND： Up-expression of RhoA after spinal cord injury is one of main reasons of neural regeneration failure. Based on our previous report that self-assembling peptide nanofiber scaffold （SAPNS） could effectively promote the repair of structure and function after spinal cord injury, the complex of SAPNS and RhoA-siRNA was constructed to repair the injured spinal cord. OBJECTIVE： To explore the promotion of SAPNS-mediated siRNA interference RhoA expression on repair of spinal cord injury. METHODS： A total of 54 Kunming mice were randomly divided into 4 groups： sham group, saline group, SAPNS group and siRNA＋SAPNS group. After transection spinal cord injury model was prepared by removed 1mm spinal cord tissue, saline, SAPNS or the complex of siRNA＋SAPNS was filled into the lesion cavities in saline group, SAPNS group and siRNA＋SAPNS group. Spinal cord was exposed in sham group.Then, the siRNA transfection efficiency was detected by FAM fluorescent signal; the RhoA expression was detected by immnohistochemistry; the axonal regeneration was examined by neurofilament （NF） immnohistochemistry and behavioral test. RESULTS AND CONCLUSION： After transplantation of FAM-siRNA＋SAPNS, the FAM fluorescent signal could be detected in the nerve fiber in the spinal cord and the neuronal bodies in motor cortex, which indicated the siRNA could be released from the complex and successfully entered into the target cells. Compared with the SAPNS group and saline group, RhoA-siRNA＋SAPNS transplantation could significantly reduce the RhoA expression in the neurons, increase the densities of NF positive nerve fiber in the spinal cord injury area, and improve the function of the hindlimbs of the spinal cord injured mice. The results indicated that mediated by SAPNS, RhoA-specific siRNA can effectively interfere the expression of RhoA, in order to promote the repair of spinal cord injury.