摘要
背景:外泌体对神经细胞具有调控修复作用,从而促进神经细胞增生,减轻脊髓损伤,但外泌体对脊髓损伤水肿作用的相关研究较少。目的:探讨人脐带间充质干细胞来源外泌体减轻氧糖剥夺/复氧损伤大鼠脊髓星形胶质细胞水肿的作用及机制。方法:超高速离心法分离提取人脐带间充质干细胞外泌体,采用胰酶消化法提取新生SD大鼠脊髓星形胶质细胞。第一部分实验分组:①对照组,细胞正常培养,未进行氧糖剥夺/复氧处理;②模型组:氧糖剥夺6 h/复氧24 h;③人脐带间充质干细胞外泌体组:氧糖剥夺6 h/复氧24 h,在复氧过程的培养基中分别加入30,60,90μg人脐带间充质干细胞外泌体,作用24 h。Western blot检测细胞AQP4蛋白表达,活细胞工作站检测细胞体积,透射电镜观察细胞水肿超微结构变化。第二部分实验分组:①对照组:细胞正常培养,未进行氧糖剥夺/复氧处理;②模型组:氧糖剥夺6 h/复氧24 h;③人脐带间充质干细胞外泌体组:氧糖剥夺6 h/复氧24h,在复氧过程的培养基中加入90μg人脐带间充质干细胞外泌体,作用24h;④JNK受体抑制剂SP600125组:氧糖剥夺6 h/复氧24 h,在复氧过程的培养基中加入5μmol/L JNK受体抑制剂SP600125,作用24 h。Western blot检测细胞AQP4及p-JNK蛋白表达,活细胞工作站检测细胞体积。结果与结论:①与模型组比较,人脐带间充质干细胞外泌体干预24 h后,透射电镜可见线粒体、内质网水肿减轻,溶酶体数量减少,细胞水肿体积显著降低(P <0.05),AQP4蛋白的表达量减少(P <0.05);②与模型组比较,人脐带间充质干细胞外泌体与JNK抑制剂SP600125干预后,细胞中AQP4、p-JNK表达明显降低(P <0.05),细胞水肿体积显著降低(P <0.05);③结果表明,人脐带间充质干细胞外泌体可通过抑制JNK信号通路降低氧糖剥夺/复氧损伤后脊髓星形胶质细胞AQP4蛋白的表达,减轻细胞水肿。
BACKGROUND: Exosomes can regulate and repair nerve cells, thus promoting the proliferation of nerve cells to alleviate spinal cord injury.Exosomes have been less reported to alleviate edema after spinal cord injury.OBJECTIVE: To study the effect and mechanism by which human umbilical cord mesenchymal stem cell-derived exosomes(hucMSCs-exo)alleviate the edema of spinal astrocytes induced by oxygen-glucose deprivation/reoxygenation.METHODS: Ultrahigh speed centrifugation method was used to isolate and extract hucMSCs-exo. Spinal astrocytes were extracted from newborn Sprague-Dawley rats by trypsin digestion. Part One: The cells were(1) cultured normally in normal control group,(2) subjected to 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in model group, or(3) cultured in medium containing 30, 60, and 90 μg of hucMSCs-exo for 24 hours following 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in hucMSCs-exo group. Western blot assay was performed to detect the expression of AQP4. Living cell workstation was used to detect cell volume. Transmission electron microscope was used to observe the ultrastructure of intracellular edema. Part Two: The cells were(1) cultured normally in normal control group,(2) subjected to 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in model group,(3) cultured in medium containing 90 μg of hucMSCs-exo for 24 hours following 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in hucMSCs-exo group, or(4) cultured in medium containing 5 μmol/L JNK inhibitor sp600125 for 24 hours following 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in JNK inhibitor sp600125 group. AQP4 expression and p-JNK protein expression were measured by western blot assay. Living cell workstation was used to detect cell volume RESULTS AND CONCLUSION:(1) After 24-hour hucMSCs-exo intervention, mitochondrial and endoplasmic reticular edemas were alleviated, the number of lysosomes decreased, the volume of intracellular cell edema was significantly reduced, and the expression of AQP4 in the cells was significantly lowered as compared with the model group(P < 0. 05).(2) Intervention with hucMSCs-exo and JNK inhibitor SP600125 significantly reduced the expression of AQP4 and p-JNK(P < 0.05) and the volume of intracellular edema(P < 0.05) as compared with the model group. Therefore, hucMSCs-exo can reduce the expression of AQP4 protein in spinal astrocytes after oxygen-glucose deprivation/reoxygenation by inhibiting JNK signaling pathway, thereby alleviating cell edema.
作者
张勇
马迅
孙麟
张丽
关晓明
吕聪
陈旭
Zhang Yong;Ma Xun;Sun Lin;Zhang Li;Guan Xiaoming;Lu Cong;Chen Xu(Department of Orthopedics, Shanxi Dayi HospitalAffiliated to Shanxi Medical University, Taiyuan 030032, Shanxi Province, China;Department of Orthopedics, Xiangning County People'sHospital, Linfen 042100, Shanxi Province, China)
出处
《中国组织工程研究》
CAS
北大核心
2019年第25期4011-4017,共7页
Chinese Journal of Tissue Engineering Research
基金
山西省科技成果转化引导专项(201604D132044),项目负责人:马迅
山西省卫生和计划生育委员会青年项目(2015001),项目负责人:张丽
山西省卫生和计划生育委员会青年项目(201601012),项目负责人:关晓明~~
关键词
人脐带间充质干细胞外泌体
星形胶质细胞
脊髓损伤
氧糖剥夺/复氧损伤
细胞水肿
AQP4
JNK受体抑制剂
human umbilical cord mesenchymal stem cell-derived exosomes
astrocytes
spinal cord injury
oxygen-glucose deprivation/reoxygenation injury
intracellular edema
AQP4
JNK receptor inhibitor
