高糖诱导大鼠海马神经元细胞模型建立及细胞凋亡的影响
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摘要
目的通过不同葡萄糖浓度和不同作用时间诱导SD大鼠海马神经元,建立稳定的糖尿病脑病细胞模型,探讨高糖对海马神经元凋亡情况的影响。方法 (1)海马神经元的原代培养及纯度鉴定。(2)最适高糖浓度探索:CCK-8法检测海马神经元各组细胞活性。(3)最适浓度下高糖作用时间的探索:Western blot检测海马神经元细胞Bcl-2、Bax的表达情况。(4)最适浓度和作用时间下,流式细胞术检测细胞的凋亡率。结果 (1)成功培养海马神经元,神经元细胞经鉴定纯度达85%以上。(2) 45 mmol/L组海马神经元存活率均> 80%。(3) 45 mmol/L-48 h为最适作用时间组,细胞内Bcl-2表达下降、Bax表达升高(P <0. 01)。(4)最适高糖组细胞凋亡率显著升高(P <0. 01)。结论高糖浓度为45 mmol/L、作用时间48 h为理想的糖尿病脑病细胞模型,且高糖通过影响Bcl-2、Bax的表达导致海马神经元凋亡率升高。
Objective To induce hippocampal neurons in SD rats by different glucose concentrations and different time of action,and establish a stable cell model of diabetic encephalopathy to investigate the effect of high glucose on hippocampal neuronal apoptosis. Methods( 1) Primary culture and purity identification of hippocampal neurons.( 2) The optimal high glucose concentration was explored; CCK-8 method was used to detect the cell viability of hippocampal neurons.( 3) Exploration of high glucose action time under optimal concentration; Western blot was used to detect the expression of Bcl-2 and Bax.( 4) Apoptosis rate of cells was detected by flow cytometry under optimal concentration and time. Results( 1) The hippocampal neurons were successfully cultured,and the purity of the neurons was 85%.( 2) The survival rate of hippocampal neurons in the 45 mmol/L group was > 80%.( 3) 45 mmol/L-48 h was the optimal time group,the expression of Bcl-2 was decreased and the expression of Bax was increased( P < 0. 01).( 4) The apoptosis rate of the optimal high glucose group was significantly increased( P < 0. 01). Conclusion The high glucose concentration of 45 mmmol/L and the action time of 48 hours are ideal cell models for diabetic encephalopathy,and high glucose can increase the apoptosis rate of hippocampal neurons by affecting the expression of Bcl-2 and Bax.
Objective To induce hippocampal neurons in SD rats by different glucose concentrations and different time of action,and establish a stable cell model of diabetic encephalopathy to investigate the effect of high glucose on hippocampal neuronal apoptosis. Methods( 1) Primary culture and purity identification of hippocampal neurons.( 2) The optimal high glucose concentration was explored; CCK-8 method was used to detect the cell viability of hippocampal neurons.( 3) Exploration of high glucose action time under optimal concentration; Western blot was used to detect the expression of Bcl-2 and Bax.( 4) Apoptosis rate of cells was detected by flow cytometry under optimal concentration and time. Results( 1) The hippocampal neurons were successfully cultured,and the purity of the neurons was 85%.( 2) The survival rate of hippocampal neurons in the 45 mmol/L group was > 80%.( 3) 45 mmol/L-48 h was the optimal time group,the expression of Bcl-2 was decreased and the expression of Bax was increased( P < 0. 01).( 4) The apoptosis rate of the optimal high glucose group was significantly increased( P < 0. 01). Conclusion The high glucose concentration of 45 mmmol/L and the action time of 48 hours are ideal cell models for diabetic encephalopathy,and high glucose can increase the apoptosis rate of hippocampal neurons by affecting the expression of Bcl-2 and Bax.
引文
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[12]Gaspar JM,Castilhoá,Baptista FI,et al. Long-term exposure to high glucose induces changes in the content and distribution of some exocytotic proteins in cultured hippocampal neurons[J]. Neuroscience,2010,171(4):981-992.
[13]蔺心敬,李吕力,杨德刚.血管性痴呆大鼠海马神经元调亡和蛋白表达实验研究[J].中国现代医学杂志,2007,17(9):2326-2329.
[14]Fan L,Zhang HF,Li XB,et al. Emodin protects hyperglycemia-induced injury in PC-12cells by up-regulation of miR-9[J]. Mol Cell Endocrinol,2018,474:194-200.
[15]闫斌,王靖博,张宏,等.槲皮素对高糖培养海马神经元凋亡及Akt、p-Akt、Bcl-2、Bax蛋白表达的影响[J].中国康复理论与实践,2017,23(12):1390-1396.
[2]张佳,王小丽,于东升.高糖诱导血管内皮细胞凋亡机制的研究[J].现代生物医学进展,2018,18(11):2012-2018.
[3]Liu F,Jiang YJ,Zhao HJ,et al. Electroacupuncture ameliorates cognitive impairment and regulates the expression of apoptosis-related genes Bcl-2 and Bax in rats with cerebral ischaemia-reperfusion injury[J]. Acupunct Med,2015,33(6):478-484.
[4]USP XXII,NF XVII[S]. United States Pharmacopeial Convention,Inc,1990,2069.
[5]Chu X,Zhao Y,Liu F. Rapidly raise blood sugar will aggravate brain damage after severe hypoglycemia in rats[J]. Cell Biochem Biophys,2014,69(1):131-139.
[6]章秋,代芳.关注糖尿病患者的认知功能障碍[J].中国全科医学,2016,19(2):130-134.
[7]Gold SM,Dziobek I,Sweat V. Hippoeampal damage and memory impairments as possible early brain complications of type 2 diabetes[J]. Diabetologia,2007,50(4):711-719.
[8]Brewer GJ,Torricelli JR,Evege EK,et al. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal,a new serum-free medium combination[J]. J Neurosci Res,1993,35(5):567-576.
[9]Beren SA,Russell JW. Metabotropic glutamate receptor3 protects neurons from glucose-induced oxidative injury by increasing intracellular glutathione concentration[J]. J Neurochem,2007,101(2):342-354.
[10]凌俊辉,钟珠,蔡斯俏,等.高糖培养对乳鼠海马神经元存活及c-fos蛋白表达影响[J].广东医学院学报,2013,31(3):256-259.
[11]张贝贝,刘文洪,李俊峰,等.铁皮石斛多糖对高糖诱导的血管内皮细胞Bax、Bcl-2表达的影响[J].中国药理学通报,2015,31(1):64-70.
[12]Gaspar JM,Castilhoá,Baptista FI,et al. Long-term exposure to high glucose induces changes in the content and distribution of some exocytotic proteins in cultured hippocampal neurons[J]. Neuroscience,2010,171(4):981-992.
[13]蔺心敬,李吕力,杨德刚.血管性痴呆大鼠海马神经元调亡和蛋白表达实验研究[J].中国现代医学杂志,2007,17(9):2326-2329.
[14]Fan L,Zhang HF,Li XB,et al. Emodin protects hyperglycemia-induced injury in PC-12cells by up-regulation of miR-9[J]. Mol Cell Endocrinol,2018,474:194-200.
[15]闫斌,王靖博,张宏,等.槲皮素对高糖培养海马神经元凋亡及Akt、p-Akt、Bcl-2、Bax蛋白表达的影响[J].中国康复理论与实践,2017,23(12):1390-1396.