砷暴露对大鼠原代星形胶质细胞分泌胶质细胞源性递质的影响

Effects of arsenite on gliotransmitter release from primary cultured astrocytes

目的 观察砷暴露对大鼠原代星形胶质细胞（astrocyte,AST）分泌胶质细胞源性递质的影响,探讨砷对学习记忆功能损伤的作用机制.方法 出生1～3 d的Wistar大鼠仔鼠,取双侧大脑半球,经处理获得原代培养脑AST,并通过胶质纤维酸性蛋白免疫荧光染色鉴定.AST分别在含0.0、2.5、5.0、10.0μmol/L亚砷酸钠的培养液中培养24h,荧光双波长分光光度计法检测细胞内游离钙离子浓度（[Ca2＋]i）;高效液相色谱（HPLC）法检测细胞培养液谷氨酸、D-丝氨酸、甘氨酸和γ-氨基丁酸含量.结果 胶质纤维酸性蛋白免疫荧光染色,AST纯度＞95％.不同浓度砷暴露组间AST内[Ca2＋]i比较差异有统计学意义（F=20.030,P＜0.05）,其中10.0 μmol/L砷暴露组AST内[Ca2＋]i[（263.27±14.80）nmol/L]明显高于0.0、2.5、5.0μmol/L砷暴露组[（204.24±27.21）、（214.49±21.85）、（232.74±23.14）nmol/L,P均＜0.05].不同浓度砷暴露组间AST分泌的D-丝氨酸、甘氨酸和γ-氨基丁酸比较差异均有统计学意义（F值分别为26.599、33.539、5.599,P均＜0.05）,其中2.5、5.0、10.0 μmol/L砷暴露组AST分泌的D-丝氨酸[（21.580±1.313）、（21.936±1.539）、（23.401±1.648）μmol/L]、甘氨酸[（26.353±2.449）、（29.711±1.530）、（29.234±2.057）μmol/L]和γ-氨基丁酸[（27.277±3.421）、（30.213±2.098）、（29.364±2.588） μmol/L]均高于0.0 μmol/L砷暴露组[（16.017±1.046）、（16.763±3.007）、（22.736±4.139） μmol/L,P均＜0.05].结论 砷暴露可引起原代AST分泌胶质细胞源性递质增加,可能会损伤学习记忆功能.

Objective To investigate the impairment mechanism of learning and memory function induced by arsenite exposure through studying the effects of sodium arsenite on gliotransmitter release from astrocytes.Methods Primary cultured astrocytes were isolated from neonatal （0-3 days） Wistar rats and determined by glial fibrillary acidic protein （GFAP） immunofluorescence staining.The primary cultured astrocytes were randomly divided into four groups,in which astrocytes were exposed to 0.0,2.5,5.0,or 10.0 μmol/L sodium arsenite,respectively,for 24 h.Intracellular free Ca2＋ concentration （[Ca2＋]i） in astrocytes was measured by fluorescence dual wavelength spectrophotometer;,concentrations of glutamate,D-serine,glycine and γ-aminobutyric acid were measured by high performance liquid chromatography （HPLC）.Results More than 95% cells were positive for GFAP immunofluorescence staining.The difference of [Ca2＋]i among groups treated with sodium arsenite was statistically significant （F =20.030,P 〈 0.05）.[Ca2＋]i increased significantly in group treated with 10.0 μmol/L sodium arsenite [（263.27 ± 14.80）nmol/L] compared with those in groups treated with 0.0,2.5,5.0 μmol/L sodium arsenite [（204.24 ± 27.21）,（214.49 ± 21.85）,（232.74 ± 23.14）nmol/L,all P 〈 0.05].The differences of the levels of D-serine,glycine and γ-aminobutyric acidamong groups treated with sodium arsenite were significant （F =26.599,33.539,5.599,all P 〈 0.05）.The levels of D-serine [（21.580 ± 1.313）,（21.936 ± 1.539）,（23.401 ± 1.648）μmol/L],glycine [（26.353 ± 2.449）,（29.711 ± 1.530）,（29.234 ± 2.057）μmol/L] and γ-aminobutyric acid [（27.277 ± 3.421）,（30.213 ± 2.098）,（29.364 ± 2.588）μmol/L] released by astrocytes increased significantly in groups treated with 2.5,5.0,10.0 μmol/L sodium arsenite compared with those in groups treated with 0.0 μmol/L sodium arsenite [（16.017 ± 1.046）,（16.763 ± 3.007）,（22.736 ± 4.139）μmol/L,all P 〈 0.05].Conclusion Arsenite could affect gliotransmitter release from astrocytes,and further impair learning and memory function.