成体大鼠心肌细胞微管解聚对线粒体分布及能量代谢的影响

目的 了解成体大鼠心肌细胞微管解聚对线粒体分布、线粒体活性及细胞能量代谢的影响. 方法 分离培养成体SD大鼠及SD大鼠乳鼠心肌细胞,按随机数字表法分为:大(乳)鼠对照组(常规培养,不加任何刺激因素)、大(乳)鼠微管解聚剂组(用含终浓度8μmol/L秋水仙碱的培养液培养,作用30 min).(1)用蛋白质印迹法检测各组大鼠和乳鼠心肌细胞聚合态β微管蛋白表达量.(2)取2组大鼠心肌细胞,用蛋白质印迹法检测细胞色素c表达量;免疫荧光染色法观察细胞聚合态β微管蛋白、电压依赖型阴离子通道(VDAC)分布情况;免疫细胞化学法检测细胞线粒体内膜电位;噻唑蓝法测量细胞活性;采用高效液相色谱法,检测细胞ATP、腺苷二磷酸(ADP)、腺苷一磷酸(AMP)含量及能荷. 结果 (1)聚合态β微管蛋白表达量:大鼠微管解聚剂组为0.52±0.07,较大鼠对照组1.25±0.12明显减少(F=31.002,P=0.000);乳鼠微管解聚剂组为0.76±0.12,较乳鼠对照组1.11±0.24显著减少(F=31.002,P=0.000),但明显高于大鼠微管解聚剂组(F=31.002,P=0.009).(2)细胞色素c表达量:大鼠对照组为0.26±0.03,明显低于大鼠微管解聚剂组(1.55±0.13,t=-24.056,P=0.000).(3)免疫荧光染色:大鼠对照组心肌细胞微管多呈线性管状、与心肌纤维平行分布;VDAC着色显示线粒体呈颗粒状与微管同向分布.大鼠微管解聚剂组微管正常排列规律被破坏,表现为免疫荧光强度减弱,微管结构不清晰、连续性丧失、粗糙;线粒体分布散乱.(4)线粒体内膜电位:大鼠对照组荧光强度为1288±84,明显高于大鼠微管解聚剂组(331±27,t=26.508,P=0.000).(5)细胞活性:大鼠对照组吸光度值为1.75±0.11;大鼠微管解聚剂组为0.81±0.07,较前者明显降低(t=17.348,P=0.000).(6)能量代谢:与大鼠对照组比较,大鼠微管解聚剂组心肌细胞ATP含量下降,ADP、AMP含量上升,ATP/ADP值与能荷均降低. 结论 在正常成体大鼠心肌细胞内,微管与线粒体分布方向一致.微管解聚后心肌细胞线粒体排列紊乱,细胞色素c从线粒体漏出,线粒体内膜电位下降、能量供应降低,细胞活性下降.

Influence of microtubule depolymerization of myocardial cells on mitochondria distribution and energy metabolism in adult rats

Objective To investigate the influence of microtubule depolymerization of myocardial cells on distribution and activity of mitochondria, and energy metabolism of cells in adult rats. Methods Myocardial cells of SD adult rats and SD suckling rats were isolated and cultured. They were divided into a-dult 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 μmol/L colchicine containing nutrient solution for 30 minutes) according to the random number table. (1) The ex-pression of polymerized β 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 polymerizedβtubulin 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. Results (1) The ex-pression of polymerized β tubulin: in AMDA group it was 0.52±0.07, which was obviously lower than that (1.25±0.12) in AC group (F=31.002, P=0.000); in SMDA group it was 0.76±0.12, which was significantly lower than that (1.11±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±0.03, which was obviously lower than that (1.55±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 mito-chondria were in granular form, distributed in the same direction as microtubules. In AMDA group, the nor-mal distribution regularity of microtubules was destroyed, with weakened immune fluorescence intensity, mi-crotubules structure indistinct, continuity lost, rough in appearance, and the distribution of mitochondria be-came disrupted. (4) Mitochondrial inner membrane potential in AC group fluorescent intensity was 1288±84, which was obviously higher than that (331±27) in AMDA group (t=26.508, P=0.000). (5) Cel-lular activity: in AC group absorbance value was 1.75±0.11, which was obviously lower than that (0.81±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. Conclusions Microtubules and mitochondria distribute in the same di-rection in normal myocardial cells in adult rats. After microtubule depolymerization, mitochondria are ar-ranged in disorder fashion ; cytochrome c leaks from mitochondria; mitochondrial membrane potential, energy supply, and cellular activity decrease in the myocardial cells.