过表达微小RNA-223脊髓来源神经干细胞移植治疗大鼠脊髓损伤

Transplantation of microRNA-223 over-expression spinal cord-derived neural stem cells for spinal cord injury in rats

目的 探讨微小RNA（miRNA,miR）-223是否通过靶基因RhoB调控脊髓继发炎症性损伤,观察过表达miR-223脊髓来源神经干细胞（NSCs）移植的治疗作用.方法 以最佳感染复数（MOI）为10的慢病毒载体系统转染NSCs并移植治疗大鼠脊髓损伤模型,分为过表达miR-223-NSCs移植组（实验Ⅰ组）、空白载体NSCs移植组（实验Ⅱ组）和对照组（仅造模）.移植后12、24h、3d及7d,分别行实时荧光定量聚合酶链反应（FQ-PCR）及原位杂交检测miR-223和RhoB表达;酶联免疫吸附试验（ELISA）检测肿瘤坏死因子-α（TNF-α）和IL-6表达水平.结果 慢病毒转染后,NSCs表达miR-223倍数上升至6.9±1.9.FQ-PCR结果显示：移植治疗后,实验Ⅰ组在脊髓损伤部位表达miR-223逐渐上升,3d出现表达高峰（11.64±1.82）.各时间点实验Ⅱ组及对照组表达均较低,且两者间差异无统计学意义（P＞0.05）.RhoB在各时间点的表达趋势与miR-223相反：移植治疗后3d,表达下降至最低（0.31 ±0.08）.原位杂交直观地印证了FQ-PCR结果.ELISA结果显示：移植后12h,TNF-α和IL-6表达均达到高峰.TNF-α随着时间表达下降,各时间点均以实验Ⅰ组的表达最低.IL-6的表达在对照组及实验Ⅱ组损伤后3d出现第2个高峰,而在实验Ⅰ组则持续下降.结论 miR-223通过靶基因RhoB调控炎性因子TNF-α和IL-6的表达,过表达miR-223-NSCs移植可能具备脊髓损伤的治疗作用.

Objective To assess whether microRNA （miRNA,miR）-223 regulates secondary spinal cord inflammatory injury via the target gene of RhoB,and to evaluate the effects of transplantation of miR-223 over-expressed spinal cord-derived neural stem cells （NSCs） for spinal cord injury in rats.Methods NSCs were transfected by lentiviral vector system with optimal multiplicity of infection （MOI） being 10）,and then transplanted to treat spinal cord injury in rats.The experiment was divided into 3 groups,including group Ⅰ （NSCs over-expressed miR-223）,group Ⅱ （NSCs transfected by blank vector） and the control group （injury model only）.After 12 h,24 h,3 days and 7 days of transplantation,real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） and hybridization in situ （ISH） were performed to analyze the expression levels of miR-223 and RhoB.Meanwhile,enzyme-linked immunosorbent assay （ELISA） was used to detect the expression of tumor necrosis factor-α （TNF-α） and interleukin （IL）-6 in 3 groups.Results After transfection,the folds of miR-223 expression increased to 6.9 ± 1.9.In group Ⅰ,the expression of miR-223 in the site of spinal cord injury was increased with time,and reached the peak at day 3 （11.64 ± 1.82） by FQ-PCR analysis.There was no difference in the miR-223 expression between group Ⅱ and the control group （P 〉 0.05） at all time points.Both The expression of miR-223 in group Ⅱ and control group was lower than in group Ⅰ.On the other hand,the trend of RhoB expression was opposite to miR-223.After 3 days of transplantation,the fold change of expression was declined to the lowest （0.31 ±0.08）.ISH confirmed the FQ-PCR results intuitively.The results from ELISA showed that the expression levels of TNF-α and IL-6 reached the peak after 12 h of transplantation.The expression level of TNF-α was declined with time.It was the lowest in group Ⅰ at each time poing.After 3 days of transplantation,the expression of IL-6 reached the second peak in group Ⅱ and control group,while it was continually declined in group Ⅰ.Conclusion MiR-223 regulates the expression of TNF-α and IL-6 via the target gene of RhoB.Transplantation of miR-223 over-expressed NSCs might be useful to treat spinal cord injury in rats.