三维培养间充质干细胞来源的分泌组物对海水浸泡合并创伤小鼠海马神经元的保护作用  

Protective effect of secreted secretomes derived from three-dimensional cultured mesenchymal stem cells on mouse hippocampal neurons with seawater immersion and trauma

作　　者：兰晓娟 谢胜强 王秉贤 马文静 陈巧汨 程岗 吕文英 张剑宁 Lan Xiao-Juan;Xie Sheng-Qiang;Wang Bing-Xian;Ma Wen-Jing;Chen Qiao-Mi;Cheng Gang;Lyu Wen-Ying;Zhang Jian-Ning(Naval Clinical College,the Fifth Clinical Medical School,Anhui Medical University,Hefei,Anhui 230032,China;Department of Neurosurgery,the Sixth Medical Center of Chinese PLA General Hospital,Beijing 100048,China;Department of Neurosurgery,the First Medical Center of Chinese PLA General Hospital,Beijing 100853,China;Institute of Military Cognition and Brain Sciences,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Hengyang Medical College,University of South China,Hengyang,Hunan 421001,China;School of Basic Medicine,Anhui Medical University,Hefei,Anhui 230032,China)

机构地区：[1]安徽医科大学第五临床医学院海军临床学院,安徽合肥230032 [2]解放军总医院第六医学中心神经外科,北京100048 [3]解放军总医院第一医学中心神经外科医学部,北京100853 [4]军事科学院军事医学研究院军事认知与脑科学研究所,北京100850 [5]南华大学衡阳医学院,湖南衡阳421001 [6]安徽医科大学基础医学院,安徽合肥230032

出　　处：《解放军医学杂志》2025年第3期324-331,共8页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(T2394483)。

摘　　要：目的 探究三维培养的间充质干细胞(MSCs)来源的分泌组物对海水浸泡(SW)合并拉伸损伤(SI)小鼠海马神经元的保护作用,为SW合并创伤性脑损伤(TBI)后的神经元修复提供新思路。方法 采用悬滴法培养MSCs,收集含有MSCs来源的分泌组物的条件培养基(CM)。采用小鼠海马神经元(HT22细胞)建立SW合并SI细胞模型。将HT22细胞随机分为对照组、SI组、SI+SW组、SI+CM组与SI+SW+CM组。采用CCK-8法检测各组细胞活力,流式细胞术检测细胞凋亡率,细胞划痕实验检测细胞迁移能力,Western blotting检测凋亡相关蛋白Bcl-2、Bcl-2关联X蛋白(Bax)、铁死亡相关蛋白长链酰基辅酶A合酶4(ACSL4)、环氧合酶-2(COX-2)的表达水平。结果 15%浓度海水浸泡12 h后,HT22细胞活力明显下降(P<0.05)。CCK-8法检测结果显示,处理12 h后,与对照组比较,SI组和SI+SW组HT22细胞活力均明显下降(P<0.0001);经MSCs分泌组物的CM处理后,SI+CM组细胞活力明显高于SI组(P<0.0001),且SW+SI+CM组细胞活力明显高于SI+SW组(P<0.001)。流式细胞术检测结果显示,SI组和SI+SW组细胞凋亡率明显高于对照组(P<0.05或P<0.001),而SI+CM组细胞凋亡率明显低于SI组(P<0.05),SI+SW+CM组细胞凋亡率明显低于SI+SW组(P<0.05)。Western blotting检测结果显示,与对照组比较,SI组和SI+SW组Bcl-2表达水平明显降低(P<0.01或P<0.0001),Bax、ACSL4和COX-2表达水平明显升高(P<0.01或P<0.001或P<0.0001);与SI组比较,SI+CM组Bcl-2蛋白表达水平明显升高(P<0.05),而Bax、ACSL4和COX-2蛋白表达水平明显降低(P<0.05);与SI+SW组比较,SI+SW+CM组Bcl-2表达水平明显升高(P<0.01),Bax、ACSL4和COX-2蛋白表达水平明显降低(P<0.01或P<0.001)。细胞划痕实验结果显示,12 h和24 h时,SI组和SI+SW组细胞迁移率明显低于对照组(P<0.01或P<0.0001),而SI+CM组细胞迁移率明显高于SI组(P<0.0001或P<0.01),SI+SW+CM组细胞迁移率明显高于SI+SW组(P<0.0001)。结论 悬滴法培养的MSCs分泌组物可�Objective To investigate the protective effects of secretomes released by three-dimensional cultured mesenchymal stem cells(MSCs)on neurons subjected to seawater immersion(SW)and stretch injury(SI),and to provide new insights into neuronal repair following SW combined with traumatic brain injury(TBI).Methods MSCs were cultured using the hanging drop method,and the conditioned medium(CM)containing MSCs secretomes was collected.A cellular model combining SW with SI was established using mouse hippocampal neuronal cells(HT22 cells).HT22 cells were randomly assigned to five groups:control,SI,SI+SW,SI+CM,and SI+SW+CM groups.Cell viability was assessed using the CCK-8 assay,apoptosis rate was measured by flow cytometry,cell migration ability was evaluated by scratch assay,and the expression levels of apoptosis-related proteins Bcl-2 and Bcl-2-associated protein(Bax),and ferroptosis-related proteins long-chain acyl-CoA synthetase 4(ACSL4)and cyclooxygenase-2(COX-2)were detected by Western blotting.Results Immersion in 15%seawater for 12 h significantly decreased HT22 cell viability(P<0.05).The CCK-8 assay indicated that cell viability in both the SI and SI+SW groups was significantly lower than that in control group after 12 h of treatment(P<0.05).Treatment with CM containing MSCs secretomes significantly increased cell viability in SI+CM group compared to SI group(P<0.0001),and in SI+SW+CM group compared to SI+SW group(P<0.001).Flow cytometry results revealed that the apoptosis rate in SI and SI+SW groups was significantly higher than that in control group(P<0.05 or P<0.001),while in SI+CM group was lower than that in SI group(P<0.05),and in SI+SW+CM group was lower than that in SI+SW group(P<0.05).Western blotting showed that compared to control group,SI and SI+SW groups exhibited reduced Bcl-2 expression level(P<0.01 or P<0.0001)and increased expression levels of Bax,ACSL4,and COX-2(P<0.01 or P<0.0001).Compared to SI group,the SI+CM group displayed increased Bcl-2 expression level(P<0.05)and decreased expression leve

关 键 词：间充质干细胞 分泌组物 海水浸泡 创伤性脑损伤 

分 类 号：R651.15[医药卫生—外科学]