【作者】 付泽；

【导师】 彭双清；

【作者基本信息】 中国人民解放军军事医学科学院 ， 卫生毒理学， 2010， 硕士

【摘要】 阿霉素(doxorubicin,DOX)属蒽醌类抗生素,作为一种高效抗肿瘤药物广泛应用于临床治疗白血病、乳腺癌等。然而在用药过程中,DOX严重的心脏毒性限制了它的临床应用。研究表明,DOX的心脏毒性主要与其体内代谢过程中活性氧(reactiveoxygenspecies,ROS)的大量生成有关,过量的ROS攻击心肌细胞,从而引起心脏的氧化损伤。金属硫蛋白(metallothionein,MT)是一类广泛存在于机体内的低分子量、富含巯基的金属结合蛋白。MT具有多种生物学效应,其清除自由基和抗氧化的功能尤其引人关注。我们前期的体内研究利用MT-I/II型基因敲除(MT-/-)和野生型(MT+/+)小鼠研究MT不同表达水平对DOX心脏毒性损伤的影响。结果显示,MT-/-小鼠由于MT基因的缺失对DOX诱发的心脏毒性更加敏感,由此说明MT在对DOX心脏毒性的保护效应中发挥了关键作用,但其具体作用机制尚不清楚。本研究采用MT+/+和MT-/-乳鼠的心肌组织分别构建体外细胞培养模型,观察DOX对这两种小鼠心肌细胞的毒性作用,并且从氧化损伤角度探讨MT对DOX致培养心肌细胞损伤的保护作用机制。应用镉-血红素饱和法检测两种小鼠心肌细胞内MT的含量,结果显示MT+/+小鼠心肌细胞中MT含量约为MT-/-小鼠的2.5倍,由此验证了实验模型的可靠性。应用不同浓度DOX对MT+/+和MT-/-小鼠心肌细胞分别进行染毒,观察细胞形态变化,检测心肌细胞乳酸脱氢酶(lactatedehydrogenase,LDH)漏出、细胞存活率、细胞搏动频率以及细胞凋亡率,评价MT对DOX所致心肌细胞毒性的影响。结果显示,DOX能够产生明显的心肌细胞毒性,剂量依赖性地引起细胞形态学改变、增加LDH漏出和细胞凋亡率、降低细胞存活率和搏动频率。MT-/-小鼠心肌细胞的损伤程度要明显高于MT+/+小鼠,该结果表明MT的缺失使心肌细胞对DOX的毒性作用更为敏感,由此可见MT在对DOX心肌细胞毒性的保护效应中发挥了重要作用。大量研究表明DOX的心脏毒性主要与ROS有关,本实验分别检测了DOX对MT+/+和MT-/-小鼠心肌细胞内ROS生成以及各种抗氧化酶活力的影响,以此来评价MT对DOX所致心肌细胞氧化应激的作用。结果显示,DOX能剂量和时间依赖性地增加心肌细胞内ROS的生成,降低抗氧化酶超氧化物歧化酶(superoxidedismutase,SOD)、过氧化氢酶(catalase,CAT)的活性,并且这一作用在MT-/-小鼠心肌细胞更为显著。该结果表明,MT的缺失使得心肌细胞对DOX所致的氧化应激更为敏感,MT对DOX心肌细胞毒性的保护作用可能与其抗氧化损伤有关。细胞内过量产生的ROS能攻击脂质、蛋白质以及DNA等生物大分子,从而造成心肌细胞氧化损伤。采用TBA显色法、单细胞凝胶电泳法(彗星试验)和8-OHdG免疫组化法、DNPH比色法分别检测了DOX对MT+/+和MT-/-小鼠心肌细胞脂质过氧化、DNA损伤以及蛋白质羰基化的影响,以此评价MT对DOX所致心肌细胞氧化损伤的影响。结果显示,DOX能引起明显的心肌细胞氧化损伤,表现为脂质过氧化产物硫代巴比妥酸反应产物(thiobarbituricacidreactivesubstance,TBARS)生成的增多、DNA断裂及8-OHdG生成的增多,以及蛋白羰基含量增多。同样地,MT-/-小鼠心肌细胞的氧化损伤程度更为显著。结果表明,MT对DOX心肌细胞毒性的保护作用可能与其降低心肌细胞氧化损伤程度有关。外源性抗氧化剂NAC和还原型谷胱甘肽(reducedglutathione,GSH)能够完全抑制DOX引起的MT+/+小鼠心肌细胞存活率降低、ROS生成增加以及抗氧化酶活性降低,使上述指标恢复甚至超过正常水平。然而对于MT-/-小鼠心肌细胞,NAC和GSH仅能起到部分抑制的作用。该结果进一步表明基础水平的MT对DOX所致的心肌细胞氧化损伤具有重要的保护效应。目前认为,线粒体功能紊乱在多种心脏疾病中发挥了关键作用。因此,本实验进一步从线粒体氧化损伤的角度进行了研究。利用MitoSoxRed和Rhodamine123荧光探针,分别检测了DOX对MT+/+和MT-/-小鼠心肌细胞线粒体超氧阴离子(O2?-)和线粒体膜电位(ΔΨ)的影响,评价MT对DOX所致心肌细胞线粒体氧化损伤的作用。结果显示,DOX能引起明显的心肌细胞线粒体损伤,表现为线粒体O2?-生成增多和线粒体ΔΨ降低,并且MT-/-小鼠心肌细胞的线粒体损伤程度更为显著。此外,还发现NAC和GSH对DOX引起的MT+/+小鼠心肌细胞线粒体损伤也能够起到完全抑制的作用,而对MT-/-小鼠心肌细胞NAC和GSH仅能起到部分抑制作用。该结果进一步表明,MT对DOX心肌细胞毒性的保护作用可能与其降低心肌细胞线粒体氧化损伤程度有关。综上所述,本研究利用体外培养的MT+/+和MT-/-小鼠心肌细胞证明了MT对DOX所致心肌细胞毒性具有明显的保护作用,其机制可能为减少心肌细胞ROS蓄积、抑制心肌细胞氧化应激和线粒体氧化损伤,从而减轻心肌细胞的凋亡和死亡,具体作用机制仍需要深入研究。更多还原

【Abstract】 Doxorubicin （DOX） is a highly effective anticancer drug that is frequently employed totreat hematological and solid tumors including leukemia, breast cancer, and soft tissuesarcomas. However, the clinical use of DOX is limited by its concurrent dose-dependentcardiotoxicity. It has been reported that the mechanism of Dox-induced cardiotoxicity ismainly attributed to the formation of reactive oxygen species （ROS） and promotion ofmyocardial oxidative stress. Metallothionein （MT） is a ubiquitous, low-molecular-weight,and thiol-rich protein which is highly inducible in response to oxidative stress. MT canfunction as antioxidant on scavenging free radicals though the precise biological function ofMT still remains indistinct and controversial. We previously found metallothionein-I/II（MT-I/II） null mice are more vulnerable to Dox cardiomyopathy, but it is unknown whetherdepletion of MT would sensitize cardiomyocytes to DOX toxicity in vitro. To this end and toexamine directly the effects of MT deficiency on Dox-induced toxicity in cardiomyocytes,the present study was undertaken to establish a primary cardiomyocyte culture system fromMT-I/II null （MT-/-） and corresponding wild type （MT+/+） neonatal mice, and to investigatethe possible mechanism of MT against Dox-induced cytotoxicity focusing on oxidativedamage.At first, MT concentrations in neonatal mice cardiomyocytes was determined by thecadmium-hemoglobin affinity assay to confirm the experimental model. It was shown thatMT concentrations in the MT-/- cardiomyocytes were about 2.5-fold lower than those inMT+/+ cardioymyocytes. Cardiomyocytes from MT-I/II null （MT-/-） and corresponding wildtype （MT+/+） neonatal mice were treated with DOX at concentrations of 0, 0.1, 1, 10μM,respectively, and then cardiomyocytes morphological alterations, LDH leakage, cell viability, beating frequency and apoptosis were observed. The results showed DOXconcentration-dependently induced cytotoxicity in neonatal mice cardiomyocytes, increasedLDH leakage and cell apotosis, decreased cell viability and beating frequency, all of theseeffect were much more servere in MT-/- cardiomyocytes.Then we observed DOX-induced cardiomyocytes oxidative stress by measuring thegeneration of intracellular ROS and the activity of antioxidant enzymes. It was observedthat DOX induced a concentration- and time-dependent increase in ROS production, whichwas exaggerated in MT-/- cardiomyocytes. Furthermore, the activity of antioxidant enzymesin both two type cardiomyocytes, such as superoxide dismutase （SOD） and catalase （CAT）,were decreased after treatment with DOX. These effect was also much more servere in MT-/-cardiomyocytes, indicating that DOX induces more ROS accumulation and oxidative stressin cardiomyocytes when MT is deficient.It has been widely accepted that intracellular ROS accumulation may injury thebiological macromolecules, such as lipid, DNA and protein, which eventually leads to cellnecrosis as well as apoptosis. So we used TBA chromogenic reaction to measure the lipidperoxidation, used the comet assay and immunoperoxidase staining for 8-OHdG to detectthe DNA damage, and used the DNPH colorimetric test to determine the proteincarbonylation. It was showned that DOX induced a significant oxidative damage in bothMT+/+ and MT-/- cardiomyocytes, including lipid peroxidation, DNA damage and proteincarbonylation. Consistently, the damage to the MT-/- cardiomyocytes were more severe thanthe damage to the MT+/+ cardiomyocytes, which indicated that the protective effect of MT toDOX-induced cardiomyocytes injury may act by alleviating the oxidative damage.Furthermore, additional antioxidant N-acetylcysteine （NAC） and glutathione （GSH）significantly rescued MT+/+ but not MT-/-cardiomyocytes from DOX-induced cell death,ROS generation, and oxidative stress. These findings suggest that basal MT provideprotection against Dox-induced toxicity in cardiomyocytes, particularly highlight theimportant role of MT as a cellular antioxidant on scavenging ROS.It is now well recognized that mitochondrial dysfunction plays a crucial role in thepathogenesis of multiple cardiac diseases. So we used relevant fluorescent probe, such asMitoSox Red and Rhodamine123, to detect the ROS （O₂ -） generation and transmembranepotential in mitochondrion. The photofluorograms were then analyzed and showed as meanfluorescence intensity. The results showed DOX significantly increased mitochondrialproduction of O₂ -, while decreased the transmembrane potential of mitochondrion in bothMT+/+ and MT-/-cardiomyocytes, the latter were much more sensitive to these effect. We alsofound NAC and GSH could completely inhibit DOX-induced mitochondrial injury in MT+/+but not MT-/-cardiomyocytes, which indicated that the protective effect of MT to DOX-induced cardiomyocytes injury may act by alleviating the mitochondrial oxidativedamage.In summary, the present study clearly demonstrates that Dox-induced oxidativedamage are enhanced in the primary cultures of cardiomyocytes derived from neonatalMT-I/II null mice, and MT as a cellular antioxidant protected against these effect byscavenging intracellular and mitochondrial ROS.KeywordKeywords: Metallothionein, doxorubicin, MT-I/II null mice, cardiomyocytes, oxidativedamageSupported by grant of National Natural Science Foundation of China （30873130）.更多还原