NT-3-HUMSCs联合基因沉默SOCS3治疗SD大鼠脊髓损伤后的神经再生修复

目的 NT-3-HUMSCs联合基因沉默SOCS3治疗SD大鼠脊髓损伤, 以期促进损伤神经再生修复.方法 (1) 用贴壁法体外培养人脐带间充质细胞 (HUMSC) , 同时进行分离, 提纯和鉴定; (2) 构建NT-3基因真核表达载体, 利用基因转染技术将其转入HUMSC, 构建NT-3-HUMSC细胞, 体外检测其存活情况及NT-3表达情况; (3) 筛选作用于SOCS3的特异性靶点, 进行序列同源性分析, 设立阴性对照, 设计并合成SOCS3-siRNA, 同时在体外检测功能; (4) 建立SD大鼠脊髓损伤模型分为为:I.假手术组10只;Ⅱ.T12全脊髓横断损伤模型40只, 随机分为4组, 生理盐水治疗组10只;siRNA+NT-3-HUMSCs治疗组10只;NT-3-HUMSCs治疗组10只;SOCS3-siRNA治疗组10只.以上各组造模成功后, 分别存活12周进行神经电生理监测; (5) 对SD大鼠进行灌注固定和取材, 观察局部胶质疤痕降解情况和轴突再生情况, 同时运用生物素化葡聚糖 (BDA) 荧光顺行追踪取损伤移植区-宿主交界处头尾侧脊髓组织, 镜下观察皮质脊髓束再生的情况.结果 (1) siRNA+NT-3-HUMSCs治疗组较生理盐水治疗组横断脊髓空洞明显缩小, 差异有统计学意义 (P<0.05) ; (2) BDA顺行追踪结果表明siRNA+NT-3-HUMSCs治疗组较生理盐水治疗组神经轴突生长明显; (3) 神经电生理检测损伤12周后治疗组P40潜伏期较假手术组缩短, siRNA+NT-3-HUMSCs治疗组较生理盐水治疗组比较潜伏期缩短明显, 波幅升高明显, 差异有统计学意义 (P<0.05) .结论 NT-3-HUMSCs联合基因沉默SOCS3治疗SD大鼠脊髓损伤, 可以促进损伤神经再生修复.

Joint Therapy by NT-3-HUMSCs and SOCS3 Gene Dilencing in Nerve Regeneration Repair after Spinal Cord Injury in SD Rats

Objective To investigate the effect of joint therapy by NT-3-HUMSCs and SOCS3 gene silencing in promoting the injury nerve regeneration repair after spinal cord injury in SD rats. Methods (1) Adherence method was used to culture human umbilical cord-derived mesenchymal cells (HUMSC) in vitro for separation, purification and identification. (2) We constructed NT-3 gene eukaryotic expression vector, and used gene transfection technology into its HUMSC, and tested the survival of NT-3-HUMSC cells and NT-3 expression in cells. (3) We screened specific targets of SOCS3, made sequence homology analysis, and set a negative control, designed and synthesized siRNA and detected the function. (4) SD rats model of spinal cordinjury were established and divided into: 1. sham group 10; 2.T12 whole spinal cord injury model 40, were randomly divided into four groups, respectively; saline treatment group 10; siRNA + NT-3-HUMSCs treatment group 10; NT-3-HUMSCs treatment group 10; siRNA treated group 10. After each group above modeling success, they received respectively the neural electrophysiological monitoring for 12 weeks survival. (5) We perfused SD rats for fixation and collect samples, and observed the local glial scar degradation situation and axon regeneration, meanwhile, used biotin glucan fluorescent (BDA) anterograde tracing. The injury transplant area-host junction spinal cord tissues were collected to observe the corticospinal tract regeneration under microscope. Results (1) In siRNA + NT-3-HUMSCs treatment group, the transection syringomyelia was significantly reduced as compared with normal saline group (P < 0.05) . (2) BDA anterograde tracing results showed that in the siRNA + NT-3-HUMSCs treatment group, neural axon grew significantly compared with the normal saline group. (3) Neural electrophysiological testing 12 weeks after injury: in the treatment group, the incubation period P40 was shorter as compared with control group; in siRNA + NT-3-HUMSCs treatment group, the incubation period was shorter obviously than normal saline, but the amplitude increased obviously (P < 0.05) . Conclusion NT-3-HUMSCs joint with SOCS3 gene silencing can promote the injury nerve regeneration repair in the treatment of SD rat spinal cord injury.