CAJ | 학술논문

目的在体外细胞实验中探讨高渗盐水(hypertonic saline,HS)是否影响小胶质细胞的激活及释放单核细胞趋化蛋白-1 (monocyte chemotactic protein 1,MCP-1),以及其潜在的机制.方法体外混合培养原代胶质细胞,利用摇床震摇的方法纯化分离小胶质细胞,分为对照组、缺氧+无糖培养基组(简称缺氧组)、缺氧+无糖培养基+ 100 mmol/L HS组(简称HS组)及SB203580组(p38信号通路特异抑制剂).除对照组外,其他各组均进行氧糖剥夺(oxygen glucose deprivation,OGD),利用免疫荧光及ELISA方法检测HS对小胶质细胞生成及释放MCP-1的影响,并使用Western blot方法检测HS是否可影响p38丝裂原活化蛋白激酶(p38 MAPK)蛋白的磷酸化水平.数据采用单因素方差分析,用LSD法进行组间两两比较,P＜0.05为差异具有统计学意义.结果免疫荧光实验显示缺氧组与对照组比较,MCP-1表达明显增高;HS与缺氧组比较,MCP-1表达明显减少;ELISA检测发现缺氧4h后,SB203580组及HS组与缺氧组比较,细胞培养基上清中MCP-1含量(pg/mL)明显减少;SB203580组与HS组间差异无统计学意义(对照组:107.956±8.921;缺氧组:173.467±9.027; HS组:145.957±10.952; SB203580组:136.279±15.63; P＜0.05);western blot显示HS组各时间点与缺氧组各相应时间点相比,p38磷酸化水平均明显降低(对照组:0.590±0.105;缺氧组:30 min,1.553±0.215;1 h,1.212±0.161;2 h,1.179±0.105;4 h,1.178±0.122.HS组:30 min,1.028±0.168;1 h,0.852±0.112;2 h,0.927±0.121; 4h,0.815±0.107; P＜0.05).结论 100 mmol/L HS可能通过抑制p38信号通路的激活来抑制小胶质细胞的激活,从而减少小胶质细胞释放趋化因子MCP-1.
목적 재체외세포실험중탐토고삼염수(hypertonic saline,HS)시부영향소효질세포적격활급석방단핵세포추화단백-1 (monocyte chemotactic protein 1,MCP-1),이급기잠재적궤제.방법 체외혼합배양원대효질세포,이용요상진요적방법순화분리소효질세포,분위대조조、결양+무당배양기조(간칭결양조)、결양+무당배양기+ 100 mmol/L HS조(간칭HS조)급SB203580조(p38신호통로특이억제제).제대조조외,기타각조균진행양당박탈(oxygen glucose deprivation,OGD),이용면역형광급ELISA방법검측HS대소효질세포생성급석방MCP-1적영향,병사용Western blot방법검측HS시부가영향p38사렬원활화단백격매(p38 MAPK)단백적린산화수평.수거채용단인소방차분석,용LSD법진행조간량량비교,P＜0.05위차이구유통계학의의.결과 면역형광실험현시결양조여대조조비교,MCP-1표체명현증고;HS여결양조비교,MCP-1표체명현감소;ELISA검측발현결양4h후,SB203580조급HS조여결양조비교,세포배양기상청중MCP-1함량(pg/mL)명현감소;SB203580조여HS조간차이무통계학의의(대조조:107.956±8.921;결양조:173.467±9.027; HS조:145.957±10.952; SB203580조:136.279±15.63; P＜0.05);western blot현시HS조각시간점여결양조각상응시간점상비,p38린산화수평균명현강저(대조조:0.590±0.105;결양조:30 min,1.553±0.215;1 h,1.212±0.161;2 h,1.179±0.105;4 h,1.178±0.122.HS조:30 min,1.028±0.168;1 h,0.852±0.112;2 h,0.927±0.121; 4h,0.815±0.107; P＜0.05).결론 100 mmol/L HS가능통과억제p38신호통로적격활래억제소효질세포적격활,종이감소소효질세포석방추화인자MCP-1.
Objective To explore whether hypertonic saline (HS) could affect the microglial activation and release of monocyte chemotactic protein 1 (MCP-1) in vitro experiments,and to study the mechanism.Methods Microglia isolated from primary mixed glia cells by shaking method were divided into four groups:control group,hypoxia + glucose free medium group (hypoxia group),100 mmol/L HS + hypoxia + glucose free medium group (HS group) and SB203580 (a specific inhibitor of p38 MAPK) group.Oxygen-glucose deprivation (OGD) was performed in all experiment groups but control group.Double immunofluorescence and ELISA were used to determine whether HS could decrease the production of MCP-1 after OGD,and western blot was employed to determine the effect of HS on the level of phosphorylated p38 MAPK.Results Double immunofluorescence showed that 100 mmol/L HS attenuated MCP-1 level in HS group after treatment with 100 mmol/L HS compared with hypoxia group,and the level of MCP-1 in cultured medium released by microglia was significantly decreased after 100 mmol/L HS or SB203580 treatment for 4 h.There was no significant difference in MCP-1 between HS group and SB203580 group (P ＞ 0.05),but compared with control group (107.956 ± 8.921),there were higher levels of MCP-1 in hypoxia group (173.467 ±9.027),HS group:(145.957 ± 10.952),and SB203580 group (136.279 ± 15.63),P ＜0.05.At 30 min,1 h,2 h and 4 h after hypoxia,western blot showed that the levels of phosphorylated p38 MAPK markedly decreased in HS group as compared to the hypoxia group at corresponding intervals (control group:0.590±0.105.Hypoxia group:30 min,1.553 ±0.215; 1 h,1.212±0.161; 2 h,1.179 ± 0.105; 4 h,1.178 ±0.122.HS group:30 min,1.028 ±0.168; 1 h,0.852 ±0.112; 2 h,0.927 ±0.121; 4 h,0.815 ± 0.107; P ＜ 0.05).Conclusions HS decreasing MCP-1 level in microglia after hypoxia may be attributed to the inhibition of p38 MAPK signaling pathway.