超短波对脊髓损伤后内源性神经干细胞存活和分化的影响

关键词：脊髓损伤  超短波干预  内源性神经干细胞  BBB  Nestin  NeuN  GFAP

Keywords:spinal cord injury  ultrashort wave  endogenous neural stem cells  BBB  HE  Nestin  NeuN GFAP

摘要：

      摘要 目的：观察脊髓损伤后内源性神经干细胞的存活和分化的变化，并且观察超短波对这一变化的影响。 方法：用46只SD大鼠，被随机分成3组：假手术组,只暴露硬脊膜,不给予打击；对照组，给予Allen打击，并不做任何处理；干预组，给予Allen打击，并在SCI后24h开始给予小剂量超短波(11W)干预，每日1次7min直至取材前。大鼠的运动功能用BBB评分进行评定,大鼠取材后分别对组织进行横向切片，分别进行HE和免疫荧光染色，来检测脊髓形态和Nesin、GFAP、NeuN在组织内的表达部位和表达量，以观察内源性神经干细胞在脊髓损伤后并超短波干预后存活和分化为星形胶质细胞的情况。 结果：①BBB评分显示，3天后后肢功能评分逐渐恢复。干预组与对照组相比，3天、5天和7天的BBB评分并没有显著的差异，直到14天，干预组的后肢功能BBB评分高于对照组，并且差异显著（P<0.05）；②Nestin、GFAP、NeuN免疫荧光染色：在脊髓中央管，Nestin阳性细胞在损伤后第3天表达最多，并且随着损伤后时间的推迟，Nestin阳性细胞的表达量却是逐渐减弱的，到第14天表达水平与假手术组相当。GFAP阳性细胞在第7天表达最强，并且与Nestin共染的区域（黄色荧光）也是最多的。在损伤周围区白质、损伤头端和尾端的表达变化不一样，脊髓损伤对于脊髓头端和尾端的干细胞存活影响不一致，头端的存活高峰在第3天，尾端则在第7天，并且干预组Nestin阳性细胞存活的数量大于对照组的时间，头端在第3天，尾端在第14天；而分化为星形胶质细胞趋势，头端和尾端却是一致的，分化的高峰都在第7天，并且此时损伤尾端干预组Nestin阳性细胞数/GFAP阳性细胞数大于对照组；在损伤后第14天，干预组头端NeuN阳性细胞个数大于对照组，且差异有显著性意义。 结论：超短波能够减少脊髓损伤早期坏死面积、提高大鼠运动功能评分，有利于损伤后3天和14天神经干细胞的存活，但是损伤头端和尾端表现不尽相同。超短波干预也能够减少损伤后7天神经干细胞分化为星形胶质细胞数量。

Abstract:

      Abstract Objective:To observe the survival and differentiation of endogenous neural stem cells after spinal cord injury, and to observe the effects of ultrashort wave on this changes. Method: Totally 46 SD rats were randomly divided into 3 groups. The sham group was treated with spinal dura exposure and without shock. The control group was treated with spinal dura exposure and shock but without any treatment. Treatment group was treated with the same as the control group and with small dose of ultrashort wave therapy 24h after injury, 7min once daily until?sacrificed. Locomotor functional was assessed every wee with BBB score. At the end of the study, the segments of spinal cord encompassing the injury site were removed and the slices were taken either in the vertical or horizonal axis, for the following HE, immunochemistry and immunofluorescence of Nestin and GFAP, to observe the proliferation, migration and differentiation of the stem cells. Result: ①The BBB score in the ultrashort wave group was higher than that in the control group until the day 14, (P<0.05). ②Immunofluorescence in the central canal of spinal cord showed that Nesitn have the most high density at day 3 after injury ,whereas GFAP at the day 7 In the white matter, the numbers of Nestin+ and Nestin+/GFAP+ in the rostral and caudal of spinal cord are inconformity. On the 14th day after the injury, the number of NeuN+ cells in the treatment group was greater than that of the control group, and the difference was statistically significant. Conclusion：Spinal cord injury can promote the proliferation of stem cells in the central canal and migration of neural stem cells to epicenter. Small dose ultrashort wave therapy can promote neural functional recovery, reduce the necrosis area after damage.