CAJ | 학술논문

目的探讨染铅星形胶质细胞条件培养液(ACM)对神经元突触形成影响,为揭示铅神经毒性的机制提供参考数据.方法星形胶质细胞在含50、100、200、400和800 μmol/L醋酸铅的培养液中培养72 h后,AlamarBlue法测细胞活力并收集ACM.将原代培养的神经元分成纯培养组、非谷氨酸诱导的ACM处理组、谷氨酸诱导无染铅ACM处理组和谷氨酸诱导的50、100和200 μmol/L染铅ACM处理组,分别在培养的24、48和72 h后,收集神经元,用蛋白质印迹法(Western blot)法检测神经元内突触素蛋白含量,采用免疫荧光法测定培养72 h后神经元内SYP的表达.结果各染铅组的细胞活力随染铅浓度的升高而明显下降,差异有统计学意义(P＜0.05).Western blot法检测分析结果显示,与纯培养组比较,各时段的非谷氨酸诱导的ACM处理组和谷氨酸诱导无染铅ACM处理组的神经元内突触素蛋白含量明显增加,差异有统计学意义(P＜0.01),与非谷氨酸诱导的ACM处理组比较,谷氨酸诱导的ACM处理组神经元内突触素蛋白表达显著减少,差异有统计学意义(P＜0.05),与谷氨酸诱导无染铅ACM处理组比较,培养72 h的各铅暴露的ACM处理组的神经元内突触素蛋白含量随染铅剂量升高而明显降低,差异有统计学意义(P＜0.01),培养48 h的200 μmol/L染铅ACM处理组的神经元内突触素蛋白含量明显降低,差异有统计学意义(P＜0.01),培养24 h的各染铅的ACM处理组的神经元内突触素蛋白含量未见明显改变.突触素免疫荧光双标染色结果显示,培养72 h的各染铅的ACM处理组的神经元内突触素荧光颗粒数明显减少,差异有统计学意义(P＜0.05).结论星形胶质细胞对神经元的突触形成具有促进作用,染铅可抑制星形胶质细胞对突触形成的促进作用.
목적 탐토염연성형효질세포조건배양액(ACM)대신경원돌촉형성영향,위게시연신경독성적궤제제공삼고수거.방법 성형효질세포재함50、100、200、400화800 μmol/L작산연적배양액중배양72 h후,AlamarBlue법측세포활력병수집ACM.장원대배양적신경원분성순배양조、비곡안산유도적ACM처리조、곡안산유도무염연ACM처리조화곡안산유도적50、100화200 μmol/L염연ACM처리조,분별재배양적24、48화72 h후,수집신경원,용단백질인적법(Western blot)법검측신경원내돌촉소단백함량,채용면역형광법측정배양72 h후신경원내SYP적표체.결과 각염연조적세포활력수염연농도적승고이명현하강,차이유통계학의의(P＜0.05).Western blot법검측분석결과현시,여순배양조비교,각시단적비곡안산유도적ACM처리조화곡안산유도무염연ACM처리조적신경원내돌촉소단백함량명현증가,차이유통계학의의(P＜0.01),여비곡안산유도적ACM처리조비교,곡안산유도적ACM처리조신경원내돌촉소단백표체현저감소,차이유통계학의의(P＜0.05),여곡안산유도무염연ACM처리조비교,배양72 h적각연폭로적ACM처리조적신경원내돌촉소단백함량수염연제량승고이명현강저,차이유통계학의의(P＜0.01),배양48 h적200 μmol/L염연ACM처리조적신경원내돌촉소단백함량명현강저,차이유통계학의의(P＜0.01),배양24 h적각염연적ACM처리조적신경원내돌촉소단백함량미견명현개변.돌촉소면역형광쌍표염색결과현시,배양72 h적각염연적ACM처리조적신경원내돌촉소형광과립수명현감소,차이유통계학의의(P＜0.05).결론 성형효질세포대신경원적돌촉형성구유촉진작용,염연가억제성형효질세포대돌촉형성적촉진작용.
Objective To investigate the effect of lead-exposed astrocyte conditioned medium (ACM) on the synaptic formation of neurons and to provide reference for the mechanism of lead neurotoxicity.Methods Astrocytes were cultured in the medium containing 50,100,200,400,and 800 μmol/L lead acetate for 72 h.Alamar Blue was used to assess the cell viability of astrocytes,and then ACM was collected.Primarily cultured neurons were divided into six groups:pure culture group,non-glutamic acid (Glu)-induced ACM treatment group,Glu-induced lead-free ACM treatment group,and Glu-induced 50,100,and 200 μmol/L lead acetateexposed ACM treatment groups.Neurons were collected after being cultured in ACM for 24,48,or 72 h.The content of synaptophysin (SYP) in neurons was determined by Western blot.The SYP expression in neurons was measured by immunofluorescence after being cultured in ACM for 72 h.Results In all lead-exposed groups,the cell viability of astrocytes declined with increasing concentration of lead (P＜0.05).The Western blot showed that compared with the pure culture group,the non-Glu-induced ACM treatment group and Glu-induced leadfree ACM treatment group had significantly increased content of SYP in neurons (P＜0.01); compared with the non-Glu-induced ACM treatment group,the Glu-induced ACM treatment groups had significantly reduced SYP expression in neurons (P＜0.05); compared with the Glu-induced lead-free ACM treatment group,all leadexposed ACM treatment groups had the content of SYP in neurons significantly reduced with increasing concentration of lead after 72-h culture (P＜0.01),the 200 μmol/L lead-exposed ACM treatment group had significantly reduced content of SYP in neurons after 48-h culture (P＜0.01),and all lead-exposed ACM treatment groups showed no significant changes in the content of SYP in neurons after 24-h culture.Doublelabeling immunofluorescence of SYP showed that all lead-exposed ACM treatment groups had a significant decrease in the number of SYP-fluorescent particles after 72-h culture (P＜0.05).Conclusion Astrocytes promote synaptic formation of neurons,which may be inhibited during lead exposure.