【作者】 邓海燕；

【导师】 黄国英；

【作者基本信息】 复旦大学 ， 儿科学， 2008， 博士

【摘要】 蒽环类抗生素(anthracycline,ANTH)是目前广泛应用于临床治疗造血系统肿瘤和实体瘤最为有效的一类抗肿瘤药物,但是其存在剂量累积性的心脏毒性,尤其是迟发性药物性心肌病,最终可导致心力衰竭。随着很多肿瘤预后的大大改善,生存率的提高,患者的远期生存质量越来越受到关注。对ANTH所致心脏毒性的研究也日渐深入。目前临床上采用心电图和超声心动图监测患者有无心脏异常的发生,并制定个体化的化疗方案,以减轻ANTH所致的心脏毒性。此外,心脏保护剂的开发也是研究热点之一。基于ANTH心脏毒性发生机制的氧化应激和自由基损伤学说,目前的研究主要集中在自由基清除剂和铁离子螯合剂方面。右丙亚胺(dexrazoxane,DZR)是其中研究最为深入而成熟的代表药物,已被美国FDA批准用于ANTH累积剂量超过300mg/m~2的晚期乳腺癌患者。但DZR会加重患者的骨髓抑制,有可能影响ANTH的抗肿瘤效果,因此临床应用仍存顾虑。促血小板生成素(thrombopoietin,TPO)是一种造血生长因子,可以促进造血干细胞增殖分化和血小板生成;近来有研究表明TPO还具有促血管生成和抗凋亡作用。而ANTH的心脏毒性与心肌细胞的凋亡有关。因此,我们设想TPO是否可以通过抗凋亡作用发挥心脏保护效应。本课题将对曾经接受过ANTH化疗的患儿进行回顾性研究,分析这些患儿有无心功能异常;并探讨建立ANTH慢性心肌损害即阿霉素心肌病动物模型的最佳方法,进一步研究TPO是否可以预防阿霉素心肌病的发生以及可能的作用机制。第一部分蒽环类抗生素所致心脏毒性的临床观察研究目的了解曾经接受过ANTH化疗的患儿是否存在心功能异常,探讨其危险因素。材料和方法回顾性分析110例白血病和淋巴瘤化疗停药患儿的超声心动图随访结果,分析的指标包括左室舒张末期内径(LVID_d)、左室收缩末期内径(LVID_s)、左室射血分数(EF)、左室短轴缩短分数(FS)、心输出量(CO)、二尖瓣瓣口血流E峰/A峰流速比值(MEV/MAV)、三尖瓣瓣口血流E峰/A峰流速比值(TEV/TAV)、等容舒张时间(IRT)、左室射血时间(LVET)、心率(HR)、R-R间期、室间隔厚度(IVS)、左室后壁收缩末期厚度(LVPW_s)和左室后壁舒张末期厚度(LVPW_d)。采用STATA 7统计软件,对比相应年龄的正常儿童参考值,分析这些接受ANTH治疗的患儿以上指标是否存在异常,并分析心功能指标与初治年龄、性别、柔红霉素(DNR)累积剂量和随访时间等因素的关系,筛查心功能下降的高危因素。结果与相应年龄的正常儿童比较,接受过ANTH化疗的患儿超声心动图检查存在多个参数的异常,包括:LVID_d减小,LVID_s增大,LVPW_d和LVPW_s变薄,心率增快,FS、EF、CO值下降,MEV/MAV比值下降(p＜0.05)。32%的患儿出现EF值低于60%。多元线性回归分析表明,DNR累积剂量越高,LVID_d和LVID_s越大,而FS和EF值越低。以210mg/m~2的DNR累积剂量为分界点,超过该剂量者与小于等于该剂量的患者比较,LVID_d、LVID_s、FS和EF的差异具有显著性(p＜0.05),且前者出现EF＜60%的风险是后者的5.66倍(OR=5.66,95%CI=1.5～31.2)。结论接受过ANTH治疗的患儿存在亚临床的心功能异常。DNR累积剂量是发生心功能异常的独立危险因素,累积剂量超过210mg/m~2的患儿出现左室扩大和心功能减退的风险增大。这些患儿需要长期随访,以便早期发现隐匿的阿霉素心肌病,尽早进行干预。第二部分阿霉素心肌病大鼠模型的建立及其超声评价研究目的探讨建立阿霉素(doxorubicin,DOX)心肌病大鼠模型的最佳给药方案及超声评价大鼠心功能的可靠性,为阿霉素心肌损伤的发病机制和保护药物的研究提供理想的动物模型和检测方法。材料和方法采用雄性S-D大鼠通过不同的DOX给药方案建立三组不同的模型:急性静脉给药模型(n=10),DOX 20mg/kg一次性尾静脉注射;慢性静脉给药模型(n=10),DOX 2.5mg/kg尾静脉注射,每周一次,连续六周;慢性腹腔给药模型(n=10),DOX 2.5mg/kg腹腔注射,每周一次,连续六周。三组模型分别设立相应对照组,即CON1组(n=10),CON2组(n=6)和CON3组(n=8)。对照组给予相应实验组等量的生理盐水尾静脉注射或腹腔注射。对三组大鼠给药前和急性模型给药后第五天,慢性模型给药第六周和第十一周处死大鼠前,进行超声心动图检查,评价心功能变化;大鼠处死后心脏行病理切片观察心肌组织学变化,并在光镜下进行半定量病理评分。结果与对照组比较,三组模型中给药的大鼠均出现体重增长抑制。心脏超声检查显示,慢性静脉给药的大鼠第十一周心功能指标FS、EF值下降最为明显(分别为31.7±3.5%和65.6±5.1%),且LVID_s大于对照组(分别为0.463±0.047cm和0.412±0.020 cm)。急性静脉给药的大鼠第五天FS、EF值较给药前和相应的对照组也有显著下降(分别为39.3±2.6%和76.1±2.7%),但高于慢性静脉给药的大鼠(p＜0.05)。慢性腹腔给药的大鼠较早出现大量血性腹水,甚至死亡,但FS、EF值无显著降低(分别为45.7±6.3%和81.8±6.3%)。心肌组织病理学检查及光镜下的半定量评分结果证实,慢性静脉给药的大鼠心肌病变最严重,光镜下表现为肌原纤维排列紊乱、缺失,胞浆空泡化;急性静脉给药的大鼠心肌病变程度较轻,而慢性腹腔给药的大鼠光镜下心肌组织未见显著病变。心脏超声所测得的FS、EF值与光镜下病理评分呈显著负相关(r值分别为-0.64和-0.57)。结论阿霉素小剂量间断静脉给药共六周,累计15mg/kg的慢性给药方案能模拟了临床化疗用药过程,并造成大鼠阿霉素心肌病改变,是阿霉素心肌病理想的动物模型。超声心动图能准确测量大鼠心脏腔径,所得心功能指标与病理改变有良好的相关性。第三部分促血小板生成素对阿霉素心肌病大鼠心肌损害的保护作用的研究研究目的探讨促血小板生成素(TPO)对于大鼠阿霉素心肌病是否具有心肌保护作用及其可能的机制。材料和方法雄性S-D大鼠60只,随机分成阿霉素组(DOX组)18只,TPO干预组(DT组)18只,作为阳性对照的右丙亚胺(DZR)干预组(DD组)14只以及正常对照组(CON组)10只。除正常对照组外,其余三组大鼠均予以DOX 2.5mg/kg尾静脉注射,每周一次,连续六周。对照组大鼠根据体重予以等量的生理盐水尾静脉注射。DT组大鼠予以TPO10μg/kg腹腔注射,每周三次,分别在DOX给药前一次,给药后两次,一共六周。DD组大鼠DZR的干预方法是每次DOX给药前三十分钟予以DZR 50mg/kg腹腔注射,每周一次,一共六周。记录大鼠体重增长情况,给药前和第十一周采血进行血细胞计数。给药前、给药第六周以及第十一周处死大鼠前进行超声心动图检查,评估心功能变化。大鼠处死后心脏进行病理切片,光镜下观察心肌组织学变化。部分心肌标本在电镜下观察超微结构变化。采用Western Blot法检测大鼠心肌组织中Akt和ERK的磷酸化水平。结果DOX给药的三组大鼠体重增长减退,血细胞计数下降。第十一周DOX组大鼠仍然存在骨髓抑制,而TPO干预的大鼠各系造血恢复良好。DOX组大鼠出现了左室扩张(LVID_d/BW和LVID_s增大)和心功能(FS和EF)的显著下降(p＜0.05);光镜下见阿霉素心肌病的典型改变;电镜下显示心肌细胞线粒体肿胀,空泡变性等。而经过TPO或DZR干预的大鼠FS和EF值显著高于DOX组,与对照组比较无显著差异(p＞0.05)。光镜下的病理评分结果显示,DD组大鼠心肌病变较DT组轻,DT组较DOX组轻,差别具有统计学意义(p＜0.05)。DOX组大鼠心肌组织中Akt和ERK的磷酸化水平被显著抑制,DZR可以上调Akt和ERK的磷酸化水平,而TPO仅显著上调了Akt的磷酸化水平(p＜0.05)。结论Akt和ERK磷酸化水平的抑制可能与DOX所致的慢性心肌损害过程有关。TPO可以减轻阿霉素所致的大鼠心肌病变,改善心功能;其机制可能与上调Akt磷酸化水平及相关的抗凋亡信号通路有关。而DZR可以上调Akt和ERK的磷酸化水平,也可能是其心肌保护作用机制之一。总结1.接受过ANTH治疗的患儿与正常儿童比较,存在亚临床心功能损害。DNR累积剂量是发生心功能异常的独立危险因素,累积剂量超过210mg/m~2的患儿发生左室扩张和EF值下降的风险增大,需要长期随访。2.利用S-D大鼠经尾静脉小剂量间断注射阿霉素的慢性给药方案,可以模拟临床化疗的用药过程,并造成阿霉素心肌病的大鼠模型。超声心动图评估大鼠的心功能,与心肌的病理改变有显著的相关性。3.Akt和ERK磷酸化水平的抑制可能与DOX所致的慢性心肌损害有关。TPO可以减轻阿霉素所致的大鼠心肌病变,改善心功能;其作用机制可能与Akt磷酸化水平的上调及相关的抗凋亡信号通路有关。DZR可以上调Akt和ERK的磷酸化水平,这可能是其心肌保护作用机制之一。更多还原

【Abstract】 Anthracycline(ANTH) is the most effective antineoplastic agents developed to treat solid tumors and hematologic malignancies.However,ANTH may result in dose-dependent cardiotoxicity.As outcomes of many cancers have improved greatly with the development of modern multi-drug chemotherapy,a major concern for this growing number of survivors is the risks of late effects of treatment. Electrocardiography and echocardiography examination as well as optimal chemotherapy schedule for every individual could probably reduce the cardiotoxicity induced by ANTH.In addition,using new protective drugs is also a promising therapeutic option.Based on oxidation stress and free radical injury hypothesis, attentions are focused on antioxidative drugs and iron chelators including dexrazoxane(DZR),the only cardiac protectant approved by FDA for clinical use. However,DZR could aggravate myelosuppression and people worry if it would interfere with antitumor effect of ANTH.So it is necessary to explore new protective drugs.Thrombopoietin(TPO),as a haematopoietic growth factor,can stimulate proliferation and differentiation of megakaryocyte.Recently,TPO was reported to have antiapoptotic and angiogenesis effects.We hypothesize that TPO may protect against cardiotoxicity induced by doxorubicin(DOX).In this study,we retrospectively analized echocardiogram data of leukemia or lymphoma survivors to find the latent abnormality of heart function.A proper rat model was established and was utilized to investigate the effect of TPO on DOX-induced cardiomyopathy and underlying mechanisms. PartⅠClinical Investigation of Anthracycline-induced CardiotoxicityPurposeThis study aimed to determine whether latent cardiac dysfunction is present in children treated with anthracycline because of leukemia or lymphoma and screen for risk factors of cardiac abnormalities.Patients and MethodsSerial echocardiogram data of 110 pediatric survivors with acute leukemia or lymphoma treated with anthracycline were retrospectively reviewed.Measurements of these patients were compared with those of normal children at the same age. Associations between age at diagnosis,cumulative dose of daunorubicin(DNR),sex, length of follow-up,and deviations from normal values in M-mode echocardiograms were evaluated using multivariate linear regression analysis.ResultsCompared to age-matched normal children,the patient group had thinner left ventricle posterior wall(LVPW),increased left ventricular internal dimension at endsystole (LVID_s) and heart rate(HR),decreased left ventricular internal dimension at end-diastole(LVID_d),fractional shortening(FS),ejection fraction(EF),cardiac output(CO) and ratio of mitral E velocity to mitral A velocity(MEV/MAV). Thirty-five of the patients(32%) had EF below 60%.Survivors who received DNR cumulative doses above 210 mg/m~2 had a 5.6-fold excess risk of reduced EF(95%CI, 1.5 to 31.2) compared with those received less than 210 mg/m~2.DNR cumulative dose was correlated with dilated LVID and reduced FS and EF.ConclusionsThis study suggested that patients received anthracycline therapy had decreased heart function and DNR cumulative dose was an independent risk factor for dilated LVID and reduced FS and EF.Survivors treated with DNR doses above 210 mg/m~2 are at higher risk for reduced EF and dilated left ventricle.These patients need long-term follow-up for signs of cardiomyopathy. PartⅡEstablishment of Doxorubicin-induced Cardiomyopathy Model in Rat and Echocardiographic AssessmentObjectiveTo explore the best way of establishing a rat model of doxorubicin-induced cardiomyopathy and assess the value of transthoracic echocardiography in assessment of heart function in rats.MethodsWe established three models of cardiotoxicity induced by doxorubicin in S-D rats. Rats were injected with DOX at one dose of 20 mg/kg intravenously for an acute intravenous model,2.5mg/kg intravenously weekly for 6 weeks for a chronic intravenous model and 2.5mg/kg intraperitoneally weekly for 6 weeks for a chronic intraperitoneal model.Each model had a control group which received equal volume of saline.Transthoracic echocardiography was performed in rats at baseline and before sacrifice and additionally at 6 weeks after beginning of DOX adminiatration for those two chronic models to measure left ventricular dimensions and calculate ejection fraction.Histopathological evaluation was performed by a blinded investigator after rats were sacrificed at week 11.The frequency and severity of myocardial lesions were assessed semi-quantitatively using a light microscope.ResultsThe body weight gain of DOX-treated rats was inhibited seriously compared to control group.At week 11,more dilated LVID_s in DOX-treated rats was found compared to control rats(0.463±0.047 vs.0.412±0.020 cm),as well as significantly decreased FS and EF(31.7±3.5%and 65.6±5.1%,repectively) in chronic intravenous model.Rats which received one large dose of DOX in acute intravenous model had significantly lower FS and EF(39.3±2.6%and 76.1±2.7%,repectively) at day 5 compared to control or baseline,but higher than rats in chronic intravenous model(p＜0.05).No significant decreased FS and EF(45.7±6.3%and 81.8±6.3%, repectively) was shown in rats which received doxorubicin intraperitoneally and had plenty of ascites.Histological examination demonstrated that myocardial injury of DOX-treated rats in chronic intravenous model was more serious than that in acute model.Similarly,no obvious cardiac lesion was found in chronic intraperitoneal group.Analysis of data revealed a significant negative correlation between cardiomyopathy score and FS and EF measured by echocardiography(r=-0.64 and -0.57;p＜0.005).ConclusionsIntravenous injection with 2.5mg/kg of doxorubicin weekly for 6 weeks up to 15 mg/kg is a reliable method to establish doxorubicin-induced cardiomyopathy animal model.Echocardiography appears to be a valuable non-invasive approach to assess heart function and has significant correlation with histological findings.PartⅢThrombopoietin Protects Against Doxorubicin-induced Cardiomyopathy in Rat ModelObjectiveThis study was designed to explore the protective effect of thrombopoietin(TPO) on doxorubicin-induced cardiomyopathy in rat model and related possible mechanisms.MethodsSixty S-D male rats were utilized in tatal.Rats were injected with DOX 2.5mg/kg intravenously once a week for 6 weeks with or without TPO 10μg/kg intraperitoneal pretreatment three times a week till last dose of doxorubicin.As a positive control group,DZR was administered at a dose ratio of 20:1 intraperitoneally 30 min prior every dose of DOX.Ten rats received saline as a normal control group.Left ventricular dimensions were measured and FS,EF was calculated automatically by transthoracic echocardiography in all surviving rats at baseline and week 6 and before sacrifice.Heart morphology was studied by light and electron microscopy to evaluate the severity of cardiac damage in rats.Western blot analysis was used to measure the phophorylation of Akt and ERK in heart tissue.ResultsDOX treatment caused depression in body weight and blood cell counts.TPO resulted in profound stimulation of blood cell production.Rats which received doxorubicin only had dilated left ventricle,significantly decreased FS and EF,severe cardiac damage including mitochondrial swelling and vacuolization under light and electron microsopy.TPO or DZR pretreatment exerted significant cardioprotective effects in terms of improved fractional shortening,ejection fraction and cardiomyopathy score.We also found that phosphorylation of Akt and ERK levels in heart tissue of DOX-treated rats were significantly inhibited and DZR increased both of them.TPO pretreatment also increased the level of Akt activation.ConclusionsOur results suggested that activation of the Akt and ERK signaling pathways was involved in the course of DOX-induced cardiomyopathy and TPO could exert cardiac protective effects probably through upregulating Akt phosphorylation in a rat model. And upregulation of Akt and ERK phosphorylation may also play an important role in cardioprotective effect of DZR.Summary1.Patients received anthracycline therapy had latent decreased heart function and DNR cumulative dose was an independent risk factor for cardiotoxicity.Those survivors treated with DNR doses above 210 mg/m~2 are at higher risk for reduced EF and dilated left ventricle and need long-term follow-up.2.Intermittent intravenous administration with repeated low doses of DOX could result in cardiomyopathy in rats and mimic clinical chemotherapy situation well. Echocardiography was a valuable approach to assess heart function of rats and had significant correlation with histological findings.3.Activation of the Akt and ERK signaling pathways was involved in the course of DOX-induced cardiomyopathy.TPO could exert cardiac protective effects in rat model probably by upregulating Akt phosphorylation level and related antiapoptotic pathway.Upregulation of Akt and ERK phosphorylation may also play a role in cardioprotective effect of DZR.更多还原