人胚胎神经干细胞延迟移植对脊髓损伤大鼠运动功能的影响及其机制

The influence and mechanism of delayed transplantation of human fetal neural stem cells on locomotor recovery after experimental spinal cord injury in rats

中文关键词:  脊髓损伤  神经干细胞  移植  功能恢复  大鼠

英文关键词:Spinal cord injury  Neural stem cells  Implantation  Functional recovery  Rat

中文摘要:

  【摘要】 目的：初步探讨人胚胎神经干细胞（human fetal neural stem cells，hfNSCs）延迟移植对脊髓损伤大鼠运动功能的影响及其机制。方法：体外分离、培养和鉴定hfNSCs，选取200~250g的Wistar大鼠28只建立T9~T11节段脊髓挫伤模型，损伤后第9天取20只BBB评分为4~5分的大鼠随机分为对照组及移植组（每组10只），同时在T10水平脊髓中线两侧1.5mm处分别给予2μl不含hfNSCs的杜氏磷酸缓冲液（DPBS）及2μl含有105 个体外培养第2代hfNSCs的DPBS，细胞移植后第7、14、28、42、56天用BBB评分评价两组后肢运动功能，移植后第28天及第56天用免疫组织化学法检测移植组大鼠损伤脊髓局部神经胶质纤维酸性蛋白（GFAP）、微管相关蛋白2（MAP2）和2′，3′-环腺苷酸-3′-磷酸二酯酶（CNPase）的表达情况，以评价大鼠损伤脊髓局部hfNSCs的存活及分化情况。移植后第56天髓磷脂碱性蛋白（MBP）染色及透射电子显微镜扫描观察移植组与对照组损伤局部髓鞘形成情况。结果：体外培养的hfNSCs表达Nestin，并可分化为星形胶质细胞[（61.2±1.6）%]、神经元[（27.6±3.4）%]及少突胶质细胞[（6.0±5.5）%]。移植前及移植后第7、14天两组BBB评分比较无统计学差异（P＞0.05），移植组在移植后第28、42、56天评分（分别为8.30±1.07分，11.30±1.05分，15.10±1.12分）较对照组（分别为7.00±0.98分，8.30±1.02分，9.10±0.96分）明显提高（P<0.05）。在移植后第28天hfNSCs可分化为GFAP阳性的星形胶质细胞、MAP2阳性的神经元和CNPase阳性的少突胶质细胞，在移植后第56天hfNSCs仍在损伤局部存活并可分化为星形胶质细胞和少突胶质细胞，但未见神经元分化；移植后第56天星形胶质细胞的积分光密度（IOD）值（1502.31±131.92）大于移植后第28天（628.98±119.31）（P<0.05），而少突胶质细胞在移植后第28天及第56天的变化无统计学差异。移植后第56天MBP染色证实人源性少突胶质细胞参与损伤区髓鞘的形成，透射电子显微镜扫描结果显示移植组有完整和连续的髓鞘形成，对照组髓鞘呈断裂及畸形表现。结论：hfNSCs延迟移植可促进脊髓损伤大鼠运动功能的恢复，其机制可能与促进损伤局部髓鞘的形成有关。

英文摘要:

  【Abstract】 Objective：To discuss the effect and mechanism of delayed transplantation of human fetal neural stem cells（hfNSCs） on the locomotor recovery of spinal cord injury.Method：The hfNSCs were separated，cultured and identified from abortive fetus in vitro.Then rat spinal cord injury（SCI） modelｓ（200-250g，n=28） wｅｒｅ established.At ｔｈｅ　9th day after injury，twenty rats with BBB scores between 4 and 5 were divided randomly into two groups with each group of 10 rats.2μl Dulbecco′s phosphate buffered saline（DPBS） with or without 105 2ｎｄ generation hfNSCs wａｓ　transplanted into the experimental and control groups（injection site：1.5mm lateral of central T10 spinal cord）.BBB score was used to evaluate the locomotor improvement between control and experimental group at the 7th，14th，28th，42th，56th day after transplantation.The expressionｓ of glial fibrillary acidic protein（GFAP），microtubule-associated protein 2（MAP2） and CNPase were evaluated by imｍunohistochemistry to observe the survival and differentiation of hfNSCs in the injured area.At ｔｈｅ　56th day after transplantation，the remyelination of two groups wａｓ evaluated by myelin basic protein（MBP） imｍunohistochemistry and transmission electron microscope.Result：The hfNSCs cultured in vitro expressed Nestin and differentiated into astrocytes[（61.2±1.6）%]，neurons[（27.6±3.4）%] and oligodendrocytes[（6.0±5.5）%] respectively.Experimental group（BBB scores：8.3±1.07 at 28th day and 15.1±1.12 at 56th day） had a better locomotor recovery than control group（BBB scores：7.0±0.98 at 28th day and 9.1±0.96 at 56th day）（P<0.05）.Immunohistochemistry showed presence of hfNSCs and differentiation of them into astrocytes and oligocytes on the 56th day after transplantation，yet no hfNSCs-derived neurons were found at that time.There were more astrocytes in ｔｈｅ　experimental group at the 56th daｙ（1502.31±131.92） than 28th days（628.98±119.31）.The numbers of oligocytes between ｔｈｅ　28th day and ｔｈｅ　56th day had no significant difference.The MBP stain at 56th day showed that hfNSCs-derived oligodendrocytes ｍｉｇｈｔ take part in the remyelination in injured area.According to the results of transmission electron microscope，compared with the control group which had broken and deformed myelin sheath，the experimental group displayed formation of more integrate and continous myelin sheath.Conclusion：Delayed hfNSCs transplantation may improve locomotor recovery by remyelination after SCI in experimental rat.