Dang Yong-ming, Fang Ya-dong, Hu Jiong-yu, Zhang Jia-ping, Song Hua-pei, Zhang Yi-ming, Zhang Qiong, Huang Yue-sheng
Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, the Third Military Medical University, Chongqing 400038, China.
Zhonghua Shao Shang Za Zhi. 2010 Feb;26(1):18-22.
To investigate the influence of microtubule depolymerization of myocardial cells on distribution and activity of mitochondria, and energy metabolism of cells in adult rats.
Myocardial cells of SD adult rats and SD suckling rats were isolated and cultured. They were divided into adult and suckling rats control groups (AC and SC, normally cultured without any stimulating factor), adult and suckling rats microtubule depolymerization agent groups (AMDA and SMDA, cultured with 8 micromol/L colchicine containing nutrient solution for 30 minutes) according to the random number table. (1) The expression of polymerized beta tubulin in myocardial cells of adult and suckling rats was detected with Western blot. (2) Myocardial cells of rats in AC and AMDA groups were collected. The expression of cytochrome c was detected with Western blot. Distribution of voltage-dependent anion channels (VDAC) and polymerized beta tubulin in myocardial cells were observed with immunofluorescent staining. Mitochondrial inner membrane potential was determined with immunocytochemical method. Activity of myocardial cells was detected with MTT method. Contents of ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) and energy charge of cells were determined with high performance liquid chromatography.
(1) The expression of polymerized beta tubulin:in AMDA group it was 0.52 + or - 0.07, which was obviously lower than that (1.25 + or - 0.12) in AC group (F = 31.002, P = 0.000); in SMDA group it was 0.76 + or - 0.12, which was significantly lower than that (1.11 + or - 0.24) in SC group (F = 31.002, P = 0.000), but was obviously higher than that in AMDA group (F = 31.002, P = 0.009). (2) The expression of cytochrome c in AC group was 0.26 + or - 0.03, which was obviously lower than that (1.55 + or - 0.13) in AMDA group (t = -24.056, P = 0.000). (3) Immunofluorescent staining result: in AC group, microtubules of myocardial cells were in linear tubiform, distributed in parallel with myocardial fiber; VDAC staining result showed that mitochondria were in granular form, distributed in the same direction as microtubules. In AMDA group, the normal distribution regularity of microtubules was destroyed, with weakened immune fluorescence intensity, microtubules structure indistinct, continuity lost, rough in appearance, and the distribution of mitochondria became disrupted. (4) Mitochondrial inner membrane potential in AC group fluorescent intensity was 1288 + or - 84, which was obviously higher than that (331 + or - 27) in AMDA group (t = 26.508, P = 0.000). (5) Cellular activity: in AC group absorbance value was 1.75 + or - 0.11, which was obviously lower than that (0.81 + or - 0.07) in AMDA group (t = 17.348, P = 0.000). (6) Energy metabolism: compared with those in AC group, content of ATP decreased, contents of ADP and AMP increased, and ATP/ADP value and energy charge decreased in AMDA group.
Microtubules and mitochondria distribute in the same direction in normal myocardial cells in adult rats. After microtubule depolymerization, mitochondria are arranged in disorder fashion; cytochrome c leaks from mitochondria; mitochondrial membrane potential, energy supply, and cellular activity decrease in the myocardial cells.
探讨成年大鼠心肌细胞微管解聚对线粒体分布与活性及细胞能量代谢的影响。
分离培养SD成年大鼠和SD乳鼠的心肌细胞。按随机数字表法将其分为成年和乳鼠对照组（AC和SC，正常培养，无任何刺激因素）、成年和乳鼠微管解聚剂组（AMDA和SMDA，用含8 μmol/L秋水仙碱的培养液培养30分钟）。（1）采用蛋白质免疫印迹法检测成年和乳鼠心肌细胞中聚合态β微管蛋白的表达。（2）收集AC组和AMDA组大鼠的心肌细胞。采用蛋白质免疫印迹法检测细胞色素c的表达。用免疫荧光染色观察心肌细胞中电压依赖性阴离子通道（VDAC）和聚合态β微管蛋白的分布。采用免疫细胞化学方法测定线粒体膜电位。用MTT法检测心肌细胞活性。采用高效液相色谱法测定ATP、二磷酸腺苷（ADP）、一磷酸腺苷（AMP）含量及细胞能荷。
（1）聚合态β微管蛋白的表达：AMDA组为0.52±0.07，明显低于AC组的1.25±0.12（F=31.002，P=0.000）；SMDA组为0.76±0.12，显著低于SC组的1.11±0.24（F=31.002，P=0.000），但明显高于AMDA组（F=31.002，P=0.009）。（2）AC组细胞色素c的表达为0.26±0.03，明显低于AMDA组的1.55±0.13（t=-24.056，P=0.000）。（3）免疫荧光染色结果：AC组心肌细胞微管呈线性管状，与心肌纤维平行分布；VDAC染色结果显示线粒体呈颗粒状，与微管分布方向一致。AMDA组微管正常分布规律被破坏，免疫荧光强度减弱，微管结构不清,连续性丧失，外观粗糙，线粒体分布紊乱。（4）AC组线粒体膜电位荧光强度为1288±84，明显高于AMDA组的331±27（t=26.508，P=0.000）。（5）细胞活性：AC组吸光度值为1.75±0.11，明显低于AMDA组的0.81±0.07（t=17.348，P=0.000）。（6）能量代谢：与AC组相比，AMDA组ATP含量降低，ADP和AMP含量增加，ATP/ADP值和能荷降低。
成年大鼠正常心肌细胞中微管与线粒体同向分布。微管解聚后，线粒体排列紊乱；细胞色素c从线粒体泄漏；心肌细胞线粒体膜电位、能量供应及细胞活性降低。
