【摘要】 背景:有研究显示舒芬太尼联合神经干细胞移植联合治疗可以提高新生的神经纤维数量。目的:观察舒芬太尼干预对神经干细胞移植治疗脊髓损伤大鼠后肢功能恢复的影响。方法:（1）将100只成年雌性SD大鼠随机分为5组:正常对照组、模型组、舒芬太尼组、神经干细胞移植组和舒芬太尼+神经干细胞移植联合治疗组,每组20只。正常对照组大鼠不实施任何干预,其他4组参照改良的Allen’s重物打击法建立大鼠脊髓损伤模型;（2）造模6 h后,联合治疗组大鼠蛛网膜下腔注射10μL细胞浓度为1×10¹⁰ L^-1的神经干细胞悬液,腹腔注射100μL舒芬太尼（150μg/kg）;神经干细胞移植组大鼠蛛网膜下腔注射10μL细胞浓度为1×10¹⁰ L^-1的神经干细胞悬液,腹腔注射100μL生理盐水;舒芬太尼组蛛网膜下腔注射10μL的神经干细胞培养液,腹腔注射100μL舒芬太尼（150μg/kg）;模型组大鼠蛛网膜下腔和腹腔各注射10μL神经干细胞培养液和100μL生理盐水;（3）造模后72 h,RT-PCR检测脊髓损伤区周围AQP4、MMP9 mRNA的表达,TUNEL法检测细胞凋亡情况;（4）造模后第1,3天和第1,2,3,4周通过BBB评分、斜板实验进行运动功能评定;（5）造模后4周取材行苏木精-伊红染色,荧光显微镜观测CM-Dil标记的神经干细胞存活情况,荧光金逆行追踪观察脊髓神经纤维的再生与分布情况。结果与结论:（1）造模后72 h,与模型组、舒芬太尼组、神经干细胞移植组比较,联合治疗组脊髓损伤区AQP4、MMP9 mRNA表达和细胞凋亡率显著降低（P<0.05）;（2）造模2周后,联合治疗组运动功能评分优于舒芬太尼组和神经干细胞移植组（P<0.05）,舒芬太尼组和神经干细胞移植组优于模型组（P<0.05）;（3）造模后4周,模型组可见脊髓空洞形成,舒芬太尼组和神经干细胞移植组损伤区脊髓空洞较小,联合治疗组脊髓空洞几乎消失;（4）联合治疗组CM-Dil阳性细胞最多,神经干细胞移植组较多,舒芬太尼组和模型组未见阳性细胞;（5）联合治疗组荧光金阳性神经纤维数最多,舒芬太尼组和神经干细胞移植组次之,模型组最少;（6）结果表明,舒芬太尼通过促进移植神经干细胞的增殖,减少大鼠脊髓损伤区AQP4、MMP9表达和局部神经细胞凋亡,进而改善后肢运动功能。更多还原

【Abstract】 BACKGROUND: Studies have shown that the combined use of sufentanil and neural stem cell（NSC） transplantation can increase the number of newborn nerve fibers.OBJECTIVE: To investigate the effect of sufentanil on the hind limb function of rats with spinal cord injury after neural stem cell transplantation. METHODS:（1） Eighty adult female Sprague-Dawley rats were used to build spine cord injury model according to the modified Allen’s method and divided into model group, sufentanil group, NSCs transplantation group and sufentanil combined with NSCs transplantation group（combined group）. Extra 20 adult female Sprague-Dawley rats were not conducted any treatment as normal control group.（2） After 6 days of modeling, the model rats were subjected to subarachnoid injection of 10 μL of NSC culture medium and intraperitoneal injection of 100 μL of saline in the model group; subarachnoid injection of 10 μL of NSC culture medium and intraperitoneal injection of 100 μL of sufentanil（150 μg/kg） in the sufentanil group; subarachnoid injection of 10 μL of 1×10¹⁰/L NSCs suspension and intraperitoneal injection of 100 μL of saline in the NSCs transplantation group; and subarachnoid injection of 10 μL of 1×10¹⁰/L NSCs suspension and intraperitoneal injection of 100 μL of sufentanil（150 μg/kg） in the combined group.（3） After 72 hours of modeling, the AQP4 and MMP9 gene expression levels were detected by RT-PCR, and the cell apoptosis changes around the spine cord injury area were determined with TUNEL staining method.（4） The motor functions of rats were tested by Basso, Beattie and Bresnahan score and inclined plane test after 1, 3 days and 1, 2, 3 and 4 weeks of modeling.（5） After 4 weeks of modeling, the histopatholgical changes in the area of spine cord injury were observed by hematoxylin-eosin staining method. The survival changes of NSCs labeled by CM-Dil were determined by fluorescence microscope. The regenerations and distributions of spinal nerve fibers were observed by fluorescein gold retrograde tagging. RESULTS AND CONCLUSION:（1） After 72 hours of modeling, the AQP4 and MMP9 gene expression levels as well as the cell apoptotic rate in the combined group were significantly lower than those in the model, sufentanil and NSCs transplantation groups（P < 0.05）.（2） After 2 weeks of modeling, the combined treatment significantly improved the hind limb motor functions of rats compared with the sufentanil and NSCs transplantation groups（P < 0.05）, and the recovery of motor function was better in the sufentanil and NSCs transplantation groups than in the model group（P < 0.05）.（3） After 4 weeks of modeling, the results of hematoxylin-eosin staining manifested that the spinal cord tissues lost and the magnified syringomyelias occurred in the model group. The syringomyelias in the sufentanil and NSCs transplantation groups were significantly smaller than that in the model group, and the syringomyelias almost disappeared in the combined group.（4） The number of positive cells was the most in the combined group, more in the NSCs transplantation group, but there were no positive cells labeled by CM-Dil in the sufentanil and negative control groups.（5） The number of positive neural fibers in the combined group was the highest followed by the sufentanil and NSCs transplantation groups, and the number of positive neural fibers in negative control group was the lowest. To conclude, sufentanil can improve the recovery of hind limb motor function by reducing the AQP4 and MMP9 expression levels in the injury area, promoting the survival of transplanted NSCs, and decreasing the local NSCs apoptosis after spinal cord injury.更多还原