Chinese Journal of Tissue Engineering Research ›› 2023, Vol. 27 ›› Issue (15): 2363-2370.doi: 10.12307/2023.371
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Lin Yanchen1, Li Jingjing2, Lu Guangxu3, Dai Erqing1, Chen Jing4, Zhong Shijiang4
Received:
2022-05-09
Accepted:
2022-06-27
Online:
2023-05-28
Published:
2022-10-18
Contact:
Chen Jing, Master, Associate chief physician, Department of Neurology, Characteristic Medical Center of the Armed Police, Tianjin 300162, China
Zhong Shijiang, MD, Chief physician, Department of Neurology, Characteristic Medical Center of the Armed Police, Tianjin 300162, China
About author:
Lin Yanchen, Master, Attending physician, Department of Rehabilitation Medicine, Characteristic Medical Center of the Armed Police, Tianjin 300162, China
Li Jingjing, Pharmacist, Department of Pharmacy, Tianjin Fourth Central Hospital, Tianjin 314000, China
Supported by:
CLC Number:
Lin Yanchen, Li Jingjing, Lu Guangxu, Dai Erqing, Chen Jing, Zhong Shijiang. Effects of neural stem cell transplantation in different ways on neuroinflammation in amyotrophic lateral sclerosis[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(15): 2363-2370.
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2.2 神经干细胞迁移、分化情况 免疫荧光显示,移植后21 d,2个移植组的绿色荧光蛋白转染神经干细胞均可迁移入中枢神经系统,且在3个解剖部位脑室内注射组的阳性细胞数均多于静脉注射组,即第三脑室周围[(5.67±0.13) vs. (2.99±0.07)个/mm2,P < 0.05],脊髓中央管[(2.68±0.06) vs. (0.54±0.04)个/mm2,P < 0.05],腰骶膨大处脊髓前角[(5.35±0.13) vs. (4.59±0.11)个/mm2,P < 0.05]。脊髓前角绿色荧光蛋白和NeuN的荧光双标显示,两组移植的神经干细胞均能分化为神经元,但数量均较少,无统计学差异[(0.89±0.02) vs. (0.82±0.02)个/mm2,P > 0.05]。两组移植源性神经干细胞在脊髓前角向神经元分化的比例也较小[(16±3)% vs. (17±2)%,P > 0.05],见图3。"
2.3 行为学表现 2.3.1 生存分析 脑室内注射组、静脉注射组、对照组的起病时间无明显差异[(87.63±4.35),(87.00±3.93),(87.86±3.62) d,P > 0.05),见图4A。生存时间方面,脑室内注射组最长,静脉注射组次之,对照组最短,但差异无显著性意义[(122.5±6.44),(120.75±5.87),(118.5± 6.88) d,P > 0.05),见图4B。KalPan-Meier生存分析也显示出相似结果(P > 0.05),见图4D。病程时间上,脑室内注射组最长,静脉注射组次之,对照组最短,但3组间也无统计学差异[(34.88±5.13),(33.75±4.85),(30.63±4.96) d,P > 0.05),见图4C。 2.3.2 运动功能检测结果 由于旋转时间和运动功能评分不满足球形检验,采用Greenhouse-Geisser统计方法。旋转实验和改良Wrathall评分均显示脑室内注射组的运动能力下降速度较对照组慢(P < 0.05),见图4E,F。与对照组相比,静脉注射组的运动能力下降速度有减慢趋势,但差异无显著性意义(P > 0.05)。与静脉注射组相比,脑室内注射组运动能力也有下降趋势,但差异无显著性意义(P > 0.05)。"
2.4.1 RT-PCR检测结果 脑室内注射组和静脉注射组促炎细胞因子肿瘤坏死因子α、白细胞介素1β、白细胞介素6的mRNA水平均低于对照组(P < 0.05),而抗炎性细胞因子转化生长因子β的mRNA水平高于对照组(P < 0.05)。与静脉注射组相比,脑室内注射组3种促炎细胞因子肿瘤坏死因子α、白细胞介素1β、白细胞介素6的mRNA水平较低,而抗炎细胞因子转化生长因子β的mRNA水平较高(P < 0.05),见图5A。 2.4.2 ELISA检测结果 脑室内注射组和静脉注射组的促炎细胞因子肿瘤坏死因子α、白细胞介素1β、白细胞介素6蛋白水平均低于对照组(P < 0.05),而抗炎细胞因子转化生长因子β水平高于对照组(P < 0.05)。与静脉注射组相比,脑室内注射组的3种促炎细胞因子肿瘤坏死因子α、白细胞介素1β、白细胞介素6的蛋白水平也较低,而抗炎细胞因子转化生长因子β的蛋白水平较高(P < 0.05),见图5B。"
2.5 脊髓小胶质细胞极化方向及NF-κB通路活性 腰骶膨大脊髓的Western blot显示,脑室内注射组和静脉注射组M1型小胶质细胞特异性标志物iNOS的表达水平均低于对照组(P < 0.05)。与静脉注射组相比,脑室内注射组iNOS表达更低(P < 0.05)。脑室内注射组和静脉注射组M2型小胶质细胞特异性标志物CD206的表达水平均高于对照组(P < 0.05)。与静脉注射组相比,脑室内注射组CD206表达更高(P < 0.05)。脑室内注射组和静脉注射组的p-IKK/IKK和p-P65/P65比值低于对照组(P < 0.05),脑室内注射组的p-IKK/IKK和p-P65/P65比值均低于静脉注射组(P < 0.05),见图6。"
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