CR2-Crry预处理诱导神经干细胞在颅脑损伤中发挥神经保护作用

Transplantation of induced neural stem cells receiving CR2-Crry pre-treatment mediates neuroprotection following brain injury

目的探讨CR2-Crry预处理诱导神经干细胞(iNSCs)对颅脑损伤的神经保护作用。方法18只雄性成年C57BL/6小鼠制备颅脑损伤模型,神经功能缺损评分(NSS)4-8分的小鼠为颅脑损伤组,按照随机数字表法分为:CR2-Crry预处理iNSCs(CR2-Crry-iNSCs)组(6只)、磷酸盐缓冲液(PBS)预处理iNSCs(PBS-iNSCs)组(6只)和PBS组(6只)。于颅脑损伤后12 h分别将1×106个CR2-Crry-iNSCs、PBS-iNSCs或等体积PBS经立体定向移植到颅脑损伤小鼠脑内。于颅脑损伤后14 d处死小鼠,制备脑组织冰冻切片,并行双重免疫荧光和原位末端标记(TUNEL)染色,观察CR2-Crry-iNSCs和PBS-iNSCs对颅脑损伤小鼠脑内Crry、NeuN和TUNEL阳性细胞数量的影响。结果双重免疫荧光染色:与PBS组比较,PBS-iNSCs组和CR2-Crry-iNSCs组颅脑损伤小鼠脑内Crry和NeuN双阳性的神经细胞数量明显增加,差异具有统计学意义(P<0.05)。此外,与PBS-iNSCs组比较,CR2-Crry-iNSCs组颅脑损伤小鼠脑内Crry和NeuN双阳性的神经细胞数量明显增加,差异具有统计学意义(P<0.05)。TUNEL染色:与PBS组比较,PBS-iNSCs组和CR2-Crry-iNSCs组颅脑损伤小鼠脑内TUNEL和NeuN双阳性的神经细胞数量明显下降,差异具有统计学意义(P<0.05)。此外,与PBS-iNSCs组比较,CR2-Crry-iNSCs组颅脑损伤小鼠脑内TUNEL和NeuN双阳性的神经细胞数量明显下降,差异具有统计学意义(P<0.05)。结论CR2-Crry预处理iNSCs移植可增加颅脑损伤小鼠脑内神经细胞Crry表达,减轻补体介导的神经损伤。

Objective To study the effects of transplanted induced neural stem cells(iNSCs)receiving CR2-Crry pre-treatment on neuroprotection following brain injury.Methods Healthy adult male C57BL/6 mouse brain injury models were established and mice having an neurological severity scores(NSS)of 4-8 were enrolled in brain injury group(n=18).These brain injury animals were randomly divided into 3 groups:the CR2-Crry-iNSC group(n=6),the phosphate buffered solution(PBS)-iNSC group(n=6)and the PBS group(n=6).At 12 h after brain injury,CR2-Crry-iNSCs,PBS-iNSCs or PBS were separately injected into the brains of brain injury mice via stereotactic method.On day 14 after brain injury,mice were sacrificed for morphological analysis.Immunofluorescent staining and TdT-mediated dUTP nick and labeling(TUNEL)staining were utilized to determine the effects of CR2-Crry-iNSC and PBS-iNSC grafts on Crry^(+)/NeuN^(+)and NeuN^(+)/TUNEL^(+)cells in the brains of brain injury mice.Results Double-labeling experiments revealed that the level of Crry^(+)/NeuN^(+)neurons in the brains of the PBS group was significantly lower than that in CR2-Crry-iNSC and PBS-iNSC groups(P<0.05).Furthermore,the level of Crry^(+)/NeuN^(+)neurons was substantially higher in the CR2-Crry-iNSC group than in the PBS-iNSC group(P<0.05).In contrast,TUNEL staining showed that the level of NeuN^(+)/TUNEL^(+)neurons in the brains of the PBS group was significantly higher than that in CR2-Crry-iNSC and PBS-iNSC groups(P<0.05).Moreover,the level of NeuN^(+)/TUNEL^(+)neurons was substantially lower in the CR2-Crry-iNSC group than in the PBS-iNSC group(P<0.05).Conclusion Transplantation of iNSCs receiving CR2-Crry pre-treatment could increase the levels of Crry expression in neurons and alleviate complement-mediated neuronal damage following brain injury.