硫酸软骨素酶ABC及Nogo-66受体拮抗剂联合鼠胚神经干细胞移植对大鼠脊髓损伤的影响

Effects of ChABC, NEP1-40 combined with neural stem cell transplantation on the recovery of neurological function in rats with spinal cord injurey

中文关键词:  脊髓损伤  细胞移植  全反式维甲酸  神经干细胞  硫酸软骨素酶ABC  Nogo-66受体拮抗剂  大鼠

英文关键词:Spinal cord injury  ATRA  Neural stem cells  ChABC  NEP1-40  Cell transplantation  Rat

中文摘要:

  【摘要】 目的：研究硫酸软骨素酶ABC（chondroitinase ABC，ChABC）、Nogo-66受体拮抗剂[Nogo-66（1-40） antagonist peptide，NEP1-40]及鼠胚神经干细胞（neural stem cells，NSCs）联合移植对大鼠损伤脊髓功能恢复的影响。方法：体外应用全反式维甲酸（all-trans-retinoic acid，ATRA）（浓度1.0μmol/L）诱导NSCs（前期实验分离、培养并冻存的鼠胚NSCs），诱导后鉴定NSCs特异性标志物，移植前通过5-溴脱氧尿嘧啶核苷（5-Bromo-2-deoxyUridine，Brdu）标记NSCs。将60只SD大鼠随机分为假手术组（A组，n=10）、损伤对照组（B组，n=10）、NSCs治疗组（C组，n=10）、NSCs联合ChABC治疗组（D组，n=10）、NSCs联合NEP1-40治疗组（E组，n=10）、NSCs联合ChABC和NEP1-40治疗组（F组，n=10）。移植前分别对B、C、D、E及F组的大鼠制作胸段脊髓全横断损伤模型。术后3d，E和F组经留置的导管注入NEP1-40 20μl/d，连续28d；术后8d，C、D、E及F组经留置导管注入经ATRA干预和BrdU标记的NSCs 10μl；术后8d，D组和F组经留置导管注入ChABC 10μl/d，连续7d；各时间点通过留置导管注入生理盐水保持各组移植液等量。术前及术后不同时间点，用BBB评分和体感诱发电位（somatosensory evoked potentials，SEP）潜伏期对大鼠后肢神经功能进行评价。移植后8周通过HE染色观察损伤脊髓组织情况，免疫荧光染色观察移植NSCs存活、神经元分化及轴突生长情况。结果：体外应用1.0μmol/L的ATRA诱导NSCs培养，可提高NSCs向神经元分化的比例。移植后2周开始各组大鼠BBB评分、SEP潜伏期开始观察到改善，移植治疗各组均优于损伤对照组，组间BBB评分和SEP潜伏期有差异（P<0.05）；移植后2周、5周、8周各时间点，B组与C、D、E及F组比较，BBB评分和SEP潜伏期较差（P<0.05）；在移植治疗各组中，F组神经功能的恢复最明显，F组与C、D及E组比较具有较高的BBB评分和较短SEP潜伏期（P<0.05）。移植后8周，HE染色显示：A组细胞结构完整、排列规则；B组组织结构严重破坏，细胞排列紊乱，见大量较大的囊腔及胶质瘢痕形成；C、D、E及F组细胞增生明显，囊腔较少。免疫荧光染色显示：C、D、E及F组内可见橘红色Brdu标记阳性细胞，F组阳性细胞数多于C、D和E组，差异具有统计学意义（P<0.05）。C、D、E、F组微管相关蛋白-2（microtubule-associated protein 2，MAP-2）标记阳性细胞数多于B组，F组多于C、D、E组，差异有统计学意义（P<0.05）；C、D、E、F组MAP-2标记阳性细胞面积大于B组，F组大于C、D、E组，差异有统计学意义（P<0.05）。结论：ATRA诱导后NSCs移植可促进大鼠脊髓损伤功能的恢复，且联合ChABC和NEP1-40移植对损伤脊髓功能的恢复具有协同促进作用。

英文摘要:

  【Abstract】 Objectives: To investigate the effects of combined transplantation of chondroitinase ABC(ChABC), Nogo-66(1-40) antagonist peptide(NEP1-40) and rat embryonic neural stem cells(NSCs) on the recovery of neurological function in rats with spinal cord injury. Methods: The NSCs cryopreserved in our preliminary experiment were cultured and induced by all-trans-retinoic acid(ATRA)(the concentration of 1.0μmol/L) in vitro, the immunofluorescence techniques were used to detect the expressions of NSCs specific markers of glial fibrillary acidic protein(GFAP) and neuron-specific enolase(NSE). The NSCs were marked by 5-Bromo-2-deoxy Uridine(BrdU) before implantation. 60 adult Sprague-Daw female rats were randomly divided into six groups, namely, the sham operation group(group A, n=10), the injury control group(group B, n=10), the NSCs treatment group(group C, n=10), the NSCs combined with ChABC treatment group(group D, n=10), the NSCs combined with NEP1-40 treatment group(group E, n=10), and NSCs combined with ChABC and NEP1-40 treatment group(group F, n=10). Complete thoracic spinal cord transected injury models of rats were performed in all rats except group A before transplantation. On the third day after surgery, group E and group F were injected with NEP1-40 for 20μl/d through the indwelling catheter for 28 days consecutively; on the eighth day after surgery, group C, D, E and F were injected with 10μl of NSCs that were intervened with ATRA and labeled with BrdU through the indwelling catheter; and on the eighth day after surgery, group D and F were injected with ChABC for 10μl/d through the indwelling catheter for 7 days consecutively. Equal quantity of transplant liquid was kept through injection with normal saline through the indwelling catheter at all time points. Basso, Beattie, Bresnahan(BBB) score and somatosensory evoked potential(SEP) test were used to evaluate the functional changes preoperatively, postoperatively as well as 2 weeks, 5 weeks and 8 weeks after transplantation, respectively. The injured spinal cords were sampled for observing 8 weeks after transplantation and HE staining were performed to observe the morphological alteration in the injured spinal cord. The number, distribution and differentiat潩湯獮??呦栠整?瑡牮慳湰獬灡汮慴湥瑤愠瑎楓潃湳?潷晥?乥匠?獸?業湩摮略捤攠摢?戠祤??呢剬??捩慭湭?楮浯灦牬潵癯敲?瑳档敥?晣略渠捳瑴楡潩湮慩汮?爬攠换潹瘠敵牳祩?潧映?瑩档敲?却???楬湥?牡慳瑳獯??浡潴牥敤漠癰敲牯??捩潮洠戲椨湍敁摐?挲栩漠湡摮牤漠楂瑲楤湕愮猠敒?????慳渺搠?乔?偁ㄠ??ふ?桤愠獲?慭?獲祫湡敢牬杹椠獰瑲楯捭?整晥映整捨瑥?潤湩?瑦桥敲?牮整捩潡癴敩牯祮?潯晦?湎敓畃牳漠汩潮杴楯挠慮汥?晲畯湮捳琮椠漲渠?楥湥?牳愠瑡獦?睥楲琠桯?獥灲楡湴慩汯?挬漠牴摨?椠湲橥畣牯祶?ry of the BBB score and the SEP latent period were observed in SCI groups, and the recovery of all transplanted treatment groups was better than that of SCI model group. The BBB score and SEP latent period showed significant difference at any observing point after transplantation(P<0.05). BBB score of group B at each time point was significantly lower than that of group C, D, E and F, and SEP latent period was significantly longer than that of group C, D, E and F(P<0.05); the outcome of neurological functional recovery in group F remarkably surpassed than that of group C, D and E among all post-transplantation observing time points, and group F had higher BBB score and shorter SEP latent period than group C, D, E(P<0.05). 8 weeks after transplantation, HE staining in group A showed complete morphological structure and regular arrangement of cells; while the morphological structure was seriously damaged in group B, where irregular arrangement of cells as well as a large number of scar and cysts could be seen; and there were obvious cells and capillary hyperplasia and a few small cysts in group C, D, E, and group F in particular. The viable positive cells labeled by BrdU could only be observed in group C, D, E and F, and the number of Brdu-positive cells in group F were greater than that of group C, D and E(P<0.05), with the difference being statistically significant. Using Fluorescence microscope, the double positive cells for MAP-2 and BrdU could be detected in group C, D, E and F. The axonal regeneration and growth was distinctly promoted in group F. The number and surface area of positive cells labeled by MAP-2 showed significant difference(P<0.05), and group F had greater number of cells and larger surface area when compared with group A, B, C, D and E(P<0.05). Conclusi