甲强龙在骨髓间充质干细胞移植治疗大鼠脊髓损伤中的影响
发布时间：2019-02-24 19:47
【摘要】：目的利用甲强龙(MP)药理学机制和骨髓间充质干细胞(BMSCs)生物特点,探讨甲强龙(MP)在骨髓间充质干细胞治疗大鼠脊髓损伤(SCI)中作用机制及其特点。方法取雄性SD大鼠4周龄4只,分别处死并分离股骨、胫骨获得骨髓,在全贴壁法下培养并鉴定第3代BMSCs,收集备用并用Brd U染色作细胞标记。取SD大鼠6月龄40只,随机等分为4组。A组(对照组)和B组(甲强龙治疗组)、C组(BMSCs治疗组)、D组(甲强龙+BMSCs联合治疗组),按上述分组采用改良Allen法制备T10脊髓损伤雄性大鼠模型,B组造模后立即尾静脉缓慢推注甲强龙(30mg/kg),C组造模后2 h注射Brd U标记的BMSCs悬液2 m L,D组给予以上两种干预方法。采用BBB评分法评估术后1d、3d、7d、10d、14d大鼠后肢神经功能状况,14d后处死各组大鼠并取出损伤部位的脊髓样本,分别进行TNF-α、IL-1β免疫组织化学染色,Tunel染色,各组统计5个高倍视野内TNF-α、IL-1β表达平均阳性细胞数并检测细胞凋亡指数;另外,对比C组和D组中BMSC存活率。用统计软件SPSS20.0分析。结果术后1 d,四组大鼠后肢神经功能较术后均无明显提高,组间BBB评分的比较差异无统计学意义(P0.05)。术后3 d,B组和D组BBB评分在较术后1 d均升高(P0.05),C组与A组仍无明显差异,与A组比较,B组和D组评分比较差异有统计学意义(P0.05);术后7、10、14天,四组均较前一时间点升高(P0.05),其中B,C,D组明显高于A组,差异有统计学意义(P0.05);D组明显高于B,C组,差异有统计学意义(P0.05);免疫组化染色显示B组和C组的TNF-α及IL-1β平均阳性细胞数均明显低于A组;D组明显高于B组和C组的TNF-α及IL-1β平均阳性细胞数;Brd U染色显示C组BMSC的阳性率为明显低于D组,两组比较差异有统计学意义(P0.05)。结论1、甲强龙和BMSCs移植均可改善SCI大鼠的神经功能恢复,以联合作用效果最佳;2、甲强龙和BMSCs均能降低局部TNF-α、IL-1β表达水平,减少损伤部位的细胞凋亡,甲强龙作用效果强于BMSCs,二者协同作用效果明显增强;3、甲强龙有效的提升了移植BMSCs的存活率。
[Abstract]:Objective to investigate the mechanism and characteristics of methylketene (MP) in the treatment of spinal cord injury (SCI) in rats by using the pharmacological mechanism and the biological characteristics of bone marrow mesenchymal stem cells (BMSCs). Methods four male SD rats of 4 weeks old were killed and separated from femur. Bone marrow was obtained from the tibia. The third generation of BMSCs, was cultured and identified with Brd U staining. Forty SD rats aged 6 months were randomly divided into 4 groups: group A (control group) and group B (), C group) (BMSCs group), D group). The male model of T10 spinal cord injury was established by modified Allen method. In group B, the tail vein was injected with methylenolone (Brd U labeled BMSCs suspension 2 mL) 2 hours after the model was established in group B. Group D was given the above two intervention methods. The neurological function of the hind limbs of the rats was evaluated by BBB scoring method on the 1st day, 3rd day, 7th day, 10d and 14th day after operation. After 14 days, the rats in each group were killed and the spinal cord samples of the injured area were taken out. TNF- 伪, IL-1 尾 immunohistochemical staining and Tunel staining were performed respectively. The average number of TNF- 伪 and IL-1 尾 positive cells and apoptosis index were measured in each group. In addition, the survival rate of BMSC in group C and group D was compared. Statistical software SPSS20.0 was used to analyze. Results on the 1st day after operation, the nerve function of the hind limbs of the four groups was not significantly improved than that of the control group, and the difference of BBB score between the four groups was not statistically significant (P0.05). The BBB scores in group B and group D were significantly higher than those in group A on the 3rd day after operation (P0.05), C and group A still had no significant difference, compared with group A, the scores of group B and group D were significantly higher than those of group A (P0.05); After 7 days 10 to 14 days, the four groups were higher than the previous time point (P0.05), among them, group D was significantly higher than group A (P0.05), the difference was statistically significant (P0.05) in); D group was significantly higher than that in group C (P0.05). Immunohistochemical staining showed that the average number of TNF- 伪 and IL-1 尾 positive cells in group B and C was significantly lower than that in group A, and the number of TNF- 伪 and IL-1 尾 in group D was significantly higher than that in group B and C. Brd U staining showed that the positive rate of BMSC in group C was significantly lower than that in group D, and the difference between the two groups was statistically significant (P0.05). Conclusion 1. Both methylenolone and BMSCs transplantation can improve the recovery of nerve function in SCI rats, and the combined effect is the best. (2) both methylenolone and BMSCs could decrease the expression of TNF- 伪 and IL-1 尾, and decrease the apoptosis of injured site. The effect of methylenolone was stronger than that of BMSCs,. 3. Methylenolone effectively improved the survival rate of BMSCs transplantation.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2
本文编号：2429873
[Abstract]:Objective to investigate the mechanism and characteristics of methylketene (MP) in the treatment of spinal cord injury (SCI) in rats by using the pharmacological mechanism and the biological characteristics of bone marrow mesenchymal stem cells (BMSCs). Methods four male SD rats of 4 weeks old were killed and separated from femur. Bone marrow was obtained from the tibia. The third generation of BMSCs, was cultured and identified with Brd U staining. Forty SD rats aged 6 months were randomly divided into 4 groups: group A (control group) and group B (), C group) (BMSCs group), D group). The male model of T10 spinal cord injury was established by modified Allen method. In group B, the tail vein was injected with methylenolone (Brd U labeled BMSCs suspension 2 mL) 2 hours after the model was established in group B. Group D was given the above two intervention methods. The neurological function of the hind limbs of the rats was evaluated by BBB scoring method on the 1st day, 3rd day, 7th day, 10d and 14th day after operation. After 14 days, the rats in each group were killed and the spinal cord samples of the injured area were taken out. TNF- 伪, IL-1 尾 immunohistochemical staining and Tunel staining were performed respectively. The average number of TNF- 伪 and IL-1 尾 positive cells and apoptosis index were measured in each group. In addition, the survival rate of BMSC in group C and group D was compared. Statistical software SPSS20.0 was used to analyze. Results on the 1st day after operation, the nerve function of the hind limbs of the four groups was not significantly improved than that of the control group, and the difference of BBB score between the four groups was not statistically significant (P0.05). The BBB scores in group B and group D were significantly higher than those in group A on the 3rd day after operation (P0.05), C and group A still had no significant difference, compared with group A, the scores of group B and group D were significantly higher than those of group A (P0.05); After 7 days 10 to 14 days, the four groups were higher than the previous time point (P0.05), among them, group D was significantly higher than group A (P0.05), the difference was statistically significant (P0.05) in); D group was significantly higher than that in group C (P0.05). Immunohistochemical staining showed that the average number of TNF- 伪 and IL-1 尾 positive cells in group B and C was significantly lower than that in group A, and the number of TNF- 伪 and IL-1 尾 in group D was significantly higher than that in group B and C. Brd U staining showed that the positive rate of BMSC in group C was significantly lower than that in group D, and the difference between the two groups was statistically significant (P0.05). Conclusion 1. Both methylenolone and BMSCs transplantation can improve the recovery of nerve function in SCI rats, and the combined effect is the best. (2) both methylenolone and BMSCs could decrease the expression of TNF- 伪 and IL-1 尾, and decrease the apoptosis of injured site. The effect of methylenolone was stronger than that of BMSCs,. 3. Methylenolone effectively improved the survival rate of BMSCs transplantation.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2
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