活性氧激活线粒体凋亡通路介导高糖诱导心肌细胞凋亡的研究
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摘要
目的
利用原代培养的乳鼠心肌细胞为模型,研究不同浓度葡萄糖对心肌细胞凋亡的影响以及活性氧和p53在高糖诱导心肌细胞凋亡中的作用,揭示氧化应激和p53转位介导的线粒体凋亡通路与糖尿病心肌病的发发生发展的关系,对糖尿病心肌病的临床治疗提供一种方向。
方法
1、取新生的SD大鼠乳鼠进行心肌细胞培养,将其接种于培养板或培养瓶中,待细胞长成单层贴壁后,将培养的乳鼠心肌细胞随机分为4组:A组(对照组,葡萄糖浓度为5.6mmol/L);B组(葡萄糖浓度为16.7mmol/L);C组(葡萄糖浓度为25mmol/L);D组(葡萄糖浓度为33.3mmol/L)。应用噻唑盐比色法和荧光分光光度计分别检测细胞活力和活性氧(Reactive Oxygen Species,ROS)含量,观察不同浓度葡萄糖对心肌细胞的影响,明确高糖致心肌细胞凋亡的最佳浓度。
2、以高糖诱导的心肌细胞为模型,应用不同干预因素进行处理,随机分为4组:⑴对照组(甘露醇浓度为25mmol/L);⑵高糖组(葡萄糖浓度为25mmol/L);(3)高糖+抗氧化剂组(抗氧化剂终浓度为10mmol/L);(4)高糖+p53抑制剂组(p53抑制剂终浓度为50μmol/L)。应用流式细胞术检测细胞凋亡率,Western blot半定量检测各组细胞半胱氨酸天冬氨酸蛋白酶9(caspase-9)和p53的表达程度,荧光分光光度计检测细胞内的活性氧,分光光度计检测上清液中的超氧化物歧化酶(Superoxide Dismutase,SOD)的含量。
结果
1、应用混合酶一步消化法,差速贴壁,成功地培养出纯度较高的心肌细胞,α-平滑肌肌动蛋白免疫细胞化学鉴定为阳性。
2、与A组(对照组,葡萄糖浓度为5.6mmol/L)相比,B组(葡萄糖浓度为16.7mmol/L)、C组(葡萄糖浓度为25mmol/L)和D组(葡萄糖浓度为33.3mmol/L)的心肌细胞活力逐渐降低,活性氧逐渐升高,其中A组、B组、C组呈浓度依赖关系,C组与D组细胞活力和活性氧没有明显差异;从中可以得到高糖诱导心肌细胞凋亡的最佳浓度为25mmol/L。
3、与对照组相比,高糖组心肌细胞凋亡率和ROS明显升高,上清液中的SOD含量明显降低,Caspase-9和线粒体蛋白p53表达增多;与高糖组相比,高糖+抗氧化剂组心肌细胞凋亡率和ROS明显降低,上清液中SOD明显升高,Caspase-9和线粒体蛋白p53表达减少;与高糖组相比,高糖+p53抑制剂组的心肌细胞凋亡率和ROS明显降低,上清液中SOD含量升高,Caspase-9和线粒体蛋白p53表达减少。
结论
1、高糖诱导了体外培养的SD大鼠乳鼠心肌细胞的凋亡;
2、高糖诱导心肌细胞凋亡过程中,ROS介导p53转位到线粒体激活线粒体凋亡通路,引起心肌细胞凋亡;
3、p53反馈调节细胞内ROS的积累,二者相互作用,共同影响心肌细胞的凋亡。
Objective
To investigate the effect of different concentration of glucose as well as reactive oxygen species and p53 on cardiac myocytes apoptosis induced by high glucose.Reveal oxidative stress and the transposition of p53 to mitochondrion may be involved in the development of diabetic cardiomyopathy,so provide a direction to cure diabetic cardiomyopthy.
Methods
1. Cultivate cardiac myocytes of neonatal rat, devided into four groups as follows: group A(control group,glucose concentration is 5.6mmol/L)、group B(glucose concentration is 16.7mmol/L)、group C (glucose concentration is 25mmol/L)andgroup D(glucose concentration is 33.3mmol/L).The viability of cardiac myocytes was measured by MTT assay,the reactive oxygen species of cardiac myocytes was detected by fluorospectrophotometer in order to get an optimal glucose concentration.
2. High glucose-induced myocardial cell as a model,using different intervention factors ,devided into four groups as follows:control group(Mannitol 25 mmol/L)、high glucose (25 mmol/L)、high glucose+ antioxidant(10mmol/L)、high glucose+ Pifithrin-α(50μmol/L).The ROS of cardiac myocytes was measured by fluorospectrophotometer,the SOD of supernatant was measured by spectrophotometer,apoptosis was detected by flow cytometry, intracellular expression of caspase-9 and the level of p53 were analyzed by Western blotting.
Results
1. The cardiac myocytes were harvested by extraction of mixed enzymatic digestion, and highly purified by different adhesion time, They showedα-smooth muscle actin(+) by cell immunohistochemistry technique.
2. Compared with group A(control myocytes),the viability of cardiac myocytes from group B to group D was gradually degraded,while ROS was increased by degrees.Among the total,compared with group C,the viability of cardiac myocytes and ROS of group D had no difference.
3. Compared with the control group, in the high glucose group, the results suggested that the cardiomyocyte apoptosis rate, ROS as well as intracellular expression of caspase-9 and p53 increased , while SOD activity decreased significantly. However, in the antioxidant group,the cardiomyocyte apoptosis rate, ROS as well as intracellular expression of caspase-9 and p53 decreased , SOD activity increased remarkably compared with the high glucose group. The cardiomyocyte apoptosis rate,ROS as well as intracellular expression of caspase-9 and p53 decreased , SOD activity increased in the Pifithrin-αgroup in comparison with that in the high glucose group.
Conclusion
1. High glucose can induce neonatal rat cardiomyocyte apoptosis in vitro.
2.The transposition of p53 to mitochondrion induced by active oxygen activates mitochondrial apoptosic pathway in cardiac myocytes apoptosis induced by high glucose.
3. P53 had retroaction to ROS,each other that influence cardiomyocyte apoptosis.
利用原代培养的乳鼠心肌细胞为模型,研究不同浓度葡萄糖对心肌细胞凋亡的影响以及活性氧和p53在高糖诱导心肌细胞凋亡中的作用,揭示氧化应激和p53转位介导的线粒体凋亡通路与糖尿病心肌病的发发生发展的关系,对糖尿病心肌病的临床治疗提供一种方向。
方法
1、取新生的SD大鼠乳鼠进行心肌细胞培养,将其接种于培养板或培养瓶中,待细胞长成单层贴壁后,将培养的乳鼠心肌细胞随机分为4组:A组(对照组,葡萄糖浓度为5.6mmol/L);B组(葡萄糖浓度为16.7mmol/L);C组(葡萄糖浓度为25mmol/L);D组(葡萄糖浓度为33.3mmol/L)。应用噻唑盐比色法和荧光分光光度计分别检测细胞活力和活性氧(Reactive Oxygen Species,ROS)含量,观察不同浓度葡萄糖对心肌细胞的影响,明确高糖致心肌细胞凋亡的最佳浓度。
2、以高糖诱导的心肌细胞为模型,应用不同干预因素进行处理,随机分为4组:⑴对照组(甘露醇浓度为25mmol/L);⑵高糖组(葡萄糖浓度为25mmol/L);(3)高糖+抗氧化剂组(抗氧化剂终浓度为10mmol/L);(4)高糖+p53抑制剂组(p53抑制剂终浓度为50μmol/L)。应用流式细胞术检测细胞凋亡率,Western blot半定量检测各组细胞半胱氨酸天冬氨酸蛋白酶9(caspase-9)和p53的表达程度,荧光分光光度计检测细胞内的活性氧,分光光度计检测上清液中的超氧化物歧化酶(Superoxide Dismutase,SOD)的含量。
结果
1、应用混合酶一步消化法,差速贴壁,成功地培养出纯度较高的心肌细胞,α-平滑肌肌动蛋白免疫细胞化学鉴定为阳性。
2、与A组(对照组,葡萄糖浓度为5.6mmol/L)相比,B组(葡萄糖浓度为16.7mmol/L)、C组(葡萄糖浓度为25mmol/L)和D组(葡萄糖浓度为33.3mmol/L)的心肌细胞活力逐渐降低,活性氧逐渐升高,其中A组、B组、C组呈浓度依赖关系,C组与D组细胞活力和活性氧没有明显差异;从中可以得到高糖诱导心肌细胞凋亡的最佳浓度为25mmol/L。
3、与对照组相比,高糖组心肌细胞凋亡率和ROS明显升高,上清液中的SOD含量明显降低,Caspase-9和线粒体蛋白p53表达增多;与高糖组相比,高糖+抗氧化剂组心肌细胞凋亡率和ROS明显降低,上清液中SOD明显升高,Caspase-9和线粒体蛋白p53表达减少;与高糖组相比,高糖+p53抑制剂组的心肌细胞凋亡率和ROS明显降低,上清液中SOD含量升高,Caspase-9和线粒体蛋白p53表达减少。
结论
1、高糖诱导了体外培养的SD大鼠乳鼠心肌细胞的凋亡;
2、高糖诱导心肌细胞凋亡过程中,ROS介导p53转位到线粒体激活线粒体凋亡通路,引起心肌细胞凋亡;
3、p53反馈调节细胞内ROS的积累,二者相互作用,共同影响心肌细胞的凋亡。
Objective
To investigate the effect of different concentration of glucose as well as reactive oxygen species and p53 on cardiac myocytes apoptosis induced by high glucose.Reveal oxidative stress and the transposition of p53 to mitochondrion may be involved in the development of diabetic cardiomyopathy,so provide a direction to cure diabetic cardiomyopthy.
Methods
1. Cultivate cardiac myocytes of neonatal rat, devided into four groups as follows: group A(control group,glucose concentration is 5.6mmol/L)、group B(glucose concentration is 16.7mmol/L)、group C (glucose concentration is 25mmol/L)andgroup D(glucose concentration is 33.3mmol/L).The viability of cardiac myocytes was measured by MTT assay,the reactive oxygen species of cardiac myocytes was detected by fluorospectrophotometer in order to get an optimal glucose concentration.
2. High glucose-induced myocardial cell as a model,using different intervention factors ,devided into four groups as follows:control group(Mannitol 25 mmol/L)、high glucose (25 mmol/L)、high glucose+ antioxidant(10mmol/L)、high glucose+ Pifithrin-α(50μmol/L).The ROS of cardiac myocytes was measured by fluorospectrophotometer,the SOD of supernatant was measured by spectrophotometer,apoptosis was detected by flow cytometry, intracellular expression of caspase-9 and the level of p53 were analyzed by Western blotting.
Results
1. The cardiac myocytes were harvested by extraction of mixed enzymatic digestion, and highly purified by different adhesion time, They showedα-smooth muscle actin(+) by cell immunohistochemistry technique.
2. Compared with group A(control myocytes),the viability of cardiac myocytes from group B to group D was gradually degraded,while ROS was increased by degrees.Among the total,compared with group C,the viability of cardiac myocytes and ROS of group D had no difference.
3. Compared with the control group, in the high glucose group, the results suggested that the cardiomyocyte apoptosis rate, ROS as well as intracellular expression of caspase-9 and p53 increased , while SOD activity decreased significantly. However, in the antioxidant group,the cardiomyocyte apoptosis rate, ROS as well as intracellular expression of caspase-9 and p53 decreased , SOD activity increased remarkably compared with the high glucose group. The cardiomyocyte apoptosis rate,ROS as well as intracellular expression of caspase-9 and p53 decreased , SOD activity increased in the Pifithrin-αgroup in comparison with that in the high glucose group.
Conclusion
1. High glucose can induce neonatal rat cardiomyocyte apoptosis in vitro.
2.The transposition of p53 to mitochondrion induced by active oxygen activates mitochondrial apoptosic pathway in cardiac myocytes apoptosis induced by high glucose.
3. P53 had retroaction to ROS,each other that influence cardiomyocyte apoptosis.
引文
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8 Cudna RE, DicksonAJ.Endoplasmic reticulum signaling as a determinant of recombin- ant protein expression. Biotechnol Bioeng, 2003,81:56-65.
9 Jacobson J,Duchen MR.Mitochondrial oxidative stress and cell death in astrocytesreq- uirement for stored Ca2+ and sustained opening of the permeability transitionpore. J Cell Sci, 2002, 115:1175-1188.
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2 Leung AA,Eurich DT,Lamb DA,et al.Risk of heart failure in patients with recent-onset type 2 diabetes:population-based cohort study.J Card Fail,2009,15(2):152-157.
3 Bergner DW, Goldberger JJ. Diabetes mellitus and sudden cardiac death: what are the data? Cardiol J,2010,17(2):117-129.
4 Danial NN, Korsmeyer SJ. Cell death: critical control points. Cell. 2004,116: 205–219.
5 Thornberry NA, Lazebnik Y. Caspases: enemies within. Science.1998,281:1312-1316.
6 Zhai D,Yu E,Jin C,et al .Vaccinia virus protein F1L is a caspase-9 inhibitor. J Biol Chem, 2009,8:5569-5580.
7 Renatus M,Stennicke HR,Scott FL,et al.Dimer formation drives the activation of the cell death protease caspase 9.Proc Natl Acad Sci USA,2001,98:14250-14255.
8 Cudna RE, DicksonAJ.Endoplasmic reticulum signaling as a determinant of recombin- ant protein expression. Biotechnol Bioeng, 2003,81:56-65.
9 Jacobson J,Duchen MR.Mitochondrial oxidative stress and cell death in astrocytesreq- uirement for stored Ca2+ and sustained opening of the permeability transitionpore. J Cell Sci, 2002, 115:1175-1188.
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