表没食子儿茶素没食子酸酯保护阿米卡星诱导耳蜗螺旋神经节神经元的凋亡

透射电镜观察SD大鼠耳蜗螺旋神经节神经元形态变化,发现阿米卡星可以诱导耳蜗螺旋神经节神经元凋亡。免疫组化染色和RT-PCR检测发现,阿米卡星诱导耳蜗螺旋神经元Bcl-2 蛋白表达下调,Bax、Caspase-3蛋白及Caspase-6 mRNA表达增强。表没食子儿茶素没食子酸酯可以抑制耳蜗螺旋神经元Bax、Caspase-3蛋白及Caspase-6 mRNA的表达,同时增强Bcl-2蛋白表达,从而降低耳蜗螺旋神经元凋亡率。证实表没食子儿茶素没食子酸酯对阿米卡星损伤的耳蜗螺旋神经节具有保护作用。     

EGCG对H2O2诱导BV2细胞氧化损伤保护作用机制的研究

目的探讨EGCG对H2O2诱导BV2细胞氧化损伤的保护作用机制。方法以200μmol/L H2O2制备BV2细胞氧化应激损伤模型,用CCK-8法检测不同浓度EGCG(0、1、5、10、20、40、80μmol/L)的保护作用,Ho-echst33258染色法检测细胞的凋亡,Western bloting法检测caspase-9蛋白的表达。结果 10及20μmol/L的EGCG组保护效果较明显;EGCG保护组的凋亡细胞显著少于无保护组;20μmol/L EGCG保护组的caspase-9蛋白表达较无保护组明显下调(P=0.03<0.05)。结论 10及20μmol/L浓度的EGCG对H2O2诱导的BV2细胞损伤模型有保护作用,其机制可能是通过减少caspase-9蛋白的表达,进而部分阻断caspase-9所介导的caspases级联反应,减少BV2细胞的凋亡。     

血管内注射可溶性β-淀粉样蛋白1-40对毛细血管内皮细胞和胶质细胞毒性的研究

目的研究血液内较高浓度的可溶性β-淀粉样蛋白1-40(Aβ1-40)对毛细血管内皮细胞和胶质细胞的毒性,以探讨其在阿尔茨海默病发病中的作用.方法成年雄性SD大鼠90只,随机分为注射Aβ1-40组、注射生理盐水组以及正常大鼠(非手术)组,每组30只.采用右侧颈外静脉血管插管并留置的方法,通过导管向血管内中每日2次注射10μg的可溶性的Aβ1-40,连续14天后,采用免疫组化和免疫印迹(Western blot)方法观察星形胶质细胞、小胶质细胞的激活状态以及转糖蛋白-1、转糖蛋白-3表达的改变.结果注射Aβ1-40以后,毛细血管周围星形胶质细胞的胶质纤维酸性蛋白表达明显增多;小胶质细胞呈明显的激活状态;Western blot法检测到脑实质内转糖蛋白-1以及转糖蛋白-3表达明显减少.结论血管内连续注射Aβ1-40以后,对血-脑屏障的内皮细胞有明显的损害,对胶质细胞有明显的激活作用,可能与AD发病有关.     

β-淀粉样蛋白对AD大鼠脑内胶质细胞的影响作用研究

目的　探讨Meynert核注射 β 淀粉样蛋白 (Aβ)后对大鼠脑神经胶质细胞超微结构的影响及其可能机制。方法　将 1μLAB1-40 (10 μg/ μL)在立体定向仪下注入大鼠右侧Meynert核 ,分别于 1周、4周时用透射电镜观察脑组织中神经胶质细胞超微结构变化。结果　实验组Meynert核、海马区和皮层区均可见神经胶质细胞增生 ,其中以小胶质细胞为主 ,星形胶质细胞次之 ;小胶质细胞聚集并吞噬变性神经细胞以及血管周围可见增生活跃的小胶质细胞聚集 ;神经元胞体皱缩 ,胞浆浓缩 ,核染色质固缩成团块状并见边聚现象 ,核膜尚完整 ,有发生凋亡的趋势 ;正常对照组和假手术组无上述变化。结论　Aβ能引发CNS神经胶质细胞增生并活化 ,免疫炎性反应在AD记忆障碍和痴呆形成过程中可能具有重要作用。     

β-淀粉样蛋白对星形胶质细胞分泌组织蛋白酶B作用的研究

目的研究β-淀粉样蛋白(Aβ)对星形胶质细胞活性、形态学以及分泌组织蛋白酶B(CB)的影响。方法体外培养星形胶质细胞,采用免疫细胞化学方法对星形胶质细胞进行鉴定;观察加入不同浓度溶解状态的Aβ1-40后细胞形态学改变,采用四唑盐法检测细胞活性,Western blot法测定细胞上清CB表达水平。结果将不同浓度新鲜Aβ1-40加于星形胶质细胞分别培养24 h,各浓度Aβ1-40对星形胶质细胞活性均无明显影响(P0.05);随时间延长,经40～60μmol/LAβ1-40处理的星形胶质细胞逐渐出现肿胀、坏死;经15、20、25μmol/L Aβ1-40诱导的星形胶质细胞上清中检测到CB表达,随Aβ1-40浓度增高可见蛋白质显影增强且带形清晰。结论 Aβ1-40可激活星形胶质细胞并诱导其释放CB,提示Aβ激活的星形胶质细胞及其释放的CB可能在AD发病中起着一定作用。     

星形胶质细胞对脑缺血后神经元的保护作用

神经元和神经胶质细胞共同构成中枢神经系统。而其中星形胶质细胞(astrocytes,AS)在数量上占有绝对优势,其数量是神经元的10～50倍,约占脑体积的一半。AS长期以来被认为是脑组织中简单的堆积物,发     

疏血通对凝血酶诱导培养的星形胶质细胞损伤的保护作用

目的探讨疏血通对凝血酶诱导培养的星形胶质细胞损伤的保护作用。方法采用体外培养星形胶质细胞的方法,对新生Wistar大鼠大脑星形胶质细胞进行培养并纯化,用不同浓度的疏血通作用后,然后根据需要加入凝血酶干预。用免疫细胞化学染色和逆转录多聚酶链反应(RTPCR)检测培养星形胶质细胞NFκB蛋白和基因的表达;用化学反应法测定培养细胞上清液中乳酸脱氢酶活性(LDH);MTT法观察细胞生长活性的影响;流式细胞仪分析细胞周期和凋亡的变化。结果疏血通下调凝血酶作用下的体外培养星形胶质细胞上NFκB的表达;使细胞生长活性明显增加;减轻细胞G2/M期的阻滞,凋亡比例下降。结论疏血通减轻凝血酶诱导培养星形胶质细胞的损伤,可能通过其抗细胞凋亡作用。     

地塞米松对β淀粉样蛋白诱导的脑内炎症损伤的保护作用

地塞米松对β淀粉样蛋白诱导的脑内炎症损伤的保护作用@林煜!510318广州$解放军421医院神经科 @陈俊抛$第一军医大学珠江医院神经内科 @贺顺龙!510318广州$解放军421医院神经科 @苏爱武!510318广州$解放军421医院神经科~~~~~~     

星形胶质细胞对天冬氨酸特异性半胱氨酸蛋白酶介导β淀粉样蛋白早期突触毒性作用的影响

目的探讨星形胶质细胞(astrocyte,AS)对天冬氨酸特异性半胱氨酸蛋白酶(cysteinyl aspartate specific proteinase,caspase)介导β淀粉样蛋白(β-amyloid,Aβ)早期突触毒性作用的影响,以期为进一步研究与血管性痴呆(vascular dementia,Va D)的发病机制奠定基础。方法以原代培养大鼠海马纯神经元体系(NE-S)及混合培养体系(MIX-S,主要包含神经元及AS)为研究对象,各体系分为6组:对照组、caspase-8抑制剂组、caspase-9抑制剂组、Aβ处理组、caspase-8抑制剂预处理加Aβ组和caspase-9抑制剂预处理加Aβ组。免疫荧光检测各组近胞体10μm段树突中突触后密度蛋白(postsynaptic density-95,PSD95)表达量的变化。结果 1在NE-S与MIX-S中,与对照组相比,caspase-8抑制剂组、caspase-9抑制剂组PSD95的表达量均无明显差异,Aβ处理组PSD95的表达量均显著降低(P均0.001)。2在NE-S中,与Aβ处理组相比,caspase-9抑制剂预处理加Aβ组PSD95的表达量显著回升至对照组水平,caspase-8抑制剂预处理加Aβ组则无显著改变;在MIX-S中的结果则相反,即caspase-8抑制剂预处理加Aβ组PSD95的表达量显著回升至对照组水平,而caspase-9抑制剂预处理加Aβ组则无显著改变。3MIX-S与NE-S两种培养系统间相比较,对照组间及Aβ处理组间PSD95的表达量均无显著差异,而caspase-8抑制剂预处理加Aβ组间及caspase-9抑制剂预处理加Aβ组间PSD95的表达量差异有显著性。结论在Aβ早期突触毒性作用中,AS参与caspase-8介导的死亡受体通路激活过程,且参与抑制神经元的线粒体通路。     

氯胺酮对NMDA诱导大鼠脊髓背角星形胶质细胞损伤的保护作用

目的观察氯胺酮对N-甲基-D-天冬氨酸(NMDA)受体过度激活诱导大鼠脊髓背角星形胶质细胞凋亡影响,并探讨其可能的作用机制.方法取新生2～3 d wistar大鼠T11-L6脊髓背角星形胶质细胞原代纯化培养,GFAP鉴定星形胶质细胞纯度达98%后用于实验.将细胞随机分6组:对照组(C组),NMDA组(N组),氯胺酮组(K组)和三种不同浓度氯胺酮加NMDA组(0.1,0.5,1 mmol/L,标记为NK1～NK3组),再培养24 h后检测SOD活性和MDA含量,免疫组化HE复染观察Bcl-2蛋白和形态学变化,流式细胞仪检测星形胶质细胞凋亡率.结果N组细胞发生了大量凋亡,SOD活性显著降低,MDA含量明显增加,Bcl-2蛋白表达不明显;NK3组细胞凋亡被显著抑制,Bcl-2蛋白强阳性表达,SOD活性明显增加和MDA含量低.结论NMDA受体过度激活可诱导大鼠脊髓背角星形胶质细胞大量凋亡,适量氯胺酮显著抑制了细胞凋亡,其机制可能是增强了星形胶质细胞Bcl-2蛋白表达,同时抑制了自由基的产生和增强了SOD活性.     

槲皮素对氧化应激大鼠星形胶质细胞的保护作用

目的 研究槲皮素对氧化应激损伤后大鼠星形胶质细胞的保护作用. 方法 采用终浓度为2 mmol/L的H2O2作用于体外原代培养的大鼠胶质细胞6 h,以诱导氧化应激.实验分为正常对照组、H2O2组、槲皮素+H2O2组.不同浓度(0、50、100、200μmol/L)槲皮素预处理24h后,应用速率法和LIVE/DEAD检测试剂盒分别检测氧化应激胶质细胞的乳酸脱氢酶(LDH)释放率以及细胞存活率的变化. 结果 终浓度为2 mmol/L的H2O2作用6 h即可造成细胞损伤,LDH释放率由对照组的(3.89±1.89)%增至(90.27±2.68)%,较对照组明显增多,细胞存活率由对照组的(99.25±0.08)%降至(59.73%±9.92)%,较对照组明显降低,差异均有统计学意义(P<0.05).槲皮素预处理后细胞LDH释放率降低,50、100、200 μmol/L浓度的槲皮素组LDH释放率分别减少到(48.19±13.98)%、(27.81±9.33)%和(18.13±8.28)%,与H2O2组比较差异有统计学意义(P<0.05);槲皮素预处理同时能提高细胞存活率,50、100、200 μmol/L浓度的槲皮素组细胞存活率分别提高至(86.80±3.62)%、(88.32±5.77)%和(91.18±3.03)%,与H2O2组比较差异均有统计学意义(P<0.05). 结论 槲皮素预处理对氧化应激大鼠胶质细胞有一定的保护作用.     

NGF对β-淀粉样蛋白诱导海马神经细胞凋亡的保护作用

目的　研究神经生长因子 (nerve growth factor ,NGF)对β-淀粉样蛋白 (amyloid-beta protein,Aβ)诱导体外培养大鼠海马神经细胞凋亡的作用 ,从而探讨 NGF对阿尔茨海默病 (Alzheimer disease,AD)的治疗作用。方法　于无菌条件下取新生 1 d大鼠海马 ,进行原代海马神经细胞培养 ,用透射电镜、琼脂糖凝胶电泳、原位末端标记 (TUNEL)等方法观察 Aβ组和 NGF组神经元细胞的凋亡变化。结果　 Aβ1 -40可诱导海马神经细胞凋亡 ,染色质浓缩 ,凋亡小体形成 ,细胞核 DNA降解 ,TUNEL 染色阳性 ;而 NGF组细胞凋亡率明显减少 (P <0 .0 5 )。结论　 Aβ1 -40能诱导海马神经细胞凋亡 ,NGF对 Aβ诱导的海马神经细胞凋亡具有保护作用。     

表没食子儿茶素没食子酸酯通过激活核因子相关因子2保护MPP+诱导的PC12细胞氧化损伤

目的 观察表没食子儿茶素没食子酸酯(EGCG)对MPP+诱导PC12细胞氧化损伤的保护作用及其与核因子相关因子-2(NRF2)之间的关系.方法 以不同浓度EGCG预处理MPP+诱导的PC12细胞,选用细胞外调节蛋白激酶(ERK)抑制剂U120作为干预药物,噻唑蓝(MTT)法检测细胞活性;Western blot检测细胞内NRF2表达量及核内外分布;实时荧光定量PCR检测NRF2下游抗氧化酶血红素氧合成酶-1(HO-1)和醌氧化还原酶1(NQ01)在转录水平的变化.结果 MTT法显示MPP+明显降低细胞生存率,具有浓度依赖效应,而EGCG预处理能明显抑制MPP+对细胞的损伤作用;Westem blot结果显示MPP+模型组NRF2表达量为对照组的148％±5％ (t=6.102,P＜0.01),EGCG预处理组NRF2表达量为对照组的188％±6％(t=11.172,P＜0.01),U120则能抑制EGCG诱导NRF2表达增加的效应,为对照组的148％±15％(t=6.118,P＜0.01);EGCG预处理组NRF2核内蛋白量的增加更加明显,为对照组的258％±2％(t=21.995,P＜0.01),U120+ EGCG+ MPP+组核内NRF2蛋白量较EGCG预处理组明显减低,为对照组的158％±1％(f=8.058,P＜0.01).与NRF2的变化一致,实时荧光定量PCR显示EGCG预处理组抗氧化酶HO-1、NQO1 mRNA水平较其余各组明显增高,U120也可抑制EGCG对HO-1和NQO1 mRNA的诱导效应.结论 EGCG能保护MPP+诱导的PC12细胞氧化损伤,其保护作用可能与通过激活ERK-NRF2途径,诱导下游HO-1、NQO1等抗氧化酶表达有关.     

鱼藤酮对中脑星形胶质细胞的损伤及阿糖胞苷的干预作用

目的 观察鱼藤酮毒性作用及阿糖胞苷(ara-c)干预对体外培养中脑腹侧星形胶质细胞增殖、还原型谷胱甘肽(GSH)含量及胶质细胞源性神经营养因子(GDNF)表达的影响. 方法 体外培养大鼠中脑腹侧星形胶质细胞随机分成9组,分别为对照组,10、20、40及60nmol/L鱼藤酮短时程损伤组(用相应浓度鱼藤酮处理24 h),10及20 nmol/L鱼藤酮长时程损伤组(相应浓度鱼藤酮处理30 d),10及20 nmol/L鱼藤酮长时程损伤+ara-c处理组(相应浓度鱼藤酮处理30 d,500nmol/L ara-c处理6 d).增殖细胞核抗原(PCNA)免疫细胞化学染色观察细胞增殖情况,GSH检测试剂盒检测细胞GSH含量.免疫细胞化学方法 和Western blot检测GDNF的表达情况. 结果短时程损伤各组10和20 nmol/L鱼藤酮作用 24 h未能使细胞GSH含量及GDNF表达最降低,但40和60 nmol/L鱼藤酮作用24 h可使细胞GSH含量降低、GDNF表达减少.长时程损伤组10和20nmol/L鱼藤酮作用30 d后处于增殖状态的星形胶质细胞比例增高,GSH含量未见降低.但GDNF表达量减少:500nmol/L ara-c抑制细胞增殖后,可使GDNF的表达回升至接近对照组水平且GSH含量明显提高. 结论 鱼藤酮可影响中腩腹侧旱形胶质细胞的增殖和功能,恶化多巴胺能神经元的生存微环境;低浓度ara-c可通过抑制旱形胶质细胞的过度增殖,恢复GDNF表达量并明显提高GSH含量,提示ara-c对帕金森病具有潜在的治疗价值.     

鱼藤酮对中脑星形胶质细胞的损伤及阿糖胞苷的干预作用

Objective To observe the toxic effects of rotenone on the proliferation, γ-glutamylcysteinylglycine (GSH) content and the expression level of glial cell line-derived neurotrophic factor (GDNF) of rat rnidbrain astrocytes in vitro and the interventional effect of arabinoeytidine (ara-c). Methods In vitro cultured rat midbrain astrocytes were assigned randomly into 9 groups, including a normal control group, 4 short-term rotenone treatment groups exposed for 24 h to 10, 20, 40 or 60 nmol/L rotenone, 2 long-term rotenone treatment groups exposed for 30 days to 10 or 20 nmol/L rotenone, and 2 ara-c groups with 500 nmol/L ara-c treatment following exposure to 10 or 20 nmol/L rotenone for 6 days. The cell proliferation was assessed by immunocytochemical detection of the expression of proliferating cell nuclear antigen (PCNA). GSH content in the treated cells was measured by GSH detection kit, and the expression of GDNF was detected with immunocytochemistry and Western blot. Results The 24-h exposure to low-level rotenone (10 and 20 nmol/L) did not cause any changes in GSH content or GDNF expression in the cells. But at 40 and 60 nmol/L, rotenone treatment for 24 h significantly decreased the GSH content and GDNF expression. Rotenone exposure for 30 days increased the ratio of proliferating astrocytes and decreased GDNF expression level, but the GSH content remained stable. The application of 500 nmol/L ara-c to suppress the cell proliferation restored the expression level of GDNF to almost the control level and markedly increased GSH content. Conclusion Rotenone affects the proliferation and activity of rat midbrain astrocytes in vitro and deteriorates the microenvironment of dopaminergic neurons. Low-level ara-c can increase the GSH content and GDNF expression levels by suppressing the proliferation of rotenone-exposed astrocytes, suggesting its potential value in the treatment of Parkinson's disease.     

鱼藤酮对中脑星形胶质细胞的损伤及阿糖胞苷的干预作用

Objective To observe the toxic effects of rotenone on the proliferation, γ-glutamylcysteinylglycine (GSH) content and the expression level of glial cell line-derived neurotrophic factor (GDNF) of rat rnidbrain astrocytes in vitro and the interventional effect of arabinoeytidine (ara-c). Methods In vitro cultured rat midbrain astrocytes were assigned randomly into 9 groups, including a normal control group, 4 short-term rotenone treatment groups exposed for 24 h to 10, 20, 40 or 60 nmol/L rotenone, 2 long-term rotenone treatment groups exposed for 30 days to 10 or 20 nmol/L rotenone, and 2 ara-c groups with 500 nmol/L ara-c treatment following exposure to 10 or 20 nmol/L rotenone for 6 days. The cell proliferation was assessed by immunocytochemical detection of the expression of proliferating cell nuclear antigen (PCNA). GSH content in the treated cells was measured by GSH detection kit, and the expression of GDNF was detected with immunocytochemistry and Western blot. Results The 24-h exposure to low-level rotenone (10 and 20 nmol/L) did not cause any changes in GSH content or GDNF expression in the cells. But at 40 and 60 nmol/L, rotenone treatment for 24 h significantly decreased the GSH content and GDNF expression. Rotenone exposure for 30 days increased the ratio of proliferating astrocytes and decreased GDNF expression level, but the GSH content remained stable. The application of 500 nmol/L ara-c to suppress the cell proliferation restored the expression level of GDNF to almost the control level and markedly increased GSH content. Conclusion Rotenone affects the proliferation and activity of rat midbrain astrocytes in vitro and deteriorates the microenvironment of dopaminergic neurons. Low-level ara-c can increase the GSH content and GDNF expression levels by suppressing the proliferation of rotenone-exposed astrocytes, suggesting its potential value in the treatment of Parkinson's disease.     

鱼藤酮对中脑星形胶质细胞的损伤及阿糖胞苷的干预作用

Objective To observe the toxic effects of rotenone on the proliferation, γ-glutamylcysteinylglycine (GSH) content and the expression level of glial cell line-derived neurotrophic factor (GDNF) of rat rnidbrain astrocytes in vitro and the interventional effect of arabinoeytidine (ara-c). Methods In vitro cultured rat midbrain astrocytes were assigned randomly into 9 groups, including a normal control group, 4 short-term rotenone treatment groups exposed for 24 h to 10, 20, 40 or 60 nmol/L rotenone, 2 long-term rotenone treatment groups exposed for 30 days to 10 or 20 nmol/L rotenone, and 2 ara-c groups with 500 nmol/L ara-c treatment following exposure to 10 or 20 nmol/L rotenone for 6 days. The cell proliferation was assessed by immunocytochemical detection of the expression of proliferating cell nuclear antigen (PCNA). GSH content in the treated cells was measured by GSH detection kit, and the expression of GDNF was detected with immunocytochemistry and Western blot. Results The 24-h exposure to low-level rotenone (10 and 20 nmol/L) did not cause any changes in GSH content or GDNF expression in the cells. But at 40 and 60 nmol/L, rotenone treatment for 24 h significantly decreased the GSH content and GDNF expression. Rotenone exposure for 30 days increased the ratio of proliferating astrocytes and decreased GDNF expression level, but the GSH content remained stable. The application of 500 nmol/L ara-c to suppress the cell proliferation restored the expression level of GDNF to almost the control level and markedly increased GSH content. Conclusion Rotenone affects the proliferation and activity of rat midbrain astrocytes in vitro and deteriorates the microenvironment of dopaminergic neurons. Low-level ara-c can increase the GSH content and GDNF expression levels by suppressing the proliferation of rotenone-exposed astrocytes, suggesting its potential value in the treatment of Parkinson's disease.     

鱼藤酮对中脑星形胶质细胞的损伤及阿糖胞苷的干预作用

Objective To observe the toxic effects of rotenone on the proliferation, γ-glutamylcysteinylglycine (GSH) content and the expression level of glial cell line-derived neurotrophic factor (GDNF) of rat rnidbrain astrocytes in vitro and the interventional effect of arabinoeytidine (ara-c). Methods In vitro cultured rat midbrain astrocytes were assigned randomly into 9 groups, including a normal control group, 4 short-term rotenone treatment groups exposed for 24 h to 10, 20, 40 or 60 nmol/L rotenone, 2 long-term rotenone treatment groups exposed for 30 days to 10 or 20 nmol/L rotenone, and 2 ara-c groups with 500 nmol/L ara-c treatment following exposure to 10 or 20 nmol/L rotenone for 6 days. The cell proliferation was assessed by immunocytochemical detection of the expression of proliferating cell nuclear antigen (PCNA). GSH content in the treated cells was measured by GSH detection kit, and the expression of GDNF was detected with immunocytochemistry and Western blot. Results The 24-h exposure to low-level rotenone (10 and 20 nmol/L) did not cause any changes in GSH content or GDNF expression in the cells. But at 40 and 60 nmol/L, rotenone treatment for 24 h significantly decreased the GSH content and GDNF expression. Rotenone exposure for 30 days increased the ratio of proliferating astrocytes and decreased GDNF expression level, but the GSH content remained stable. The application of 500 nmol/L ara-c to suppress the cell proliferation restored the expression level of GDNF to almost the control level and markedly increased GSH content. Conclusion Rotenone affects the proliferation and activity of rat midbrain astrocytes in vitro and deteriorates the microenvironment of dopaminergic neurons. Low-level ara-c can increase the GSH content and GDNF expression levels by suppressing the proliferation of rotenone-exposed astrocytes, suggesting its potential value in the treatment of Parkinson's disease.