嗜铬细胞瘤新诊断方法的建立以及SGL-3、SGL-1治疗心力衰竭的药理学研究

【作者】 刘哲；

【导师】 惠汝太； 何作祥； 李建军； 吴海英；

【作者基本信息】 中国协和医科大学 ， 遗传学， 2009， 博士

【摘要】 背景嗜铬细胞瘤是一种多发于肾上腺髓质的罕见肿瘤,如不能及时诊断治疗常会导致严重的突发心血管疾病甚至死亡。传统的嗜铬细胞瘤生化诊断方法均存在准确性不高等问题,变肾上腺素类物质（包括变肾上腺素和变去甲肾上腺素）是目前国际公认的嗜铬细胞瘤的首选检测指标。而据了解,国内目前还没有建立24小时尿变肾上腺素类物质的检测方法。本研究的目的是运用高效液相色谱仪建立24h尿变肾上腺素类物质的检测方法,从可靠性和准确性等方面考察此方法在嗜铬细胞瘤诊断中的应用价值,确立其诊断标准和正常值范围,并通过与儿茶酚胺检测结果的比较验证变肾上腺素类物质在嗜铬细胞瘤诊断中的优越性。方法首先通过加热使结合型变肾上腺素类物质转化为游离型变肾上腺素类物质,然后使用固相萃取柱进行提取,蒸干重溶后采用高效液相色谱-电化学法进行检测。通过测定仪器变异、批内变异、批间变异和回收率来考察方法的稳定性。然后用此方法对324例高血压非嗜铬细胞瘤患者（对照组）和7例嗜铬细胞瘤患者（病例组）进行了变肾上腺素类物质的检查,运用受试者工作曲线确定诊断标准,用百分位法确定正常值范围。同时对部分病例的血浆儿茶酚胺或24h尿儿茶酚胺进行了检测。结果①变肾上腺素、变去甲肾上腺素的出峰时间均不超过10分钟;②变肾上腺素的仪器变异、组内变异、批间变异和回收率分别为2.3%、5.5%、5.1%、93.4%;变去甲肾上腺素的仪器变异、组内变异、批间变异和回收率分别为1.9%、4.8%、4.7%、87.9%;③对照组的变肾上腺素检测值为152±114μg/24h,变去甲肾上腺素检测值为294±267μg/24h,病例组的变肾上腺素检测值为1426±1956μg/24h,变去甲肾上腺素检测值为4376±2591μg/24h;④变肾上腺素的诊断标准为415μg/24h,99%正常值范围上限为419μg/24h,变去甲肾上腺素的诊断标准为1464μ∥24h,99%正常值范围上限为1184μg/24h;⑤变肾上腺素诊断的灵敏度为71.4%,特异度为98.8%,变去甲肾上腺素诊断的灵敏度为100%,特异度为100%,血浆去甲肾上腺素诊断的灵敏度为71.4%,特异度为79.0%,24小时尿去甲肾上腺素诊断的灵敏度为100%,特异度为82.4%。结论本研究建立了快速、稳定、准确的24小时尿变肾上腺素类物质的检测方法,并在国内首次确立了其诊断标准和正常值范围;相比血浆、尿儿茶酚胺等检测指标,变肾上腺素类物质是目前临床检验嗜铬细胞瘤的最佳指标。背景在过去的几十年中,尽管人们对充血性心力衰竭的认识逐渐加深,治疗手段和治疗效果也有了明显改善,但心衰患者的发病率和死亡率依然居高不下,开发新的心衰治疗药物仍具有重要而迫切的临床意义。SGL-3是一个传统中药组方,经长期临床应用证明对心力衰竭具有显著的改善作用。本研究的目的之一即通过体内、体外实验验证SGL-3对心力衰竭的治疗效果,并初步探讨其作用机制。SGL-1是一个黄酮类化合物单体,经前期实验证明具有强抗氧化性,而在心力衰竭尤其是过量应用抗癌药物阿霉素导致的心脏毒性中,活性氧的产生是最重要的作用机理,因此,本研究的另一目的是通过体外实验验证SGL-1对阿霉素所致心脏毒性的改善作用,为进一步研究SGL-1在治疗心力衰竭中的应用和机理打下基础。方法①SGL-3体外实验。采用Angiotensin-Ⅱ（1μmol/L）刺激原代培养的乳鼠心肌细胞引起心肌细胞的肥大,通过H³掺入实验和鬼笔环肽染色观察SGL-3（10μg/mL）对心肌细胞肥大的抑制作用。②SGL-3体内试验。采用20周龄雄性Wistar大鼠,对大鼠进行腹主动脉缩窄手术（AAC）或假手术,20周后,通过超声心动图检测确定术后大鼠出现的心脏功能异常。之后将动物分组,灌胃给药,SGL-3的给药浓度为:150 mg·kg^-1·d^-1、500 mg·kg^-1·d^-1和1,500 mg·kg^-1·d^-1,同时设置对照组,即卡托普利(270 mg·kg^-1·d^-1)与倍他乐克(1,800 mg·kg^-1·d^-1)合用组,给药18周。然后使用多导生理仪和超声心动图检测不同组大鼠的射血分数（EF）等心脏血流动力学和形态学参数,采用苏木素—伊红染色、masson染色及天狼猩红染色进行了病理学检查,并通过高效液相色谱和放免法测定大鼠肾素—血管紧张素—醛固酮系统和心房利钠肽（ANP）血浆浓度的变化。③SGL-1体外实验。采用噻唑蓝、Hoechst33258染色法观察SGL-1的抗细胞凋亡作用,检测乳酸脱氢酶以量化SGL-1的抗细胞损伤作用,检测活性氧、丙二醛、超氧化物歧化酶、谷胱甘肽过氧化物酶活性评价SGL-1的抗氧化作用,并通过噻唑蓝染色法考察了SGL-1对阿霉素抗肿瘤活性的影响。结果①SGL-3可以显著抑制Angiotensin-Ⅱ刺激引起的心肌细胞肥大,通过H³掺入实验可观察到蛋白合成量显著下降（减少74%±26%）,通过鬼笔环肽染色可观察到细胞表面积显著减少（下降104%±31%）。②体内实验结果显示,SGL-3能够改善心脏的收缩功能（AAC组EF值为51.8%±8.42%,假手术组EF值为75.6%±7.65%,SGL-3治疗组EF值为69.3%±7.73%,卡托普利与倍他乐克合用组的EF值为56.3%±8.41%）。病理学检查则发现SGL-3能够明显抑制AAC引起的心肌细胞肥大、心肌纤维化。通过高效液相色谱和放免法测定,发现肾素—血管紧张素—醛固酮系统和ANP血浆浓度均有一定程度的改善（与模型组相比,p＜0.05）。③SGL-1可以显著减轻阿霉素引起的细胞凋亡（82.8%vs.48.8%）和细胞损伤（1.83倍vs.1.23倍）,清除活性氧,降低丙二醛浓度（2.5倍vs.1.46倍）,使超氧化物歧化酶（58.9%vs.89.3%）、谷胱甘肽过氧化物酶（46.1%vs.87.3%）活性恢复至接近正常水平,并且不影响阿霉素的抗肿瘤活性（30.9%vs.29.5%）。结论①SGL-3可以减轻心肌细胞肥大,抑制心肌肥厚向心力衰竭发展的进程。其机制可能与SGL-3改善心功能,抑制后负荷增高引起的肾素—血管紧张素—醛固酮系统激活有关。②SGL-1可以显著减轻阿霉素引起的心肌细胞凋亡和损伤,其机制与SGL-1清除活性氧,改善细胞内氧化应激状态有关。更多还原

【Abstract】 Background Pheochromocytoma is a rare tumor of adrenal medulla.If the tumor is not diagnosed and treated in time,it could lead to various cardiovascular or cerebrovascular diseases and even death.The imaging or biochemical tests now used for detecting pheochromocytoma have been proved to have some shortages,such as low sensitivity or specificity.Recently both metanephrine and normetanephrine,two O-methylated metabolites of catecholamine,were recognized internationally as the preferred monitoring indicators for pheochromocytoma.The objectives of this study are to establish the method of measuring metanephrine and normetanephrine in 24h urine using high performance liquid chromatography,to investigate its application and reliability,and to set up the diagnostic criteria and range of normal values.Moreover,the superiority of metanephrine and normetanephrine measurement in the diagnosis of the pheochromocytoma could be validated by comparing catecholamine levels in plasma and urine.Methods In the study,solid-phase extraction cartridge is used for the extraction of metanephrine and normetanephrine from the samples,and high performance liquid chromatography is used for their measurement.The variations of equipment,intra-assay variations,inter-assay variations and recoveries of metanephrine and normetanephrine were detected to examine the stability of the method.The measurement of metanephrines in 24h urine samples of 324 hypertensive non-pheochromocytoma patients（controls） and 7 verified pheochromocytoma patients（cases） were carried out by the method.The diagnostic criteria of metanephrine and normetanephrine were established by using receiver operating characteristic curves,and their range of normal values was set up by percentile rank method.The concentration of catecholamine in plasma and 24h urine of all patients and most controls were measured.Results①Peak value of both metanephrine and normetanephrine were detected within 10 minutes.②The variations of equipment,intra-assay variations,inter-assay variations and recoveries of metanephrine were 2.3%,5.5%,5.1%and 93.4% respectively,while the corresponding values of normetanephrine were 1.9%,4.8%,4.7% and 87.9%.③The average values of metanephrine and normetanephrine in controls were 152±114μg/24h and 294±267μg/24h,while the corresponding values for cases were 1426±1956μg/24h and 4376±2591μg/24h.④The diagnostic criteria and 99% upper of normal values of metanephrine were 415μg/24h and 419μg/24h,while the relevant values for normetanephrine were 1464μg/24h and 1184μg/24h.⑤The sensitivity and specificity of metanephrine were 71.4%and 98.8%,and the corresponding figures for normetanephrine were 100%and 100%.The diagnostic sensitivity and specificity of plasma catecholamines were 71.4%and 79.0%,and the corresponding figures for 24h urine catecholamines were 100%and 82.4%.Conclusion A quick,reliable and accurate method of quantifying metanephrine and normetanephrine in 24h urine was established by using high performance liquid chromatographic assay,and the diagnostic criteria and range of normal values of these two indexes were set up first time in China.In comparison with the plasma and 24h urine catecholamines,the measurement of metanephrine and normetanephrene is now the best biochemical assay for detecting pheochromocytomas. BackgroundAlthough the treatment of congestive heart failure（CHF） has improved a lot during last decades of years,the morbidity and mortality of CHF are still at high levels.Therefore,it is still an important and necessary work to develop new anti-heart failure drugs.SGL-3 is a traditional Chinese medicine,and has been proved to be very effective in the treatment of CHF.One of the objectives of this study was to demonstrate the protective effects of SGL-3 through in vitro and in vivo experiments,and to find out its pharmacological mechanism.SGL-1 is a flavone compound which has been proved to be a powerful antioxidant.Since the excessive reactive oxygen species（ROS） plays an important role in the process of doxorubicin-induced cardiotoxicity,we tried to demonstrate the protective effects of SGL-1 against doxorubicin-induced apoptosis and injury in H9c2 cells.Methods①SGL-3 in vitro study.In cultured rat neonatal cardiac myocytes,the inhibition of angiotensin-Ⅱ-induced（1μmol/L）cardiac myocyte hypertrophy by SGL-3（10μg/mL） was observed using[³H]leucine incorporation and phalloidine assay.②SGL-3 in vivo study.In vivo,twenty-weeks-old male Wistar rats were subjected to suprarenal abdominal aorta constriction（AAC） operation（n=48） or sham operation （n=12）.Twenty weeks after surgery,heart failure was detected in AAC rats by using echocardiography,then the AAC rats were treated with either SGL-3(150 mg·kg^-1·d^-1, 500 mg·kg^-1·d^-1 and 1500 mg·kg^-1·d^-1,PO,n=12 in each group) or Captopril(270 mg·kg^-1·d^-1,PO) plus Metoprolol(1,800 mg·kg^-1·d^-1,PO)（n=12） for 18 weeks respectively.Cardiac hemodynamic and morphological parameters were obtained by using multi-functional physiology recorder and echocardiography.The pathologic changes were observed using hematoxylin and eosin staining,masson staining and sirius red staining.Moreover,the levels of plasma ANP and other indexes of renin-angiotensin-aldosterone system（RAAS） were also measured.③SGL-1 in vitro study.The 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） staining,Hoechst33258 staining and lactate dehydrogenase（LDH） measurement were used to investigate the anti-apoptosis effect of SGL-1.The reactive oxygen species were observed using 2,7-dichlorofluorescein diacetate（DCFH-DA） probe,while the activities of malondialdehyde（MDA）,superoxide dismutase（SOD） and glutathione peroxidase（GPx） were measured to investigate the antioxidative activity of SGL-1.Results①In cultured rat neonatal cardiac myocytes,SGL-3 significantly inhibited cardiac myocyte hypertrophy which was induced by angiotensin-Ⅱ,reduced[³H]leucine incorporation by 74%±26%and cell surface area by 104%±31%.②The in vivo results revealed that SGL-3 could improve cardiac systolic function more effectively（EF:51.8%±8.42%for AAC group,75.6%±7.65%for sham operation group, 69.3%±7.73%for SGL-3 treated group） than Captopril plus Metoprolol（EF: 56.3%±8.41%）.Histological examination of cardiac structure showed that the extent of cardiac myocyte hypertrophy was restrained,and in compare with vehicle,myocardial fibrosis,circulating renin-aldosterone system and ANP secreting were remarkedly inhibited in AAC rats treated with SGL-3（p＜0.05）.③In H9c2 cells,SGL-1 significantly relieved doxorubicin-induced apoptosis（82.8%vs. 48.8%） and cell injury（1.83 times compared with control vs.1.23 times）,reduced the production of ROS and MDA（2.5 times vs.1.46 times）,and raised the activities of SOD （58.9%vs.89.3%） and GPx（46.1%vs.87.3%）.Moreover,we found that SGL-1 did not influence the anticancer activity of doxorubicin（30.9%vs.29.5%）.Conclusions①SGL-3 attenuated the cardiac myocyte hypertrophy both in vitro and in vivo and inhibited the process from hypertrophy to heart failure.The major mechanisms might be the improvement of cardiac functions and the inhibition of RAAS.②SGL-1 significantly reduced the apoptosis and injury induced by doxorubicin.These effects might be related to the scavenging of ROS and the improvement of oxidative stress in cardiac myocytes.更多还原