| 线粒体细胞外裂解规律及环孢霉素A对其抑制作用的实验研究 |
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Received:July 19, 2015
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| 期刊信息：《中国骨伤》2015年28卷，第11期，第1037-1041页 |
| DOI：10.3969/j.issn.1003-0034.2015.11.013 |
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目的:观察线粒体细胞外裂解规律及环孢霉素A (cyclosporine A,CsA)对其抑制作用,探讨创伤后SIRS的发病机制与缓解策略。
方法:选取10只60～70日龄,体重240～280 g的雄性SD大鼠,用于细胞线粒体的分离。新鲜分离大鼠线粒体随机分为2组,分别在添加或不添加CsA的血浆环境下培养8 h,每30 min通过ELISA方法检测培养液中线粒体特征性蛋白COX、MDH浓度。大鼠NR8383巨噬细胞系随机分为6组,对照组(A组):常规培养液培养;NR8383+CsA共培养组(B组):培养液中含10 mmol/L CsA;NR8383+完整线粒体共培养组(C组):培养液中添加完整线粒体(mtDNA=5 μg/ml);NR8383+完整线粒体+CsA共培养组(D组):培养液中添加完整线粒体(mtDNA=5 μg/ml)与10 mmol/L CsA;NR8383+裂解线粒体共培养组(E组):培养液中添加裂解的线粒体(mtDNA=5 μg/ml);NR8383+裂解线粒体+CsA共培养组(F组):培养液中添加裂解线粒体(mtDNA=5 μg/ml)与10 mmol/L CsA.在培养1、3和 5 h时,通过ELISA方法检测上清中TNF-α及IL-6的浓度。
结果:在线粒体血浆培养中,MDH与COX水平随时间延长而升高,并在3 h与3.5 h时达到峰值,而CsA使其峰值均延迟到4.5 h;在巨噬细胞不同培养条件下,A组与B组的TNF-α与IL-6在不同时间点基本稳定,其他组间TNF-α与IL-6存在差异。培养1 h时,与C组比较,D组 的TNF-α与IL-6无显着差异,E组TNF-α与 IL-6显着增高;培养3 h时,与C组相比,D 组的TNF-α及 IL-6水平均显着降低,E组TNF-α与 IL-6水平显着增高;培养5 h时,与C组相比,D组TNF-α及 IL-6水平仍显着降低,E组TNF-α与IL-6无显着变化。F组与E组的TNF-α与IL-6水平无显着差异。
结论:线粒体在血浆中随时间而逐渐裂解,进一步激活巨噬细胞,CsA对线粒体的裂解具有抑制作用,从而抑制其对巨噬细胞的激活效应。 |
| [关键词]:线粒体 细胞分裂 环孢霉素A 创伤和损伤 动物,实验 大鼠,Sprague-Dawley |
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| Extracellular splitting pattern of mitochondria and the depressant effects of CsA on the process |
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Abstract:
Objective:To investigate extracellular splitting pattern of mitochondria and the depressant effects of CsA on the process and explore the mechanism of post-traumatic SIRS and its therapeutic strategy.
Methods:Ten male SD rats with 60 to 70 days age and 240 to 280 g weight were used for mitochondrial isolation. Freshly isolated mitochondria were randomly divided into two groups,which were cultured in blood plasma with or without CsA respectively for 8 h. COX and MDH were assayed by ELISA every 30 min. Meanwhile,Rat macrophage cell line NR8383 were treated as follows,control (group A):cultivation with normal medium;NR8383+CsA co-culture group(group B):culture medium was supplemented with CsA of 10 mmol/L;NR8383+intact mitochondria co-culture group (group C):culture medium was supplemented with intact mitochondria (mtDNA=5 μg/ml);NR8383+intact mitochondria+CsA co-culture group(group D):culture medium was supplemented with intact mitochondria (mtDNA=5 μg/ml)and CsA of 10 mmol/L;NR8383+disrupted mitochondria co-culture group (group E):culture medium was supplemented with disrupted mitochondria (mtDNA=5 μg/ml);NR8383+disrupted mitochondria+CsA co-culture group(group F):culture medium was supplemented with disrupted mitochondria(mtDNA=5 μg/ml)and CsA of 10 mmol/L. TNF-α and IL-6 concentrations in supernatant were assessed at 1,3,5 h after culture.
Results:In the mitochondria plasma cultures,MDH and COX levels were increased with the time and peaked at about 3 h and 3.5 h;CsA can delay the appearance of peak to 4.5 h. Among different treated groups,there was no significant difference in TNF-α and IL-6 between group A and group B;there was significant difference in TNF-α and IL-6 other groups. After 1 h culture,compared with group C,no significant difference of TNF-α and IL-6 was observed in group D,while TNF-α and IL-6 were significant higher in group E;after 3 h culture,compared with group C,TNF-α and IL-6 were significantly lower in group D,while TNF-α and IL-6 were significantly higher in group E;after 5 h culture,compared with group C,TNF-α and IL-6 were significantly lower in group D,while no significant difference of TNF-α and IL-6 were observed in group E. At each time point,there was no significant difference in TNF-α and IL-6 between group F and group E.
Conclusion:Mitochondria can split in serum with time,which will further activate macrophages. CsA has depressant effect to mitochondrial splitting on the process and will therefore inhibit the activation of macrophages. |
| KEYWORDS:Mitochondria Cell division Cyclosporine A Wounds and injuries Animals, laboratory Rats, Sprague-Dawley |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 常玉立,刘宏,温建民,孙天胜.线粒体细胞外裂解规律及环孢霉素A对其抑制作用的实验研究[J].中国骨伤,2015,28(11):1037~1041 |
| 英文格式: | CHANG Yu-li,LIU Hong,WEN Jian- min,SUN Tian-sheng.Extracellular splitting pattern of mitochondria and the depressant effects of CsA on the process[J].zhongguo gu shang / China J Orthop Trauma ,2015,28(11):1037~1041 |
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