大鼠肝脏线粒体的体外功能评价及对其损伤与保护的研究

发布时间：2018-02-25 15:21

本文关键词： 肝脏线粒体呼吸功能线粒体通透性孔道线粒体膜电位超氧自由基银杏酸阿霉素辅酶Q_(10)　出处：《复旦大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的: 通过五个指标(线粒体呼吸功能、线粒体通透性孔道、线粒体膜电位、线粒体活力以及线粒体超氧自由基)评价分离制备的大鼠肝脏线粒体功能,探索银杏酸和阿霉素对肝脏线粒体的损伤效应与作用机制,并筛选防治这类损伤的药物。方法: 1.肝脏线粒体的制备与质量评估: 采用分步差速离心法制备大鼠肝脏线粒体。以线粒体呼吸功能和胞浆、线粒体标志酶(乳酸脱氢酶和柠檬酸合酶)活性来评估线粒体制备质量。 2.肝脏线粒体的功能评估: (1)线粒体呼吸功能的测定(采用Clark氧电极法) (2)线粒体通透性孔道肝放的测定(采用分光光度法) (3)线粒体膜电位的测定(采用Rhodamine 123荧光法) (4)线粒体活力测定(采用MTT法) (5)线粒体超氧自由基生成的测定(采用NBT法) 3.借助上述指标,观察银含酸及阿霉素对大鼠肝脏线粒体功能的损伤作用。 4.借助上述指标,观察辅酶Q_(10)、地高辛和银杏叶提取物对GA13和阿霉素所致大鼠肝脏线粒体功能损伤的保护作用。结果: 1.线粒体的功能评价 (1)呼吸功能和标志酶(LDH/CS)活性的测定确保了所制得的线粒体相对富集、功能完好,可供本研究的各项指标测定使用。 (2)线粒体制备后8小时内功能可以保持完好。 (3)通过五个指标(线粒体呼吸功能、线粒体通透性孔道、线粒体膜电位、线粒体活力以及线粒体超氧自由基)和各类线粒体工具药,较为全面地评价了分离制备的大鼠肝脏线粒体的功能。本研究将测定细胞活力的MTT法移植于直接测定线粒体活力,取得了良好的效果。还通过NBT法为检测线粒体超氧自由基提供了简便、灵敏的方法。这些研究为线粒体体外功能评价平台的建立奠定了良好的基础。 2.GA对线粒体的损伤作用 (1)GA可剂量依赖性地抑制线粒体的呼吸功能,并显示对氧化磷酸化有解偶联作用。 (2)GA13和GA15可剂量依赖性地诱导mPTP的开放(EC_(50)值分别为2.75μM和3.20μM),且均被CysA特异性抑制,提示GA所导致的mPTP开放作用位点很可能在环亲和素D(cyclophilin-D)。 (3)GA13和GA15可剂量依赖性地破坏肝脏线粒体膜电位,其IC_(50)值分别为3.58μM和3.79μM。 (4)GA13和GA15可剂量依赖性地抑制线粒体活力(MTT信号),其IC_(50)值分别为9.77μM和7.61μM。 (5)通过上述实验,本研究首次系统阐明了GA对肝脏线粒体的损伤作用及其机制。 3.阿霉素对线粒体的损伤作用 (1)阿霉素在体外对线粒体的呼吸功能造成较为明显的损伤作用,主要表现在Ⅳ态呼吸速率提高、RCR值和ADP/O值降低。 (2)阿霉素可剂量依赖性地诱导mPTP开放,其EC_(50)值为57.4μM;但其效应相对弱于钙离子。2μM CysA对阿霉素诱导的mPTP开放只能产生50%左右的抑制,表明阿霉素诱导的mPTP开放还存在其他机制。 (3)阿霉素具有破坏线粒体膜电位的作用,其IC_(50)值为7.32μM。 (4)通过上述实验,本研究在线粒体层面探索了阿霉素的毒性作用机制,为防治阿霉素造成的毒副作用提供了重要线索。 4.CoQ_(10)对GA13和阿霉素所致线粒体损伤的保护作用本研究通过上述线粒体功能指标,评估了三种药物(CoQ_(10)、地高辛及GBE)对GA和阿霉素造成的线粒体功能损伤的作用,发现: (1)CoQ_(10)能剂量依赖性地抑制GA13对线粒体呼吸功能的损伤。 (2)CoQ_(10)能剂量依赖性地抑制阿霉素对线粒体呼吸功能的损伤。结论: 1.本研究通过对线粒体五个方面(呼吸功能、通透性孔道、膜电位、线粒体活力以及超氧自由基生成)的功能测定,较为完整地反映了线粒体的功能。 2.本研究首次系统阐明了GA对肝脏线粒体的损伤作用及其机制。 3.本研究在线粒体层面探索了阿霉素的毒性作用机制,为防治阿霉素造成的毒副作用提供了重要线索。 4.本研究通过对三种药物的筛选,发现CoQ_(10)可有效抑制GA和阿霉素造成的线粒体呼吸功能损伤,为进一步研究该药对这类损伤的防治和临床应用奠定了基础。
[Abstract]:Objective:
Through the five indicators (mitochondrial respiratory function, mitochondrial permeability pore, mitochondrial membrane potential and mitochondrial activity and mitochondrial superoxide radical) isolated rat liver mitochondrial function evaluation preparation, explore ginkgolic acid and adriamycin damage effect on liver mitochondria and the mechanism of action, and screening of drugs for prevention and treatment of this injury.
Method:
1. preparation and quality evaluation of liver mitochondria:
Mitochondria of rat liver were prepared by stepwise differential centrifugation. Mitochondrial preparation function was evaluated by mitochondrial respiratory function, activity of cytoplasmic and mitochondrial marker enzymes (lactate dehydrogenase and citrate synthase).
2. the function evaluation of liver mitochondria:
(1) determination of mitochondrial respiratory function (using Clark oxygen electrode)
(2) determination of mitochondrial permeability channel liver release (using spectrophotometric method)
(3) determination of mitochondrial membrane potential (using Rhodamine 123 fluorescence method)
(4) determination of mitochondrial activity (using MTT method)
(5) determination of the formation of mitochondrial superoxide radical (using NBT method)
3. the effects of acid and adriamycin on the mitochondrial function of rat liver were observed with the above indexes.
4. the protective effects of coenzyme Q_ (10), digoxin and Ginkgo biloba extract on mitochondrial function damage induced by GA13 and adriamycin in rat liver were observed.
Result:
Functional evaluation of 1. mitochondria
(1) determination of the activity of respiratory function and marker enzyme (LDH/CS) ensures the relative enrichment of the obtained mitochondria and the function is good, which can be used for the determination of various indexes of this study.
(2) the function can remain intact within 8 hours after the preparation of the mitochondria.
(3) through five indicators (mitochondrial respiratory function, mitochondrial permeability pore, mitochondrial membrane potential and mitochondrial activity and mitochondrial superoxide radicals) and various mitochondrial medicine, comprehensive evaluation of isolated rat liver mitochondria preparation function. This research will be the cell viability was determined by MTT method for direct determination of transplantation the mitochondrial activity, and achieved good results. The NBT method for detection of mitochondrial superoxide radical provides a simple, sensitive method. Lays a good foundation for the study of mitochondrial function in vitro to establish the evaluation platform.
Damage effect of 2.GA on mitochondria
(1) GA dose dependently inhibited the mitochondrial respiratory function, and shows the effect on the uncoupling of oxidative phosphorylation.
(2) GA13 and GA15 could induce mPTP openness in a dose-dependent manner (EC_ (50) values were 2.75 M and 3.20 M respectively), and all were inhibited by CysA, suggesting that the mPTP opening site induced by GA is probably in the presence of cyclic affinity hormone (cyclophilin-D).
(3) GA13 and GA15 can destroy the liver mitochondrial membrane potential in a dose-dependent manner, and their IC_ (50) values are 3.58 mu M and 3.79 mu M., respectively.
(4) GA13 and GA15 can inhibit mitochondrial activity (MTT signal) in a dose-dependent manner, and its IC_ (50) values are 9.77 mu M and 7.61 mu M., respectively.
(5) through these experiments, the damage and mechanism of GA on liver mitochondria were systematically elucidated in this study for the first time.
Damage effect of 3. adriamycin on mitochondria
(1) the effect of adriamycin on the respiratory function of mitochondria was obviously damaged in vitro, which mainly manifested in the increase of respiratory rate in IV state, and the decrease of RCR value and ADP/O value.
(2) doxorubicin can induce mPTP to be induced in a dose-dependent manner. Its EC_ (50) value is 57.4 M, but its effect is relatively weaker than that of calcium ion.2 mu M CysA, which only inhibits 50% of adriamycin induced mPTP opening, indicating that there are other mechanisms for adriamycin induced mPTP opening.
(3) adriamycin has the effect of destroying the mitochondrial membrane potential, and its IC_ (50) value is 7.32 mu M.
(4) through the above experiments, we explored the toxic mechanism of doxorubicin at the mitochondrial level, which provided important clues for preventing the side effects caused by adriamycin.
Protective effect of 4.CoQ_ (10) on mitochondrial damage induced by GA13 and adriamycin
In the present study, we evaluated the effects of three drugs (CoQ_ (10), digoxin and GBE) on mitochondrial dysfunction induced by GA and adriamycin through the above mitochondrial function indicators.
(1) CoQ_ (10) can inhibit the damage of GA13 to mitochondrial respiratory function in a dose-dependent manner.
(2) CoQ_ (10) can inhibit the damage of adriamycin to mitochondrial respiratory function in a dose-dependent manner.
Conclusion:
1., we studied mitochondrial function in five aspects, including respiratory function, permeability pore, membrane potential, mitochondrial activity and superoxide radical production.
2. this study was the first to systematically elucidate the damage effect of GA on liver mitochondria and its mechanism.
3. this study explored the toxicity mechanism of doxorubicin at the mitochondrial level, which provided an important clue for the prevention and treatment of adriamycin.
4., through screening three drugs, we found that CoQ_ (10) can effectively inhibit mitochondrial respiratory function injury induced by GA and adriamycin, which laid a foundation for further research on the prevention and clinical application of this drug.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R33;R96

【引证文献】