丙戊酸联合骨髓间充质干细胞修复大鼠急性脊髓损伤可行性及其机制研究

The feasibility and the mechanism study about valproic acid combination with bone marrow mesenchymal stem cells to repair the acute spinal cord injury in rats

目的 探讨丙戊酸（VPA）联合骨髓间充质干细胞（BMSCs）促进大鼠急性脊髓损伤（SCI）修复的可行性及其作用机制.方法 选取3~4周龄健康雄性SD幼鼠5只,在无菌条件下取出双侧干骺端完整的胫骨及股骨,截除干骺骨端,暴露骨髓腔,收集骨髓细胞体外分离培养BMSCs,对第3代BMSCs进行流式细胞仪检测鉴定,并调配成浓度为1×106/mL的BMSCs细胞悬液脊髓移植备用.选取8周龄SD大鼠60只,按随机数字表法分为对照组、SCI模型组、VPA治疗组、BMSCs移植组、VPA＋BMSCs联合治疗组5组,每组12只.每组又按照术后取材时间不同随机分为第7、14天2个亚组,每个亚组6只.对照组12只大鼠仅显露T11脊髓背侧面不作SCI模型,其他4组（48只）大鼠采用改良Allen法制备脊髓损伤模型.模型制备成功后,对照组、SCI模型组在相应脊髓损伤节段一次性注入0.9%氯化钠注射液1 mL,同时300 mg/kg皮下注射0.9%氯化钠注射液,每12 h 1次,直至取材;VPA治疗组、BMSCs移植组除在脊髓损伤节段一次性注入0.9%氯化钠注射液1 mL外,分别给予皮下注射300 mg/kg VPA和1×106/mL的BMSCs细胞悬液1 mL,每12 h 1次,直至取材;VPA＋BMSCs联合治疗组在脊髓损伤节段一次性注入1×106/mL的BMSCs细胞悬液1 mL,同时给予皮下注射300 mg/kg VPA,每12 h 1次,直至取材.术后第12 h、7天、14天各组大鼠采用BBB评分法对下肢运动功能进行评定比较.第7天、14天BBB评分后,切取损伤脊髓节段脊髓组织制备切片,进行HE染色,光学显微镜下测量对比各组脊髓空洞的面积;免疫组织化学染色,观察对比术后第14天各组凋亡相关蛋白Caspase-3的表达情况;TUNEL染色观察对比术后第14天各组脊髓神经细胞凋亡情况,并计算对比脊髓神经细胞凋亡指数（AI）.结果 （1）显微镜下观察显示,第3代BMSCs细胞形态均一,呈长梭形;流式细胞仪检测显示,BMSCs特异性表面标志物CD34阳性率为0.83%,CD44阳性率为99.4%,提示BMSCs.（2）组内比较：与损伤前比较,对照组损伤后BBB评分差异无统计学意义;SCI模型组、VPA治疗组、BMSCs移植组、VPA＋BMSCs联合治疗组损伤后不同时间点BBB评分均明显降低,但随着时间延长BBB评分逐渐升高,差异均有统计学意义（P值均〈0.05）.组间比较：在损伤后的不同时间点,SCI模型组、VPA治疗组、BMSCs移植组、VPA＋BMSCs联合治疗组BBB评分均明显低于对照组,VPA治疗组、BMSCs移植组、VPA＋BMSCs联合治疗组的BBB评分均高于SCI模型组,VPA＋BMSCs联合治疗组BBB评分均高于VPA治疗组、BMSCs移植组,差异均有统计学意义（P值均〈0.05）.（3）对照组脊髓组织学形态正常;VPA治疗组、BMSCs移植组、VPA＋BMSCs联合治疗组脊髓空洞面积均小于SCI模型组,VPA＋BMSCs联合治疗组脊髓空洞面积小于VPA治疗组和BMSCs移植组,差异均有统计学意义（P值均〈0.05）.（4）在损伤后不同时间点对照组偶见Caspase-3表达,SCI模型组、VPA治疗组、BMSCs移植组、VPA＋BMSCs联合治疗组Caspase-3表达明显高于对照组,VPA治疗组、BMSCs移植组、VPA＋BMSCs联合治疗组Caspase-3表达明显低于SCI模型组,VPA＋BMSCs联合治疗组Caspase-3表达明显低于VPA治疗组和BMSCs移植组,差异均有统计学意义（P值均〈0.05）.（5）TUNEL染色检测脊髓细胞凋亡结果显示：SCI模型组、VPA治疗组、BMSCs移植组、VPA＋BMSCs联合治疗组脊髓细胞凋亡指数均大于对照组,VPA治疗组、BMSCs移植组、VPA＋BMSCs联合治疗组脊髓细胞凋亡指数均小于SCI模型组,VPA＋BMSCs联合治疗组脊髓细胞凋亡指数均明显低于VPA治疗组和BMSCs移植组,差异均有统计学意义（P值均〈0.05）.结论 VPA联合BMSCs移植可促进大鼠SCI修复,特别在减少脊髓空洞的形成、下调Caspase-3的表达、抑制神经细胞凋亡以及促进运动神经功能恢复等方面具有协同作用.

Objective To investigate the feasibility of valproic acid （VPA） combined with bone marrow mesenchymal stem cells （BMSCs） to promote restoration of the rat spinal cord injury （SCI） and its mechanism of action. Methods BMSCs from Sprague-Dawley （SD） rats were cultured in vitro. The third generation BMSCs were detected by flow cytometry and collected for use. Spinal cord injury model was Made by modified Allen＇s techniqtechnique. According to the random number table method sixty adult male SD rats were divided into five groups： control group, SCI model group, VPA treated group, BMSCs transplantation group and VPA combined with MBSCs group. At BMSCs transplantation group and VPA combined with MBSCs group, 1 mL BMSCS, it＇s concentration is 1 × 106 / mL, were injected into the spinal cord of injured Segment;while control group, SCI model group and VPA treated group received the same dose of normal saline at the same time point. VPA（300 mg/ kg） was administrated in rats through hypodermic injection immediately after injury, then repeated per 12 h until killing; while control group, SCI model group and BMSCs transplantation group received the same dose of normal saline at the same time point. The recovery of the locomotor function of each group was evaluated with basso Basso-beattieBeattie-bresnahan Bresnahan （BBB） scale at 7 d, and 14 d after injury, then the rats were killed. The sections were stained with hematoxylin and eosin （HE） for Syringomyelia area. The expression of Caspase-3 protein was detected with immunohistochemistry. Then the neuronal apoptosis was observed by TUNEL staining. Results （1） By the flow cytometry instrument detection third-generation cultured BMSCs can be stable expressed CD34 and CD44. （2）The motor function of the control group was not affected. The BBB score of the three treatment groups was significantly higher than that of the SCI model group（all P values 〈 0. 05）. The score of VPA combined with MBSCs group was significantly higher than that of VPA treated group and D on the 14th day after SCI（all P values 〈 0. 05）. （3）HE staining results showed the area of the lesion area of the four groups were respectively （4. 57 ± 0. 26）, （3. 34 ± 0. 21）, （3. 51 ± 0. 18） and （2. 43 ± 0. 35）, respectively. VPA combined with MBSCs group was significantly lower than the other three group（all P values 〈 0. 05）. （4）Immunohistochemical staining showed that a small amount of Caspase-3 expression was found in control group. The expression of Caspase-3 in three treatment groups was significantly lower than that in SCI model group（P 〈 0. 05）. While the expression of Caspase-3 in VPA combined with MBSCs group was significantly lower than that in VPA treated group and D（all P values 〈 0. 05）. （5）A small number of apoptosis-positive cells were found in control group at 14 days after injury. Compared with SCI model group, the apoptosis index of the three treatment groups was lower（all P values 〈 0. 05）. The apoptosis index of VPA combined with MBSCs group was significantly lower than that of VPA treated group and BMSCs transplantation group （all P values 〈 0. 05）. Conclusions Hypodermic injection of VPA for BMSCs transplantation promote SCI repair in rats is feasible, especially in the lower expression of Caspase-3, inhibition of neural cell apoptosis and promote motor nerve functional recovery effect is remarkable.