CAJ | 학술논문

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. 目的瞭解成體大鼠心肌細胞微管解聚對線粒體分佈、線粒體活性及細胞能量代謝的影響. 方法分離培養成體SD大鼠及SD大鼠乳鼠心肌細胞,按隨機數字錶法分為:大(乳)鼠對照組(常規培養,不加任何刺激因素)、大(乳)鼠微管解聚劑組(用含終濃度8μmol/L鞦水仙堿的培養液培養,作用30 min).(1)用蛋白質印跡法檢測各組大鼠和乳鼠心肌細胞聚閤態β微管蛋白錶達量.(2)取2組大鼠心肌細胞,用蛋白質印跡法檢測細胞色素c錶達量;免疫熒光染色法觀察細胞聚閤態β微管蛋白、電壓依賴型陰離子通道(VDAC)分佈情況;免疫細胞化學法檢測細胞線粒體內膜電位;噻唑藍法測量細胞活性;採用高效液相色譜法,檢測細胞ATP、腺苷二燐痠(ADP)、腺苷一燐痠(AMP)含量及能荷. 結果 (1)聚閤態β微管蛋白錶達量:大鼠微管解聚劑組為0.52±0.07,較大鼠對照組1.25±0.12明顯減少(F=31.002,P=0.000);乳鼠微管解聚劑組為0.76±0.12,較乳鼠對照組1.11±0.24顯著減少(F=31.002,P=0.000),但明顯高于大鼠微管解聚劑組(F=31.002,P=0.009).(2)細胞色素c錶達量:大鼠對照組為0.26±0.03,明顯低于大鼠微管解聚劑組(1.55±0.13,t=-24.056,P=0.000).(3)免疫熒光染色:大鼠對照組心肌細胞微管多呈線性管狀、與心肌纖維平行分佈;VDAC著色顯示線粒體呈顆粒狀與微管同嚮分佈.大鼠微管解聚劑組微管正常排列規律被破壞,錶現為免疫熒光彊度減弱,微管結構不清晰、連續性喪失、粗糙;線粒體分佈散亂.(4)線粒體內膜電位:大鼠對照組熒光彊度為1288±84,明顯高于大鼠微管解聚劑組(331±27,t=26.508,P=0.000).(5)細胞活性:大鼠對照組吸光度值為1.75±0.11;大鼠微管解聚劑組為0.81±0.07,較前者明顯降低(t=17.348,P=0.000).(6)能量代謝:與大鼠對照組比較,大鼠微管解聚劑組心肌細胞ATP含量下降,ADP、AMP含量上升,ATP/ADP值與能荷均降低. 結論在正常成體大鼠心肌細胞內,微管與線粒體分佈方嚮一緻.微管解聚後心肌細胞線粒體排列紊亂,細胞色素c從線粒體漏齣,線粒體內膜電位下降、能量供應降低,細胞活性下降.
목적 료해성체대서심기세포미관해취대선립체분포、선립체활성급세포능량대사적영향. 방법 분리배양성체SD대서급SD대서유서심기세포,안수궤수자표법분위:대(유)서대조조(상규배양,불가임하자격인소)、대(유)서미관해취제조(용함종농도8μmol/L추수선감적배양액배양,작용30 min).(1)용단백질인적법검측각조대서화유서심기세포취합태β미관단백표체량.(2)취2조대서심기세포,용단백질인적법검측세포색소c표체량;면역형광염색법관찰세포취합태β미관단백、전압의뢰형음리자통도(VDAC)분포정황;면역세포화학법검측세포선립체내막전위;새서람법측량세포활성;채용고효액상색보법,검측세포ATP、선감이린산(ADP)、선감일린산(AMP)함량급능하. 결과 (1)취합태β미관단백표체량:대서미관해취제조위0.52±0.07,교대서대조조1.25±0.12명현감소(F=31.002,P=0.000);유서미관해취제조위0.76±0.12,교유서대조조1.11±0.24현저감소(F=31.002,P=0.000),단명현고우대서미관해취제조(F=31.002,P=0.009).(2)세포색소c표체량:대서대조조위0.26±0.03,명현저우대서미관해취제조(1.55±0.13,t=-24.056,P=0.000).(3)면역형광염색:대서대조조심기세포미관다정선성관상、여심기섬유평행분포;VDAC착색현시선립체정과립상여미관동향분포.대서미관해취제조미관정상배렬규률피파배,표현위면역형광강도감약,미관결구불청석、련속성상실、조조;선립체분포산란.(4)선립체내막전위:대서대조조형광강도위1288±84,명현고우대서미관해취제조(331±27,t=26.508,P=0.000).(5)세포활성:대서대조조흡광도치위1.75±0.11;대서미관해취제조위0.81±0.07,교전자명현강저(t=17.348,P=0.000).(6)능량대사:여대서대조조비교,대서미관해취제조심기세포ATP함량하강,ADP、AMP함량상승,ATP/ADP치여능하균강저. 결론 재정상성체대서심기세포내,미관여선립체분포방향일치.미관해취후심기세포선립체배렬문란,세포색소c종선립체루출,선립체내막전위하강、능량공응강저,세포활성하강.
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.