转化生长因子-β1 siRNA神经干细胞后期移植对大鼠脊髓损伤修复的影响

Effect of TGF-β1 Gene Silenced Neural Stem Cells Transplantation on Repair of Spinal Cord Injury in Rats

目的:探讨转化生长因子-β1(TGF-β1)siRNA鼠神经干细胞(NSCs)后期移植对大鼠脊髓损伤修复的影响。方法:TGF-β1 siRNA质粒病毒转染至NSCs,转染时分对照组、转染对照组、转染组共3组,Western blot检测NSCs被转染后TGF-β1蛋白的表达。选取成年雌性SD大鼠78只,建立大鼠急性脊髓损伤模型,随机分为对照组,NSCs移植组及TGF-β1-siRNA组。通过BBB评分、斜板试验、改良的Tarlov评分进行运动功能评定。造摸后1周通过RT-PCR、Western blot检测脊髓损伤区周围突触素及TGF-β1基因转录和蛋白的表达。造模后4周,HE染色及荧光显微镜观测CMDil标记的NSCs存活及分布情况,HRP逆行示踪评估神经纤维再生情况,透射电镜观察各组脊髓损伤区超微结构改变。结果:Western blot检测显示TGF-β1低表达可持续14 d。造模后1周起,大鼠下肢运动功能评价TGF-β1-siRNA组优于NSCs移植组,NSCs移植组优于对照组。HE染色NSCs移植组损伤区脊髓空洞较小,TGF-β1-siRNA组脊髓空洞几乎消失,对照组可见脊髓组织缺失及脊髓空洞形成。TGF-β1-siRNA组TGF-β1 mRNA表达为5.33±0.24,较对照组(10.47±0.54)和NSCs移植组(8.06±0.36)显著降低(P <0.05),突触素的表达为11.36±0.28,明显高于NSCs移植组(8.27±0.33)及对照组(5.04±0.30);移植后4周,对照组HRP阳性神经纤维数(48.66±8.94个/高倍视野)和CM-Dil阳性细胞(0±0.00个/高倍视野)最少,TGF-β1-siRNA组最多,分别为(162.46±16.65个/高倍视野)、(38.68±4.89个/高倍视野),NSCs移植组次之,分别为(85.86±11.24个/高倍视野)、(22.38±3.45个/高倍视野)。在透射电镜观察下,TGF-β1-siRNA组大鼠脊髓组织损伤区可见大量有髓神经纤维及无髓神经纤维,轴突数量较多,再生轴突髓鞘完整;NSCs移植组可见较多有髓神经纤维及无髓神经纤维;对照组损伤区可见胶质瘢痕和少量有髓神经纤维,坏死的有髓神经纤维。结论:TGF-β1siRNA神经干细胞能有效降低脊髓损伤区TGF-β1表达,促进突触素表达,可促进脊髓损伤大鼠神经突触的再生,改善大鼠肢体运动功能。

objective： To investigate the effects of TGF- β 1 gene silenced neural stem cells （NSCs） transplantation on the repair of spinal cord injury in rats. Methods The TGF- β 1 siRNA plasmid virus was tra_nsfected to NSCs, and the TGF-β 1 protein expression of NSCs after transfected was detected by Western blot. Seventy-eight adult female SD rats were selected to construct the acute spinal cord injury model, and randomly divided into control group, NSCs transplantation group and TGF- β 1-siRNA group. The exercise function was evaluated by BBB score, slant plate test and improved Tarlov score. TGF- β 1 expression of the tissue around the spinal cord injury area was detected by RT-PCR and Western blot after one week. Four weeks after model preparation, the survival and distribution of NSCs labeled CM-Dil were observed by HE staining and fluorescence microscopy. HRP retrograde tracer was used to evaluate nerve fiber regeneration and transmission electron microscopy was used to observe the ultrastructural changes in the spinal cord injury areas of each group. Results Western blot tests showed that the low expression of TGF- β 1 lasted for 14 d. The lower limb motility evaluation of TGF- β 1 -siRNA group was better than that of NSCs transplantation group, and that of NSCs transplantation group was better than that of control group. The spinal cord voids in the injured area of the NSCs transplantation group were small, and the spinal cord voids in the TGF- β 1-siRNA group almost disappeared. In the control group, spinal cord tissue loss and spinal cord voids were seen. TGF-β 1 mRNA expression of TGF-β 1-siRNA group was 5.33 ± 0.24, significantly lower than those of control group（10.47 ± 0.54） and NSCs transplantation group（8.06 ± 0.36） （P 〈0.05）.The expression of the Syn was 11.36 ± 0.28, which was significantly higher than those of the NSCs transplantation group（8.27 ± 0.33） and the control group（5.04 ± 0.30）; Four weeks after transplantation, the number of HRP-positive nerve fibers in the control group（48.66 ± 8.94 / high magnification field） and CM- Dil-positive cells（0.00 ± 0.00/high magnification field） were decreased compared with the TGF-β 1-siRNA group [（162.46 ± 16.65 /high magnification field） and （38.68 ± 4.89 /high magnification field）], and the NSCs transplantation group [（85.86 ± 11.24 / high magnification field）, and（22.38 ± 3.45 / high magnification field）]. Under transmission electron microscope observation, there were a lot of myelinated nerve fibers and nonmyelinated nerve fibers in the spinal cord tissue injury area of TGF- β 1 -siRNA rats. In NSCs transplantation group, there were more myeloid nerve fibers and no myeloid nerve fibers. The injury area of control group showed glial scar and a small amount of rnyeloid nerve fibers and necrotic myeloid nerve fibers. Conclusion TGF- β 1 gene silenced neural stem cells can effectively reduce TGF- β 1 expression and enhance Syn expression in spinal cord injury region, promote the regeneration of neural synapses and improve limb motor function in rats with spinal cord injury.