摘要
目的观察TRESK介导mTOR信号通路对谷氨酸诱导脊髓神经元细胞凋亡的影响。方法脊髓原代神经元细胞接种于培养孔或培养皿中。采用随机数字表法,将其随机分为4组,每组48孔和18皿:空白对照组(C组)、谷氨酸处理组(G组)、谷氨酸+TRESK siRNA组(Glu+T组)和谷氨酸+TRESK siRNA+mTOR特异性抑制剂组(Glu+T+m组)。C组不予任何处理;G组谷氨酸(100μmol/L)处理24 h;Glu+T组使用TRESK siRNA转染神经元细胞后,谷氨酸(100μmol/L)处理24 h;Glu+T+m组使用TRESK siRNA转染神经元细胞后,终浓度为20 nmol/L的mTOR特异性抑制剂Rapamycin(Rapa)预处理20 min后再用谷氨酸(100μmol/L)处理24 h。各组谷氨酸处理24 h后,测定神经元凋亡率、cleaved Caspase3和Bax蛋白的表达水平,测定mTOR磷酸化水平及TRESK mRNA表达。结果与C组比较,G组大鼠脊髓神经元活力降低,细胞凋亡率升高,cleaved Caspase3和Bax蛋白表达上调(P<0.05);与G组比较,Glu+T组大鼠脊髓神经元活力降低,细胞凋亡率升高,cleaved Caspase3和Bax蛋白表达增加(P<0.05);与Glu+T组比较,Glu+T+m组大鼠脊髓神经元活力升高,细胞凋亡率下降,cleaved Caspase3和Bax蛋白表达减少(P<0.05)。与C组比较,G组脊髓神经元TRESK mRNA表达明显上调(P<0.05);与G组比较,Glu+T组大鼠脊髓神经元TRESK mRNA表达明显下调(P<0.05)。与C组比较,G组脊髓神经元mTOR磷酸化水平表达明显增加(P<0.05);与G组比较,Glu+T组脊髓神经元mTOR磷酸化水平表达明显增加(P<0.05);与Glu+T组比较,Glu+T+m组脊髓神经元mTOR磷酸化水平表达明显减少(P<0.05)。结论 TRESK介导mTOR信号通路抑制谷氨酸诱导脊髓神经元凋亡。
Objective To investigate the effect of TRESK on glutamic acid - induced apoptosis of neurons in spi- nal cord via roTOR signal pathway. Methods Primary cultured spinal cord neurons of SD rats were collected and cul- tured. Using random number table, the neurons were randomly divided into 4 groups, blank control group （ Group C）, glutamie acid treatment group （Group G）, glutamic acid ＋ TRESK siRNA group （Group Glu ＋ T） and glutamate ＋ TRESK siRNA ＋ roTOR inhibitor （ Group Glu ＋ T ＋ m）. Neurons in Group C received no process; neurons in Group G were trea- ted with glutamic acid （100μmol/L） for 24 h; neurons in Group Glu ＋ T were treated with glutamic acid （100μmol/L） for 24 h after transfection with TRESK siRNA ; and neurons in Group Glu ＋ T ＋ m were pretreated with Rapamycin （ 20 nmol/L） for 20 rain and treated with glutamic acid （ 100μmol/L） for 24 h after transfection with TRESK siRNA. Cell ap- optosis rate, protein expression levels of cleaved Caspase3 and Bax, roTOR phosphorylation and TRESK mRNA expression level were assessed. Results Compared with Group C, cell activity were significant reduced, while cell apoptosis rate and protein expression of cleaved Caspase3 and Bax were significant increased in Group G （ P 〈 0. 05 ）. Compared with Group G, cell activity was significant reduced, while cell apoptosis rate and protein expression of cleaved Caspase3 andBax were significant increased in Group Glu ＋ T and Group Glu ＋ T ＋ m （P 〈 0. 05 ）. Compared with Group Glu ＋ T, cell activity was significant reduced, while cell apoptosis rate and protein expression of cleaved Caspase3 and Bax were signifi- cant increased in Group Glu ＋ T ＋ m （P 〈 0. 05 ）. Compared with Group C, neuron TRESK mRNA expression in was sig- nificantly raised in.,Group G （P 〈 0. 05）. Compared with Group G, neuron TRESK mRNA expression was significantly raised in Group Glu ＋ T and Group Glu ＋ T ＋ m （P 〈 0. 05 ）. Compared with Group C, neuron mTOR
出处
《广东医学》
CAS
北大核心
2017年第24期3762-3766,共5页
Guangdong Medical Journal
基金
国家自然科学基金资助项目(编号:81300974)
广东省自然科学基金资助项目(编号:2015A030313899)
广东省医学科研基金资助项目(编号:A2015013)
佛山市医学类科技攻关项目(编号:2015AB00327)
