人脐带间充质干细胞移植联合小剂量超短波对大鼠脊髓损伤星形胶质细胞形成的影响

关键词：脊髓损伤  内源性干细胞  脐带间充质细胞移植  小剂量超短波治疗

Keywords:spinal cord injury  endogenous stem cell  umbilical cord mesenchymal cell transplantation  low- dose ultrashort wave therapy

摘要：

      摘要 目的：探讨人脐带间充质干细胞移植联合小剂量超短波治疗对大鼠脊髓损伤（SCI）星形胶质细胞形成的影响。 方法：将90只SD大鼠随机分成5组：假手术组（n=6）、对照组（n=21）、小剂量超短波（ultrashort wave，USW）治疗组（n=21）、人脐带间充质细胞移植组（human umbilical cord mesenchymal stem cells，hUCMSCs）（n=21）、USW治疗+hUCMSCs移植组（n=21）。hUCMSCs移植组、USW治疗+hUCMSCs移植组在损伤后立即使用微量注射器刺破硬脊膜垂直缓慢注入5μl含细胞密度为1×106/μl的细胞悬液； USW治疗组、USW治疗+hUCMSCs移植组大鼠在SCI后1d起采用对置法接受每天1次、每次7min的小剂量USW治疗。大鼠的运动功能用Basso Beattie Bresnahan评分（BBB评分）进行评定，SCI大鼠取材后分别对组织进行纵向切片行HE染色、免疫荧光染色，检测移植后hUCMSCs细胞存活及向星形胶质细胞分化，损伤区域附近内源性干细胞向星形胶质细胞分化及SCI后4周时纤维化形成的情况。 结果：①SCI大鼠造模后1—2周内损伤周围组织附近，由内源性干细胞分化的星型胶质细胞形成胶质屏障，发挥保护作用。②SCI14d，移入hUCMSCs细胞位于损伤区域内，部分移入的干细胞向星形胶质细胞去分化。损伤周围的星形胶质细胞多由内源性干细胞分化而来，人源干细胞特异性标记物MAB1281在此区域不表达。③USW+hUCMSCs移植组，USW协同hUCMSCs通过旁分泌作用改善损伤区域微环境，从而促进更多内源性干细胞聚集损伤处分化为星形胶质细胞，并阻碍坏死物质及炎症扩散（P<0.01 vs对照组、小剂量USW组、hUCMSCs移植组）。④USW+hUCMSCs移植抑制SCI后巨噬细胞等细胞产生的不利于轴突生长的纤维瘢痕，hUCMSCs移植在过程中扮演重要的作用（P<0.01 vs对照组、小剂量USW组）。⑤USW+hUCMSCs移植更好促进SCI后大鼠运动恢复（P<0.01vs对照组、小剂量USW组、hUCMSCs移植组）。 结论：小剂量USW治疗协同急性期人脐带间充质干细胞移植通过旁分泌作用治疗脊髓损伤大鼠，使损伤周围聚集更多内源性干细胞并分化为星形胶质细胞形成屏障抑制坏死物质与炎症扩散，从而促进运动功能恢复。

Abstract:

      Abstract Objective：To explore the effects of human umbilical cord mesenchymal stem cell transplantation combined with low dose ultrashort wave（USW） therapy on the formation of astrocytes after spinal cord injury in rats. Method： Ninty SD rats were randomly divided into 5 groups： sham operation group (n=6)， control group (n=21)， USW treatment group (n=21)， human umbilical cord mesenchymal stem cells（hUCMSCs） transplantation group (n=21)，and USW+hUCMSCs transplantation group (n=21). Spinal cord contusion was inflicted using modified Allen's method without any treatment. Cell transplantation group and USW+hUCMSCs transplantation group were punctured with a microinjector and 5ul cell suspension with cell density of 1×106/L was injected vertically and slowly. The control group and the low-dose USW group were punctured with a microinjector and 5ul fresh cell culture fluid was injected vertically and slowly. In the sham operation group， only exposed the T10 spinal cord without contusion and injection. Rats in the USW treatment group and USW+hUCMSCs transplantation group were treated with USW treatment once a day and 7min each time one day after SCI. The motor function of the rats was evaluated using BBB score. After sacrificed， SCI rats were subjected to longitudinal sections for HE staining and IF staining to detect the survival and differentiation of hUCMSCs cells after transplantation， the differentiation of endogenous stem cells into astrocytes near the injury area， and the formation of fibrosis at 4w after SCI. Result： ①Within 1-2w after modeling，the astrocytes differentiated by endogenous stem cells formed a glial barrier and played a protective role SCI rats around the injured area. ②SCI 14d， transplanted hUCMSCs cells located in the damaged area，and some transplanted stem cells differentiated into astrocytes. The astrocytes around the injury area were mostly differentiated from endogenous stem cells， and the specific marker MAB1281 of human stem cells was not expressed in this region. ③In the USW+hUCMSCs transplantation group， USW combined with hUCMSCs improved the microenvironment of the damaged area through paracrine， so as to promote more endogenous stem cell into damaged area and differentiate into astrocytes that hindered the accumulation of necrotic material and inflammation (P＜0.01 vs control group， low-dose USW group， and hUCMSCs transplantation group). ④After USW+hUCMSCs transplantation，the results showed the inhibition of macrophages and subsequent fibrous scar that was not conductive to axon growth. HUCMSCs transplantation played an important role in the process (P＜0.01 vs control group and low-dose USW group). ⑤USW+hUCMSCs transplantation could promote the motor recovery of rats after SCI (P＜0.01 vs control group， low-dose USW group， and hUCMSCs transplantation group). Conclusion：The rats with spinal cord injury are treated by low-dose USW therapy in combination with acute human umbilical cord mesenchymal stem cell transplantation by paracrine， which results in more endogenous stem cells gathering around the damaged area and differentiating into astrocytes that form a barrier to inhibit the proliferation of necrotic substances and inflammation， thus promoting the recovery of motor function.