高渗刺激引起培养的大鼠下丘脑星形胶质细胞和C6细胞合成并释放谷氨酸

目的观察高渗刺激对培养的大鼠下丘脑星形胶质细胞或C6细胞合成、释放谷氨酸的影响。方法获取一日龄SD大鼠下丘脑组织，进行星形胶质细胞培养、纯化和鉴定。培养细胞随机分五组：（1）等渗组：用新鲜等渗培养液置换原先的培养液，将细胞分成5个亚组，分别孵育1、3、5、10和15min。（2）高渗组：用320mosMNaCl高渗溶液置换原先的培养液，将细胞分成5个亚组，分别孵育1、3、5、10和15min。（3）甘伯酸（carbenoxolone，CBX，一种缝隙连接阻断剂）＋等渗组和（4）CBX＋高渗组：用含有CBX（终浓度为100mmol／L）的等渗培养液作用1h，后分别用等渗或高渗培养液置换出原培养液，将细胞分成5个亚组，分别孵育1、3、5、10和15min。（5）Ca2＋高渗组：用含有Ca2＋（1000μmol／L）的等渗培养液作用1h，后用高渗培养液置换出原培养液，将细胞分成5个亚组，分别孵育1、3、5、10和15min。每个亚组5个培养皿。收集各组的细胞，进行抗谷氨酸和抗胶质原纤维酸性蛋白（Glial fibrillary acidic protein，GFAP）的双重免疫荧光染色，Confocal显微镜观察。用反相高效液相色谱荧光测定法测定细胞培养液中谷氨酸的含量。培养的C6细胞分为四组：等渗组，高渗组，CBX＋等渗组和CBX＋高渗组，用于流式细胞仪（flowcytometry，FCM）定量检测细胞内谷氨酸的含量。结果星形胶质细胞内抗GFAP染色的荧光强度在五组问无明显差异。星形胶质细胞内抗谷氨酸染色的荧光强度在等渗组中各时间点无明显变化，高渗组中在刺激1min后表达增加，5min达到高峰，15min恢复到正常。CBX＋高渗组的抗谷氨酸染色荧光强度明显高于CBX＋等渗组和等渗组，一直维持到15min不下降。培养基中的谷氨酸浓度在等渗组各时间点无明显变化，高渗组中谷氨酸浓度从5min到15min明显增加。Ca2＋高渗组和CBX＋高渗组中?

Hypertonic stimulation induces synthesis and release of glutamate in cultured rat hypothalamic astrocytes and C6 cells

Objective To investigate whether hypertonic saline （HS） can induce the synthesis and release of glutamate in cultured hypothalamic astrocytes or C6 cell line. Methods Astrocytes were isolated, cultured, purified and identified from the hypothalamus of newborn rat （1 day）. The astrocytes were randomly divided into five groups： isotonic （IS） and HS groups, astrocytes were incubated by IS and HS （320 mosM NaCl） medium, respectively, for 1, 3, 5, 10 or 15 rain; carbenoxolone （CBX） ＋IS and CBX＋HS groups, astrocytes were pre-treated with CBX （100 mmol/L） for 1 h at 37℃ in a 5% CO2 / 95% atmosphere, then removed to IS and HS medium, respectively, for 1, 3, 5, 10 or 15 min; Ca2＋＋HS group, astrocytes were pre-incubated with Ca2＋ （1 000 μmol/L） for 1 h at 37℃ in a 5% CO2 / 95% atmosphere, followed by a wash with isotonic FBS/DMEM, and then removed to hypertonic saline for 1, 3, 5, 10 or 15 min. The media of five groups were collected to analyze the medium glutamate concentration with high performance liquid chromatography. The astrocytes were fixed and double immunofluorescent stained with anti-glial fibrillary acidic protein （GFAP） and anti-glutamate. The C6 cells were divided into four groups： IS, HS, CBX＋IS and CBX＋HS groups, and used for quantitative measurement of glutamate in cells by flow cytometry （FCM）. Results （1） Anti-GFAP immunofluorescent signal revealed no significant difference among various time points in each group, or among the five groups. （2） The anti-glutamate immunofluorescent signal was increased in HS group and peaked at 5 min, and decreased and returned to the level of IS group at 15 rain （P 〈 0.01 vs the 5 min of HS group）. In CBX＋HS group, the glutamate intensity was higher than that in CBX＋IS and HS groups. （3） The medium glutamate concentration had no change after treatment with HS for 1 and 3 min, while increased markedly after treatment for 5 min to 15 min （P 〈 0.01 vs 1 min and 3 min）. On the contrary, the