阻断Rho/Rock信号通路联合骨髓间充质干细胞对脊髓损伤大鼠神经功能修复的影响

Experimental study on the repair mechanism of spinal cord injury in rats with Rho/Rock pathway combined with bone marrow mesenchymal stem cells transplantation

目的探讨阻断Ras同源基因/Rock相关卷曲螺旋蛋白激酶(Rho/Rock)信号通路(采用信号通路阻断剂Fasdiual阻断)联合骨髓间充质干细胞(BMSCs)移植于脊髓损伤大鼠对神经功能修复的影响。方法选取80只8周龄SD(Sprague-Dawley)雄性大鼠,采用改良的Allen重物打击法制备脊髓损伤(SCI)大鼠模型后将大鼠随机分为4组(20只/组),根据不同干预措施分别为SCI模型组、BMSCs移植组、Fasdiual治疗组、BMSCs与Fasdiual联合治疗组。分别于SCI术后第1、7、14、21、28天采用BBB(basso-beattie-bresnahan)评分法对下肢运动功能恢复情况评分;于SCI术后第28天切取脊髓损伤节段脊髓标本制备切片,应用HE染色观察对比各组脊髓组织形态结构变化及脊髓空洞的面积;采用免疫组织化学染色及免疫荧光染色观察对比各组神经干样细胞特异性表达蛋白神经丝蛋白(NF200)、神经元性特异性烯醇化酶(NSE)和炎症表达蛋白胶质纤维酸性蛋白(GFAP)的表达情况。结果 SCI术后第1、7天各组BBB评分差异无统计学意义(P>0.05),随着观察时间的延长,各组SCI大鼠BBB评分逐渐升高;SCI术后第14、21、28天BMSCs移植组、Fasdiual治疗组及联合治疗组BBB评分均高于SCI模型组,且联合治疗组BBB评分高于BMSCs移植组及Fasdiual治疗组,差异均有统计学意义(P<0.05)。HE染色结果显示,SCI模型组脊髓空洞面积最大,BMSCs移植组、Fasdiual治疗组脊髓空洞面积小于SCI模型组,联合治疗组空洞面积最小,差异均有统计学意义(P<0.05)。免疫组化结果显示,NF200、NSE、GFAP在各组均有阳性表达。NF200、NSE在联合治疗组中阳性表达与其他各组相比差异有统计学意义(P<0.05),BMSCs移植组和Fasdiual治疗组中NF200、NSE阳性表达分别与SCI模型组相比差异均有统计学意义(P<0.05);联合治疗组中GFAP阳性表达与其余各组相比差异均有统计学意义(P<0.05),BMSCs移植组和Fasdiual治疗组中GFAP阳性表达与SCI模型组相比差异均有统计学意义(P<0.05)。免疫荧光染色显示各治疗组NF200、NSE荧光表达强于SCI模型组,以联合治疗组荧光表达最强;SCI模型组中GFAP表达均高于BMSCs移植组、Fasdiual治疗组及联合治疗组,其中联合治疗组表达强度最低。结论 Rho/Rock信号通路阻断剂Fasdiual和BMSCs移植均可促进SCI大鼠神经功能的修复,且两者联合应用促进脊髓修复方面上有协同作用,能更加有效促进SCI大鼠神经功能恢复。

Objective To explore the mechanism of blocking Rho/Rock pathway(signal pathway blocker Fasdiual) combined with bone marrow mesenchymal stem cells(BMSCs) transplanted into spinal cord injured rats. Methods Spinal cord injury(SCI) was employed on 80 SD(Sprague-Dawley) male rats using all improved simple device, imitating the Allen method, and the rats were randomly divided into 4 groups(20/group), according to the different intervention measures respectively as the SCI group, BMSCs transplantation group, Fasdiual treatment group, Fasdiual treatment group with BMSCs transplantation. After 1, 7, 14, 21 and 28 d after modeling, BBB(Basso-Beattie-Bresnahan) score was used to evaluate the motor function recovery of all rats. The spinal cord tissue morphological changes and syringomyelia area by HE staining between groups were compared. The chemical staining and immunofluorescence tissue staining were compared among the groups of neural stem cells express neurofilament protein(NF200) and neuron specific enolase(NSE) and inflammatory protein expression of glial fibrillary acidic protein(GFAP) expression. Results There was no significant difference in BBB score in SCI rats after 1 and 7 d(P>0.05). The BBB score of SCI rats in every group increased gradually with the observation time prolonged;on the 14 th, 21 st and 28 th day after SCI, the BBB scores of BMSCs transplantation group, Fasdiual treatment group and combined treatment group were higher than those of the SCI model group, and the BBB score of combined treatment group was higher than that of BMSCs transplantation group and Fasdiual treatment group, and the differences were statistically significant(P<0.05). HE staining showed that the area of the spinal cord cavity was the largest in the SCI group. The area of the spinal cord cavity in the BMSCs transplantation group and the Fasdiual treatment group were smaller than that in the SCI group. The area of the empty area in the combined treatment group was the smallest, and the difference was statistically significant compared with each group(P<0.05). The immunohistochemical results showed that NF200, NSE and GFAP were positive in all groups. The NF200 and NSE in the combined treatment group was significantly higher than that in the other groups(P<0.05). The positive expression of NF200 and NSE in the BMSCs transplantation group and Fasdiual group were significantly higher than that in the SCI model group(P<0.05). The positive expression of GFAP in the combined therapy group was significantly different from that in the other groups(P<0.05), The positive expression of GFAP in the BMSCs transplantation group and Fasdiual group were significantly different from that in the SCI group(P<0.05). Immunofluorescence staining showed that the expression of NF200 and NSE in each treatment group was stronger than that in the SCI group, and the flurescence expression in the combined treatment group was the strongest, and the expression of GFAP in the SCI group was higher than that in the BMSCs transplantation group, Fasdiual treatment group and combined treatment group, among which the expression intensity of GFAP in the combined treatment group was the lowest. Conclusion Both the Rho/Rock pathway inhibitor Fasdiual and BMSCs transplantation can promote the recovery of neural function in the SCI rats. Blocking the Rho/Rock pathway combined with bone marrow mesenchymal stem cell transplantation has synergistic effect in promoting the repair of spinal cord and more effectively promoting the recovery of neural function in the SCI rats.