不同次数移植BMSCs修复脊髓损伤的比较研究

TRANSPLANTATION OF BONE MARROW MESENCHYMAL STEM CELLS INTO SPINAL CORD INJURY:A COMPARISON OF DELIVERY DIFFERENT TIMES

目的观察经蛛网膜下腔不同次数移植的BMSCs在Wistar大鼠损伤脊髓内的存活、迁移及对大鼠脊髓损伤(spinal cord injury,SCI)修复的作用,探讨BMSCs移植的最佳次数。方法8只2月龄Wistar大鼠,体重约120g。取股骨骨髓体外分离培养第2代BMSCs,用Hoechst33342标记。另取75只成年Wistar大鼠体重约220g,采用改良Allen法制备T9、10SCI模型。随机分为5组(n=15),A组经蛛网膜下腔置管移植1次1×107个/mL BMSCs悬液0.1mL(术后1周),B组2次(术后1、3周),C组3次(术后1、3、5周),D组5次(术后1、3、5、7、9周),E组于术后1、3、5、7、9周各注入0.2mLPBS作为空白对照。SCI术后不同时间点行大鼠后肢Basso-Beattie-Bresnahan(BBB)评分评价神经功能状况,并行荧光显微镜、HE染色、免疫组织化学染色观察BMSCs在体内迁移、存活及分化情况。结果术后3周,A～D组BBB评分均有明显增加,与E组比较差异均有统计学意义(P<0.01)。术后7、9、12周,C、D组BBB评分均明显高于A、B组(P<0.01),B组高于A组(P<0.01)。各时间点C、D组间比较差异均无统计学意义(P>0.05)。荧光显微镜观察示术后3周,移植的BMSCs存活于损伤脊髓边缘,A组细胞数达高峰;随后A组细胞数逐渐减少,B、C、D组分别于术后5、7、7周细胞数达高峰后逐渐下降。12周C、D组存活细胞数显著多于A、B组,差异有统计学意义(P<0.01);各时间点C、D组间比较差异均无统计学意义(P>0.05)。HE染色示术后3周损伤脊髓形成的空洞A、B、C、D组逐渐减小,12周C、D组空洞最小,A、B组次之,E组最大。免疫组织化学染色示神经丝蛋白200(neurofilament 200,NF-200)细胞数变化趋势与BMSCs存活细胞数类似。术后12周NF-200阳性细胞数在C、D组表达较A、B组明显;神经元样细胞发出的神经丝穿过损伤区在C、D组最明显,A、B组较少。结论多次移植BMSCs更有利于SCI修复,移植以3次为最好。

Objective To investigate the influence of different transplantating times on the survival and immigration of the bone marrow mesenchymal stem cells（BMSCs）in injured spinal cord by subarachnoid administration,and to evaluate the most optimal subarachnoid administration times for BMSCs.Methods Eight adult male rats（weighing 120 g）were used to isolate BMSCs that were cultured,purified and labeled with Hoechst 33342 in vitro.Another 75 adult Wistar rats（weighing 220 g）were made the spinal cord injury（SCI）models at T9,10 level according to the improved Allen＇s method and were randomly divided into 5 groups（groups A,B,C,D,and E,n=15）.The labeled BMSCs at 1×107/mL 0.1 mL were injected into subarachnoid space of the rats via a catheters under the subarachnoid space in groups A（one time at 1 week）,B（two times at 1 and 3 weeks）,C（3 times at 1,3,and 5 weeks）and D（5 times at 1,3,5,7,and 9 weeks）and 0.2 mL phosphate-buffered saline（PBS）was injected in group E（5 times at 1,3,5,7,and 9 weeks）as blank control.The neurological functions were evaluated using the Basso-Beattie-Bresnahan（BBB）scale 1,3,5,7,9,and 12 weeks after transplantation.The migration,survival,differentiation,and histomorphological changes of BMSCs were observed by HE,immunohistochemistry,and fluorescence microscopy.Results At 3 weeks after injury,there were significant differences in the BBB scores between group E and groups A,B,C,D（P0.01）,and between groups A,B and groups C,D（P0.01）.At 7,9,and 12 weeks,the BBB scores were significantly higher in groups C and D than in groups A and B（P0.01）,and in group B than in group A（P0.01）.There were no significant differences in the BBB scores between groups C and D（P0.05）.The fluorescence microscopy showed that the transplanted BMSCs survived and grew in the injured region at 3 weeks after injury and as time went on,the transplanted cells gradually decreased in group A;in groups B,C,and D,BMSCs count reached the peak values at 5 and 7 weeks and then gradually decreased.At 12 weeks,the survival BMSCs were significantly more in groups C and D than in groups A and B（P 0.01）.HE staining showed that the formation of cavity was observed in each group at 3 weeks after injury and the area of cavity gradually decreased in groups A,B,C,and D.At 12 weeks,the area of cavity was the miximal in groups C and D,moderate in groups A and B,and the maximal in group E.The immunohistochemistry staining indicated that the expression of NF-200 was more intense in groups C and D than in groups A and B.The expression of NF-200-positive fibers was more intense in group C.Conclusion Multiple administration of BMSCs promotes the restoration of injured spinal cord and improves neurological functions,and three times for BMSCs transplantation is best.