Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equi...Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA （shRNA） for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments （shRNA） were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector （carrying shRNA） and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells （PC12）. Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 ± 0.68%, 95.08 ± 1.79% and 96.60 ± 1.14% at the mRNA level, and 72.89 ± 2.17%, 58.83 ± 1.15% and 59.88 ± 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.展开更多
Background： Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of B...Background： Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of Bcl-2/Bax protein expression and recovering from damage to mitochondria after OGD （oxygen-glucose deprivation）-reperfusion. Methods： Viability of PC 12 cells which were injured at different time of OGD injury, was quantified by measuring MTT （2-（4,5-dimethylthia-zol-2-yl）-2,5-diphenyltetrazolium bromide） staining. In addition, PC 12 cells＇ viability was also quantified after their preincubation in different concentration of edaravone for 30 min followed by （OGD）. Furthermore, apoptotic population of PC 12 cells that reinsulted from OGD-reperfusion with or without preincubation with edaravone was determined by flow cytometer analysis, electron microscope and Hoechst/Pl staining. Finally, change of Bcl-2/Bax protein expression was detected by Western blot. Results： （1） The viability of PC12 cells decreased with time （1-12 h） after OGD. We regarded the model of OGD 2 h, then replacing DMEM （Dulbecco＇s Modified Eagle＇s Medium） for another 24 h as an OGD-reperfusion in this research. Furthermore, most PC 12 cells were in the state of apoptosis after OGD-reperfusion. （2） The viability of PC 12 cells preincubated with edaravone at high concentrations （1, 0.1, 0.01 μmol/L） increased significantly with edaravone protecting PC 12 cells from apoptosis after OGD-reperfusion injury. （3） Furthermore, edaravone attenuates the damage of OGD-reperfusion on mitochondria and regulated Bcl-2/Bax protein imbalance expression after OGD-reperfusion. Conclusion： Neuroprotective effects of edaravone on ischemic or other brain injuries may be partly mediated through inhibition of Bcl-2/Bax apoptotic pathways by recovering from the damage of mitochondria.展开更多
目的：采用实时荧光定量聚合酶链式反应（polymerase chain reaction，PCR）技术，研究P2X受体在大鼠神经胶质瘤和嗜铬细胞瘤及原代培养皮层神经元和星形胶质细胞上的表达差异。方法：取新生1-2 d SD大鼠大脑皮层，分离纯化神经元和星...目的：采用实时荧光定量聚合酶链式反应（polymerase chain reaction，PCR）技术，研究P2X受体在大鼠神经胶质瘤和嗜铬细胞瘤及原代培养皮层神经元和星形胶质细胞上的表达差异。方法：取新生1-2 d SD大鼠大脑皮层，分离纯化神经元和星形胶质细胞，并采用实时荧光定量PCR技术，比较P2X受体在大鼠胶质瘤C6细胞、大鼠肾上腺嗜铬细胞瘤PC-12细胞、星形胶质细胞和皮层神经元上的表达差异。结果：C6细胞P2X2、P2X3和P2X5表达水平显著高于星形胶质细胞，P2X4、P2X6和P2X7表达水平显著低于星形胶质细胞，PC-12细胞P2X1、P2X2、P2X3和P2X6表达水平显著高于皮层神经元，P2X5和P2X7表达水平则显著低于皮层神经元。此外，还发现P2X2、P2X5和P2X6在C6和PC-12细胞上的表达水平存在显著差异。结论：大鼠胶质瘤和嗜铬细胞瘤细胞表达多种P2X受体，且与原代培养细胞存在表达差异，提示核苷酸介导的信号传递系统可能作为潜在的肿瘤治疗靶点。展开更多
基金the National Natural Science Foundation of China, No. 30672136
文摘Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA （shRNA） for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments （shRNA） were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector （carrying shRNA） and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells （PC12）. Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 ± 0.68%, 95.08 ± 1.79% and 96.60 ± 1.14% at the mRNA level, and 72.89 ± 2.17%, 58.83 ± 1.15% and 59.88 ± 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.
基金Project (No. 2005C30059) supported by the Science and TechnologyProgram of Zhejiang Province, China
文摘Background： Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of Bcl-2/Bax protein expression and recovering from damage to mitochondria after OGD （oxygen-glucose deprivation）-reperfusion. Methods： Viability of PC 12 cells which were injured at different time of OGD injury, was quantified by measuring MTT （2-（4,5-dimethylthia-zol-2-yl）-2,5-diphenyltetrazolium bromide） staining. In addition, PC 12 cells＇ viability was also quantified after their preincubation in different concentration of edaravone for 30 min followed by （OGD）. Furthermore, apoptotic population of PC 12 cells that reinsulted from OGD-reperfusion with or without preincubation with edaravone was determined by flow cytometer analysis, electron microscope and Hoechst/Pl staining. Finally, change of Bcl-2/Bax protein expression was detected by Western blot. Results： （1） The viability of PC12 cells decreased with time （1-12 h） after OGD. We regarded the model of OGD 2 h, then replacing DMEM （Dulbecco＇s Modified Eagle＇s Medium） for another 24 h as an OGD-reperfusion in this research. Furthermore, most PC 12 cells were in the state of apoptosis after OGD-reperfusion. （2） The viability of PC 12 cells preincubated with edaravone at high concentrations （1, 0.1, 0.01 μmol/L） increased significantly with edaravone protecting PC 12 cells from apoptosis after OGD-reperfusion injury. （3） Furthermore, edaravone attenuates the damage of OGD-reperfusion on mitochondria and regulated Bcl-2/Bax protein imbalance expression after OGD-reperfusion. Conclusion： Neuroprotective effects of edaravone on ischemic or other brain injuries may be partly mediated through inhibition of Bcl-2/Bax apoptotic pathways by recovering from the damage of mitochondria.
文摘目的：采用实时荧光定量聚合酶链式反应（polymerase chain reaction，PCR）技术，研究P2X受体在大鼠神经胶质瘤和嗜铬细胞瘤及原代培养皮层神经元和星形胶质细胞上的表达差异。方法：取新生1-2 d SD大鼠大脑皮层，分离纯化神经元和星形胶质细胞，并采用实时荧光定量PCR技术，比较P2X受体在大鼠胶质瘤C6细胞、大鼠肾上腺嗜铬细胞瘤PC-12细胞、星形胶质细胞和皮层神经元上的表达差异。结果：C6细胞P2X2、P2X3和P2X5表达水平显著高于星形胶质细胞，P2X4、P2X6和P2X7表达水平显著低于星形胶质细胞，PC-12细胞P2X1、P2X2、P2X3和P2X6表达水平显著高于皮层神经元，P2X5和P2X7表达水平则显著低于皮层神经元。此外，还发现P2X2、P2X5和P2X6在C6和PC-12细胞上的表达水平存在显著差异。结论：大鼠胶质瘤和嗜铬细胞瘤细胞表达多种P2X受体，且与原代培养细胞存在表达差异，提示核苷酸介导的信号传递系统可能作为潜在的肿瘤治疗靶点。
