【作者】 江山；

【导师】 饶志仁；

【作者基本信息】 第四军医大学 ， 神经生物学， 2009， 博士

【摘要】 低渗刺激可引起视上核星形胶质细胞合成释放抑制性递质牛磺酸,从而抑制神经元合成释放VP。已知脑内存在Glutamate mediated astrocyte-neuron signaling。SON是脑内的渗透压调节中枢之一,那么SON内是否存在Glutamate mediated astrocyte-neuron signaling,它在高渗刺激下是否起调节作用,其作用机制如何?为此本研究进行了三组实验(实验一、二和三)。此外,急慢性高渗刺激对胶质细胞的影响是否有所不同?我们进行了相关研究(实验四)。实验一急性静脉高渗刺激后SON神经元的反应依赖于AST的活性目的:观察急性静脉高渗(HS)刺激后,SON内AST和神经元的反应及其相互关系。方法:65只SD大鼠被分为4组:第1组正常对照组(n=5)不进行任何处理;第2组为等渗刺激组(IS, n=20),将等渗盐水(0.9% NaCl)注入尾静脉;第3组为高渗刺激组(HS, n=20),将高渗盐水(9% NaCl)注入尾静脉;第4组为氟代柠檬酸+ HS组(FCA plus HS组,n=20),预先向侧脑室缓慢注入1nmol/1μm的FCA,2h后再给高渗刺激;以上动物除第1组外,其余各组均分别存活15,45,90和180 min,每一时间点5只。常规灌流固定、切片,含有SON的切片进行抗VP,抗Fos,抗GFAP的单一或双重免疫荧光染色。同时HS刺激前以及刺激后45min,抽取各组大鼠静脉血,用放免法测定血浆VP含量。结果:1.急性静脉HS刺激后,SON内AST被激活,反应明显,表现为细胞胞体变大,突起变粗,GFAP深染,平均荧光强度增强,SON-VGL厚度增厚,Fos/GFAP双标细胞增加,以上反应在刺激后45min达到高峰。2.刺激后SON内神经元也被激活,Fos阳性胞核明显增加,VP阳性神经元的数目也显著增加,二者的高峰均为90min。3.FCA可明显干扰AST和神经元两者的反应。4.刺激后血浆VP水平显著升高,FCA预先处理再给HS刺激血浆VP水平不升高。结论:静脉HS刺激后SON内AST反应敏感,在刺激后45min达到高峰,神经元的Fos和VP表达高峰均为刺激后90min,血浆VP含量也升高。FCA是AST代谢抑制剂,结果AST和神经元的反应均被阻断;这表明SON内神经元对HS刺激的反应依赖于AST的活性。实验二静脉HS刺激后AST经Cx43 hemichannels释放Glu调节神经元活性目的:探讨HS刺激后SON内AST对神经元的调节机制。方法:1.整体动物实验:25只SD雄性大鼠被分为5组(5只/组):第1组为正常对照组;第2组为IS组;第3组为HS组;第4组为FCA plus HS组;第5组为甘珀酸+HS组(CBX plus HS组);第6组为PPADS+HS组;第7组为BBG+HS组。按实验一的方法制作动物模型,所有动物成活45min,常规固定、切片,切片进行抗NMDAR-2,抗glutamate,抗GFAP,抗Cx43,抗Fos,抗VP等单一或双重免疫荧光染色。2.培养细胞:使用2种细胞模型:(1)原代培养的AST,取18天胎鼠的下丘脑,常规分离、纯化培养AST 2周,将培养细胞分为6组:第1组为IS组(用不含Ca2+/Mg2+的DMEM培养);第2组为HS组;第3组为缝隙连接阻断剂(CBX和Gap26)+ HS组;第4组为谷氨酸转运体抑制剂(苏羟基天冬氨酸,THA)+ HS组,第5组为P2Xn受体拮抗剂(PPADS以及BBG)+ HS组,第6组为Ca2+螯合剂(BAPTA-AM)+ HS组,各组均处理1、3、5、7、9、10、12、15min。(2)C6细胞系,分组情况同上。分别收上述各组原代培养的AST培养液用于HPLC检测Glu含量。而采用流式细胞仪(FCM)测定C6细胞内Glu以及Cx43含量结果:1.整体动物实验:急性静脉HS刺激后,引起SON内下列变化: (1)AST的Glu表达明显上调,FCA干扰其上调,CBX则不干扰。(2)AST的Cx43表达明显上调,FCA干扰其上调,CBX则不干扰。(3)神经元NMDAR-2的表达明显上调,FCA和CBX均阻止其上调。(4)神经元内VP的表达上升,FCA,CBX均抑制VP的上升。2.培养细胞实验(1)HS刺激后,C6胞内Glu表达明显上升,3min达到高峰,以后下降。而原代AST培养液中Glu含量在5min达到高峰,此后呈下降趋势。(2)CBX或Gap26 + HS处理后,C6胞内Glu含量明显上升,并一直维持在高表达水平,而原代AST培养液中Glu含量与HS组相比,明显下降。(3)THA +HS组,与HS组相比,培养液中Glu含量未见明显差异。(4)PPADS或BBG + HS组,与HS组相比,细胞内和培养液中Glu含量均未见明显差异。(5)BAPTA-AM+HS组,与HS组相比,细胞内和培养液中Glu含量均未见明显差异。(6)培养细胞的Cx43表达也明显增加。结论:1.静脉HS后激活SON内AST,被激活的AST增加合成Glu,并通过Cx43 hemichannels释放,作用于SON神经元的NMDAR-2,增加VP的合成释放。2.HS激活培养的胶质细胞,增加Glu和Cx43的合成,CBX或Gap26不影响Glu的合成,但可阻断其释放;THA, PPADS,BBG和BAPTA-AM对Glu的合成、释放均无明显的影响。实验三高(低)渗刺激对胶质细胞释放递质的影响目的:探讨HS或低渗刺激后胶质细胞释放的递质是否不同?ATP是否参与对渗透压的调控。方法:1.培养细胞,本研究使用2种培养的胶质细胞模型:(1)原代培养的AST取18天胎鼠的下丘脑,常规分离、纯化培养2周;(2)C6细胞系。所有培养细胞分为3组:第1组为IS组;第2组为HS组;各组均处理1、3、5、10、15min。第3组为低渗刺激组;各组均处理1、3、5、10、15min。2.分别收上述各组培养液及细胞内液,采用HPLC测定原代培养AST细胞外液Glu以及taurine含量,而用FCM测定C6细胞内Glu以及taurine含量。3.采用荧光素-荧光素酶法检测原代培养的AST细胞内液及外液的ATP含量。结果:1.HS刺激促进AST主要合成释放Glu,而taurine未见变化2.低渗刺激促进AST主要合成释放taurine,而Glu未见变化3.与等渗组相比,渗透压发生改变后,细胞内外ATP水平未见显著变化。结论:HS刺激引起AST主要合成释放Glu,兴奋神经元释放VP。低渗刺激促进AST主要合成释放taurine,抑制神经元释放VP。而ATP未参与该调节过程。实验四急、慢性HS刺激对SON内AST和神经元影响的差异目的:探讨急、慢性HS刺激对大鼠行为学以及SON内AST和神经元影响的差异。方法:1.动物分组:25只SD雄性大鼠被分为5组(5只/组):第1组为正常对照组;第2组为2%高渗盐水喂养3d组;第3组为2%高渗盐水喂养6d组;第4组为2%高渗盐水喂养9d组;第5组为腹腔9%高渗盐水刺激组,动物成活45min。2.一般情况检测:测定大鼠体重以及进食3.测定各组大鼠的机械痛阈以及高级神经功能4.免疫荣光染色所有动物规定时间点处死,常规固定、切片,切片进行抗GFAP,抗VP,抗Fos,抗Cx43等单一,双重疫荧光染色。结果:1.急性腹腔高渗刺激45min后,SON内AST胞内GFAP与Cx43荧光强度明显上调,GFAP标记的与Cx43标记的SON-VGL厚度增加,Fos/GFAP双标记细胞数目增多,同时AST的突起增长,变粗,胞体变大,呈活化型AST形态。此外VP和Fos阳性神经元的个数也显著增多。2.喂2%高渗盐水组(1)喂高渗盐水3d后,与对照组相比,Cx43及GFAP表达显著升高,GFAP标记的与Cx43标记的SON-VGL厚度也增加,但低于急性腹腔高渗刺激组。Fos/GFAP双标记细胞数也显著增加,VP和Fos阳性神经元的个数也显著增多。此时,AST的突起有所回缩,细胞大小未见变化。(2)喂高渗盐水6d后,Cx43、GFAP的表达下降,GFAP标记的与Cx43标记的SON-VGL厚度均明显降低,Fos/GFAP双标记细胞数以及VP和Fos阳性神经元的个数也显著减少,但仍高于对照组。(3)喂高渗盐水9d后,AST的突起回缩,胞体萎缩,Cx43、GFAP的表达明显下降,Fos/GFAP双标记细胞数,VP和Fos阳性神经元的个数也显著减少,GFAP标记的与Cx43标记的SON-VGL厚度均明显降低。(4)急性腹腔高渗刺激45min后,大鼠的机械痛阈、体重未见明显变化,而喂盐水3d后,大鼠的机械痛阈逐渐升高,体重变轻,进食量减少,至9d达到峰值,提示大鼠身体机能处于衰竭状态。明暗箱测定大鼠高级神经功能发现,大鼠学习记忆能力下降。结论:急性和慢性高渗刺激对AST有所不同,急性刺激可以激活AST,从而参与调节神经元的活性,而慢性高渗刺激后,在早期(3d)AST及神经元的活性明显增强,到后期(6,9d)大鼠身体机能处于衰竭,反应机能明显减退,AST和神经元的反应也就被明显抑制。更多还原

【Abstract】 It is well known that the hypotonic stimulus induce the synthesis and release of taurine from astrocytes which inhibit the synthesis and release of VP. In brain, glutamate mediated astrocyte-neuron signaling and SON is the center of osmotic regulation. Is the glutamate mediated astrocyte-neuron signaling included in the regulation of SON under hypertonic stimulus. In order to clarify this question, we performed three experiments (first, second and third experiments). Moreover, is there difference between the acute and chronic hypertonic stimulus on the astrocytes? The fourth experiment was performed.The first experimentAcute intravenous hyperosmotic stimulus induces activation in the neurons depends on the regulation of astrocytes in supraoptic nucleus Objective To investigate the response and relationship between the astrocytes and neurons in SON induced by acute intravenous hypertonic stimulus.Methods 65 SD rats were divided into four groups: control group(n=5), Iso-osmia group(IS group,n=20), hypertonic stimulus group(HS group,n=20) and FCA plus HS group(n=20). Except the control group, the rats in other three groups were survived for 15, 45, 90 and 180 min,n=5/per time point, fixation, cut section and anti-VP, anti-Fos, anti-GFAP immunofluorescent staining were preformed by usual methods.We also detected the VP content in plasma by radioimmuno assay. Assays were performed blindly.Result 1.HS induced SON astrocytes clearly responding and revealed marked cellular hypertrophy with thickened processes, the mean fluorescent intensity of GFAP staining intensified, the mean thickness of SON-VGL thickened, the mean number of Fos/GFAP double labeled astrocytes significantly increased. At 45 min after stimulus, the response peaked.2.HS also actived the neurons in SON. The expression of Fos and VP positive neurons were increased.The responsed peak was 45min.3.The FCA inhibited the response of astrocytes and neurons after HS.4.VP level in plasma was increased after HS and this effect could be inhibited by FCA.Conclusion Intravenous hypertonic stimulus induced the response of astrocyte and neuron. This effect could be inhibited by FCA----metabolic inhibitor. It is suggested that the activation of neurons should depend on the the activation astrocytes after intravenous HS in SON.The second experiment Astrocytes regulate the activation of neurons by release glutamate through Cx43 hemichannel in SON after the intravenous hypertonic stimulusObjective To investigate mechanism of astrocytes regulation neurons in SON.Methods 1.35 SD male rats were divided into 7 groups(5 rats/per group): control group, IS group, HS group, FCA plus HS group,CBX plus HS group, PPADS plus HS group and BBG plus HS group. All rats were survived for 45min, fixation, cut section and anti- NMDAR-2, anti-glutamate, anti-GFAP, anti-Cx43, anti-Fos and anti-VP immunofluorescent staining were performed by usual methods.2. Cultured cells (1)Primary cultured astrocytes:Cultured astrocytes were divided into 6 groups: IS group, HS group, Gap junction inhibitor(CBX and Gap26) plus HS group, THA plus HS group, P2XnR antagonist(PPADS and BBG) plus HS group and Ca2+chelator(BAPTA-AM) plus HS group.Every group was treated by HS for 1、3、5、7、9、10、12、15min. (2)C6 cells:We cultured C6 cells and divided it into 6 groups as above.Culture fluid of primary cultured astrocyte was collected for detecting glutamate content by HPLC. Intracelluar glutamate content of C6 cell was determined by flow cytometry(FCM).Results 1.In vivo, FCA inhibited the increase of glutamate and Cx43 after HS while CBX had no the effect. At the same time, the expression of NMDAR-2 and VP were blocked by FCA and CBX.2.In vitro, the C6 intracelluar glutamate content or extracelluar fluid glutmamte of cultured astrocyte was increased and peaked at 3min or 5min respectively after HS. After treated by CBX or Gap26, the C6 intracelluar glutamate content kept the high expression. However, the glutamate content in extracelluar fluid was decreased. Moreover,THA, PPADS, BBG or BAPTA-AM has no effect on the intracellular or extracelluar glutamate content. HS also induced the expression of Cx43.Conclusion Intravenous HS induced the synthesis and release of glutamate from astrocyte through Cx43 hemichannel. And then the glutamate affected the NMDAR-2 and increased the synthesis and release of VP.The third experiment The effect of hypertonic(hypotonic) stimulus on the transmitter release from astrocytesObjective To investigate if the hypertonic or hypotonic stimulus induced the astrocyte to release different transmitter and ATP was included in the regulation of osmotic pressure.Methods 1.Cultured cells: Primary cultured astrocytes and C6 cells were divided into three groups: IS group, HS group and hypotonic stimulus group. HS group and hypotonic stimulus groups were both treated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14.and 15 min.2.Culture fluid of primary cultured astrocytes was collected for detecting glutamate and taurine content by HPLC. Intracelluar glutamate and taurine content of C6 cells were determined by FCM.3.Luciferin-luciferase based luminescence assay was used for detecting the ATP content.Results 1.HS induced the release of glutamate(not taurine)from astrocyte2.Hypotonic stimulus induced the release of taurine(not glutamate) from astrocyte.3.ATP had no change with the change of osmotic pressure. Conclusion HS induced the release of glutamate from astrocytes and activated the neurons to release VP. Hypotonic stimulus induced the release of taurine from astrocytes which inhibited the release of VP from neurons. ATP was not included in the regulation of osmotic pressure in SON.The fourth experiment The difference between the effects of acute and chronic hypertonic stimulus on the astrocytes and neurons in SONObjective To investigate the different between the acute and chronic HS on the astrocytes and neurons in SONMethods 1.25 SD male rats were divided into 5 groups(5 rats/group): control group, HS 3d group(feeding 2% hypertonic saline for 3d), HS 6d group(feeding 2% hypertonic saline for 6d), HS 9d group(feeding 2% hypertonic saline for 9d), acute HS group.2.Determined body weigh and foodintake.3.Detected the paw withdrawal mechanical threshold.4.The anti-GFAP, anti-Cx43, anti-Fos and anti-VP immunofluore-scent staining were preformed by usual methods.Results 1. At 45min after acute celiac HS, SON astrocytes revealed marked cellular hypertrophy with thickened processes, the mean fluorescent intensity of GFAP staining intensified, the mean thickness of SON-VGL thickened, the mean number of Fos/GFAP double labeled astrocytes and VP/Fos positive neurons significantly increased.2. Feeding 2% hypertonic saline group:(1)After feeding hypertonic 2% saline for 3d, compared with control group, the expression of Cx43 and GFAP were increased and the thickness of SON-VGL thickened.And at the same time, the Fos/GFAP double labeled astrocytes and VP/Fos positive neurons also increased. However, the changes were fewer than that in the acute hypertonic stimulus.(2)After feeding hypertonic 2% saline for 6d, the expression of Cx43 and GFAP, the thickness of SON-VGL, and the number of VP/Fos positive neurons all decreased compared with acute HS group.(3)After feeding hypertonic 2% saline for 9d, the expression of Cx43 and GFAP, the thickness of SON-VGL, and the number of VP/Fos positive neurons all obviously decreased compared with acute HS group. And the cell body exhibited atrophy with the retracted processes.(4)For the acute HS group, the paw withdrawal mechanical threshold and body weigh had no change. However, after feeding 2% saline for 3d, the paw withdrawal mechanical threshold was higher than that in the acute HS group. The body weigh and foodintake were also decreased. By the detecting of ethology, the ability of learning and memory also decreased.Conclusion Acute and chronic HS have different effect on the astrocytes.Acute HS could activate astrocytes which regulate the neurons in turn. However, after the chronic HS, the astrocytes and neurons were activated at the early stage(3d) and inhibited at the later stage(6d, 9d).The phenomenom prompted us that the rats were exhausted at the later stage of chronic HS which inhibited the astrocytes and neurons.更多还原