本文關(guān)鍵詞： 肝臟線粒體 呼吸功能 線粒體通透性孔道 線粒體膜電位 超氧自由基 銀杏酸 阿霉素 輔酶Q_(10)　出處：《復(fù)旦大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的: 通過五個(gè)指標(biāo)(線粒體呼吸功能、線粒體通透性孔道、線粒體膜電位、線粒體活力以及線粒體超氧自由基)評價(jià)分離制備的大鼠肝臟線粒體功能,探索銀杏酸和阿霉素對肝臟線粒體的損傷效應(yīng)與作用機(jī)制,并篩選防治這類損傷的藥物。 方法: 1.肝臟線粒體的制備與質(zhì)量評估: 采用分步差速離心法制備大鼠肝臟線粒體。以線粒體呼吸功能和胞漿、線粒體標(biāo)志酶(乳酸脫氫酶和檸檬酸合酶)活性來評估線粒體制備質(zhì)量。 2.肝臟線粒體的功能評估: (1)線粒體呼吸功能的測定(采用Clark氧電極法) (2)線粒體通透性孔道肝放的測定(采用分光光度法) (3)線粒體膜電位的測定(采用Rhodamine 123熒光法) (4)線粒體活力測定(采用MTT法) (5)線粒體超氧自由基生成的測定(采用NBT法) 3.借助上述指標(biāo),觀察銀含酸及阿霉素對大鼠肝臟線粒體功能的損傷作用。 4.借助上述指標(biāo),觀察輔酶Q_(10)、地高辛和銀杏葉提取物對GA13和阿霉素所致大鼠肝臟線粒體功能損傷的保護(hù)作用。 結(jié)果: 1.線粒體的功能評價(jià) (1)呼吸功能和標(biāo)志酶(LDH/CS)活性的測定確保了所制得的線粒體相對富集、功能完好,可供本研究的各項(xiàng)指標(biāo)測定使用。 (2)線粒體制備后8小時(shí)內(nèi)功能可以保持完好。 (3)通過五個(gè)指標(biāo)(線粒體呼吸功能、線粒體通透性孔道、線粒體膜電位、線粒體活力以及線粒體超氧自由基)和各類線粒體工具藥,較為全面地評價(jià)了分離制備的大鼠肝臟線粒體的功能。本研究將測定細(xì)胞活力的MTT法移植于直接測定線粒體活力,取得了良好的效果。還通過NBT法為檢測線粒體超氧自由基提供了簡便、靈敏的方法。這些研究為線粒體體外功能評價(jià)平臺(tái)的建立奠定了良好的基礎(chǔ)。 2.GA對線粒體的損傷作用 (1)GA可劑量依賴性地抑制線粒體的呼吸功能,并顯示對氧化磷酸化有解偶聯(lián)作用。 (2)GA13和GA15可劑量依賴性地誘導(dǎo)mPTP的開放(EC_(50)值分別為2.75μM和3.20μM),且均被CysA特異性抑制,提示GA所導(dǎo)致的mPTP開放作用位點(diǎn)很可能在環(huán)親和素D(cyclophilin-D)。 (3)GA13和GA15可劑量依賴性地破壞肝臟線粒體膜電位,其IC_(50)值分別為3.58μM和3.79μM。 (4)GA13和GA15可劑量依賴性地抑制線粒體活力(MTT信號),其IC_(50)值分別為9.77μM和7.61μM。 (5)通過上述實(shí)驗(yàn),本研究首次系統(tǒng)闡明了GA對肝臟線粒體的損傷作用及其機(jī)制。 3.阿霉素對線粒體的損傷作用 (1)阿霉素在體外對線粒體的呼吸功能造成較為明顯的損傷作用,主要表現(xiàn)在Ⅳ態(tài)呼吸速率提高、RCR值和ADP/O值降低。 (2)阿霉素可劑量依賴性地誘導(dǎo)mPTP開放,其EC_(50)值為57.4μM;但其效應(yīng)相對弱于鈣離子。2μM CysA對阿霉素誘導(dǎo)的mPTP開放只能產(chǎn)生50%左右的抑制,表明阿霉素誘導(dǎo)的mPTP開放還存在其他機(jī)制。 (3)阿霉素具有破壞線粒體膜電位的作用,其IC_(50)值為7.32μM。 (4)通過上述實(shí)驗(yàn),本研究在線粒體層面探索了阿霉素的毒性作用機(jī)制,為防治阿霉素造成的毒副作用提供了重要線索。 4.CoQ_(10)對GA13和阿霉素所致線粒體損傷的保護(hù)作用 本研究通過上述線粒體功能指標(biāo),評估了三種藥物(CoQ_(10)、地高辛及GBE)對GA和阿霉素造成的線粒體功能損傷的作用,發(fā)現(xiàn): (1)CoQ_(10)能劑量依賴性地抑制GA13對線粒體呼吸功能的損傷。 (2)CoQ_(10)能劑量依賴性地抑制阿霉素對線粒體呼吸功能的損傷。 結(jié)論: 1.本研究通過對線粒體五個(gè)方面(呼吸功能、通透性孔道、膜電位、線粒體活力以及超氧自由基生成)的功能測定,較為完整地反映了線粒體的功能。 2.本研究首次系統(tǒng)闡明了GA對肝臟線粒體的損傷作用及其機(jī)制。 3.本研究在線粒體層面探索了阿霉素的毒性作用機(jī)制,為防治阿霉素造成的毒副作用提供了重要線索。 4.本研究通過對三種藥物的篩選,發(fā)現(xiàn)CoQ_(10)可有效抑制GA和阿霉素造成的線粒體呼吸功能損傷,為進(jìn)一步研究該藥對這類損傷的防治和臨床應(yīng)用奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R33;R96

【引證文獻(xiàn)】

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本文編號：1534080