【作者】 王冉；

【导师】 陈学良；

【作者基本信息】 山东大学 ， 内科学， 2008， 博士

【摘要】 目的:急性髓系白血病（AML）是一种造血系统细胞异常增殖的恶性血液肿瘤。其发生和发展与白血病细胞凋亡异常密切相关。细胞具有增殖、分化和凋亡三方面的特征,在维持正常组织的生长平衡过程中三者互相协调、共同调节。一旦细胞凋亡受阻,打破细胞的平衡调节,造成明显的生长优势,就为肿瘤的发生奠定基础。因此诱导凋亡疗法是目前治疗白血病的最重要方法之一。三氧化二砷（As₂O₃）作为经典的凋亡诱导剂,不仅可使初治的APL患者有较高完全缓解率,对全反式维甲酸和化疗耐受的复发患者及其他类型的白血病也有良好的治疗作用,并且不会导致骨髓抑制。但是其毒副作用在一定程度上限制了As₂O₃在临床的应用。有研究表明As₂O₃的毒性呈剂量依赖性,因此,寻找少毒副作用增敏剂,与As₂O₃联合应用,增强其诱导凋亡的作用,降低其使用剂量,减轻其毒副作用,是拓宽As₂O₃临床应用范围的重要手段。鼠尾草酸（Carnosic acid,CA）是迷迭香提取物中的抗氧化多酚,作为无毒防腐添加剂,广泛应用于食品加工行业。研究发现CA具有多种药理作用,如抗氧化、抗肿瘤、抗血栓、抗炎等。近年来,最新研究证实CA能够增强全反式维甲酸和维生素D衍生物诱导白血病细胞分化的能力。CA在体内和体外是否具有诱导白血病细胞凋亡的能力?CA与As₂O₃联合是否具有协同作用?其内在的分子机制又是什么?针对上述问题,本实验着重探讨CA自身及联合As₂O₃诱导HL-60细胞（人急性髓系白血病细胞株）及人白血病小鼠模型体内白血病细胞凋亡的作用,并采用各种分子生物学技术,研究其内在的分子机制。方法:1、体外实验:选用人急性髓系白血病细胞株HL-60细胞为研究对象,CA自身及联合As₂O₃作用,采用台盼蓝染色,细胞计数仪计数,观察药物对细胞生长情况的影响;用MTT法检测细胞活性的改变;瑞氏-吉姆萨染色观察药物作用后细胞形态学改变;碘化丙锭（PI）染色,流式细胞术分析细胞周期改变;AnnexinV/FITC和PI染色计算细胞凋亡率;应用Western blot检测细胞周期、细胞凋亡及信号转导途径的相关蛋白caspase-9、BAD、p-BAD、p27、PTEN、Akt、p-Akt的表达:采用半定量逆转录聚合酶链反应（RT-PCR）检测PTEN mRNA的表达。2、体内实验:（1）HL-60/SCID小鼠动物模型的建立及鉴定:选取5周龄的联合免疫缺陷（SCID）小鼠,鼠尾静脉注射对数生长期的HL-60细胞1×10⁷个,建立HL-60/SCID小鼠人白血病模型。观察小鼠的一般情况及生存时间;每周检查血常规,进行白细胞计数及血涂片检测;取濒死小鼠的脑、肝、脾、肾、骨髓及淋巴结肿块,进行组织病理学及细胞学检测;取濒死小鼠的骨髓细胞制成染色体标本,应用G显带法进行染色体核型分析。（2）剂量-效应实验:取接种1周后的SCID小鼠,随机分为3组,每天分别给与CA 200mg/Kg、300mg/Kg、500mg/Kg,i.g.,治疗4周后记录生存时间。（3）CA自身及与As₂O₃联合治疗HL-60/SCID小鼠的疗效观察:取接种1周后的SCID小鼠,随机分为4组:①对照组:每日给与橄榄油,i.g.+生理盐水,i.p.;②CA组:每日给与CA 500mg/Kg,i.g.+生理盐水,i.p.;③As₂O₃组:每日给与橄榄油,i.g.+As₂O₃ 3mg/Kg,i.p.:④CA+As₂O₃组:每日给与CA 500mg/Kg,i.g.+As₂O₃ 3mg/Kg,i.p.。给药4周。观察小鼠的一般情况及生存时间。取接种4周后的SCID小鼠,分组给药同（3）。给药10天后处死,取脾细胞制成单个细胞悬液,流式细胞术分析细胞周期改变和检测细胞凋亡率。取小鼠的脑、肝、脾、肾、骨髓及淋巴结肿块,进行组织病理学及细胞学检测,免疫组化检测caspase-3、p27、PTEN的表达改变。结果:1、体外实验:台盼蓝染色,细胞计数仪计数,和MTT法示,CA对HL-60细胞生长的抑制呈剂量、时间相关性,并且CA显著增强As₂O₃对HL-60细胞活性的抑制作用,差异有统计学意义（P＜0.05）。瑞氏-吉姆萨染色,油镜下观察可见治疗组细胞有凋亡现象出现。单用CA组、As₂O₃组和CA+As₂O₃组作用48h后,处于G1期的细胞百分率（%）分别为:49.69±2.66、52.93±2.18和77.59±1.40,CA显著增强As₂O₃引起的细胞G1期停滞,差异有统计学意义（P＜0.05）。Westrenblot示,与细胞周期改变一致,联合用药组p27蛋白表达明显高于单药组。表明p27参与了以上药物引起的G1期停滞。Annexin V-FITC/PI染色法显示加入15μM CA,使得凋亡率（%）从As₂O₃组的42.35±2.51上升到CA+As₂O₃组的75.5±2.45,差异有统计学意义（P＜0.05）。说明CA能够诱导HL-60细胞凋亡,其作用随着浓度的增加而增强;并且能够加强As₂O₃诱导凋亡的作用。Westren blot示48h后,cleaved caspase-9在15μM CA组,4μM As₂O₃组和15μM CA+4μMAs₂O₃组的表达依次增强,均强于对照组;BAD的表达没有明显变化;p-BAD的表达依次减弱,均弱于对照组。说明药物诱导凋亡的作用与激活caspase-9,抑制BAD磷酸化有关。PTEN蛋白和mRNA的表达在15μM CA组,4μM As₂O₃组和15μM CA+4μM As₂O₃组依次增强,均强于对照组。Akt在15μM CA组,4μM As₂O₃组和15μM CA+4μM As₂O₃组的表达没有明显变化;p-Akt的表达依次减弱,均弱于对照组。提示CA、As₂O₃能够促进PTEN的表达,抑制Akt的磷酸化。2、体内实验:（1）HL-60/SCID小鼠动物模型的建立及鉴定:接种后14天外周血涂片可见HL-60细胞,发病晚期濒死小鼠白细胞计数数倍于正常,血涂片中HL-60细胞可占90%。白细胞计数第14、21、和28天WBC（10⁹/L）分别为3.9±1.2、5.4±0.7和16.1±3.9。生存期为24—32天。取濒死小鼠解剖后,可见肝、脾、肾、睾丸肿大,并且肝、脾、肾及腹膜弥漫存在粟粒样白色结节（白血病细胞浸润）,颈项、四肢、眼、淋巴结可见肿块。脑、肝、脾、肾、骨髓及淋巴结肿块组织病理切片和细胞涂片显示有大量白血病细胞浸润。染色体核型分析示动物体内白血病细胞与体外培养的HL-60细胞染色体核型一致。（2）剂量一效应实验:经过4周治疗后,生存期（天）分别为:200mg/Kg:34.9±3.28,300mg/Kg:42.6±5.82,500mg/Kg:47.0±6.17,提示小鼠生存期呈剂量相关性,差异有统计学意义（P＜0.05）。（3）CA自身及与As₂O₃联合治疗HL-60/SCID小鼠的疗效观察:经过4周治疗后,生存期（天）分别为:对照组:27.0±2.49,CA组:50.8±3.12,As₂O₃组:62.2±2.66,CA+As₂O₃组:80.1±5.02,提示与单药相比,CA和As₂O₃联合生存期明显延长（P＜0.05）。流式细胞术分析细胞停滞在G1期的细胞百分率（%）为:对照组:36.0±4.53,CA组:46.4±6.47,As₂O₃组:51.0±3.54,CA+As₂O₃组:75.2±8.53,说明CA显著增强As₂O₃引起的细胞G1期停滞,差异有统计学意义（P＜0.05）。流式细胞术分析药物引起的细胞凋亡率（%）分别为:对照组:4.8±1.79,CA组:13.2±3.19,As₂O₃组:58.2±5.97,CA+As₂O₃组:76.2±4.87,CA能够促进As₂O₃诱导凋亡的能力（P＜0.05）。组织病理切片和细胞涂片示:对照组可见大量的白血病细胞成团聚集,治疗组均可见不同数量的凋亡的白血病细胞。免疫组化示:联合用药组caspase-3、p27、PTEN的表达均高于单药组。结论:CA在体外实验中能够抑制HL-60细胞的增殖,具有一定的诱导细胞凋亡和细胞周期停滞的能力,并且呈时间、剂量依赖性。在体内实验中,CA亦能引起HL-60细胞凋亡,并能够延长白血病小鼠的生存时间,呈剂量依赖性。更为重要的是,CA与As₂O₃联合应用,不但能够在体外显著增加白血病细胞的凋亡率,引起细胞周期阻滞于G1期,并且在体内也能显著增强上述作用,并能显著延长小鼠生存时间。其协同效果明显高于单药组。在诱导凋亡作用机制的研究中,我们发现CA自身及联合As₂O₃,在体外能促进p27的表达,激活caspase-9,抑制BAD的磷酸化。且PTEN/Akt信号通路亦参与了各药物诱导的细胞凋亡和细胞周期的停滞。在体内研究中,CA自身及联合As₂O₃能够活化caspase-3,增强FTEN、p27的表达。综上所述,体内、体外实验证明CA能够增强As₂O₃抗白血病的作用,使得应用较小剂量的As₂O₃就能达到较好的治疗效果,降低其使用剂量,也就减轻了毒副作用,并且CA本身安全无毒,又具有一定的抑制白血病的效果,因此,CA有希望作为辅佐剂在As₂O₃联合疗法治疗白血病中发挥作用。更多还原

【Abstract】 Objective:Acute myeloid leukemia（AML）is an aggressive hematologic malignancy. The development and progression of the leukemic disease invariably involve deregulation in the apoptotic response.Therefore,apoptosis-inducing therapy is one of the current primary treatments for AML.As₂O₃,as an apoptosis inducer,has shown substantial efficacy in treating both newly diagnosed and relapsed/refractory patients with acute promyelocytie leukemia（APL,a subset of AML）.Moreover,As₂O₃ has clinical efficacy in patients with all-trans retinoic acid（ATRA）-resistant APL,for there seems to be no obvious cross-resistance between ATRA and As₂O₃.In addition, use of As₂O₃ generally does not lead to myelosuppression,providing a potential advantage over use of conventional cytotoxic agents.However,the reported chronic toxicities and carcinogenicity of As₂O₃ have hampered its acceptance as a first-choice drug.The major clinical limitation is a pronounced toxicity,which is dose dependent. Hence,one approach to overcoming the problem is combination therapy,which may allow for administration of lower doses of arsenic trioxide,minimizing toxicity and potential drug antagonism.Thus,it is of great importance to develop adjuvant agents, which have low toxicity,increase the apoptotic action of As₂O₃ and reduce the drug dosage to physiologically tolerable concentrations（＜5μM）.Carnosic acid,the major rosemary polyphenol,is an antioxidant food additive with non-toxicity.It has many pharmacological effects,such as antioxidant,antitumor,antithrombotic and antiinflammatory effect.The purpose of our study was to evaluate the effects of carnosic acid on apoptosis,to specifically concentrate on the cooperated effects of carnosic acid with low concentration of As₂O₃ in HL-60 human myeloid leukemia cells,and to further investigate the molecular mechanisms involved in these effects in vitro and in vivo.Methods:In vitro:Cell growth was determined by counting cells using a Coulter counter.Cellular viability was measured by MTT assay,and cell cycle distribution and apoptosis were monitored by flow cytometry（FCM）.The protein expression of cleaved caspase-9,BAD,p-BAD,p27,PTEN,Akt,p-Akt were assessed by western blot analysis and the lever of PTEN mRNA was tested by RT-PCR analysis. In vivo:（1）Establishment and Evaluation of Human Acute myeloid Leukemia HL-60 Model in SCID Mice.SCID mice were transplanted by tail vein injection with 1×10⁷ HL-60 cells.Leukemic cells were detected by chromosome karyotype analysis and histopathologic methods.（2）In dose-response experiments,mice were treated for 4 w with daily i.g.of CA（200mg/Kg,300mg/Kg,500mg/Kg）,after 7 days of leukemia engraftment.The average survival time was observed.（3）Treatment of carnosie acid and carnosie aeid-As₂O₃ combination.Mice were divided into 4 groups（①control group,②CA group,③As₂O₃ group,④CA+ As₂O₃ group） to receive treatment for 4 w after 7 days of leukemia engraftment.The changes of common condition and the average survival time were observed.After 4 w of leukemia engraftment,the mice were divided into 4 group（①control group,②CA group,③As₂O₃ group,④CA+ As₂O₃ group）to receive treatment for 10 d.At the end of the experiment,all mice were killed by the institutionally approved method. Spleen,liver,long bone and lymph node metastasis were excised and examined by microscopy and immunohistochemistry.The cell cycle progression and the apoptosis of spleen cells were examined using FCM.Results:In vitro:Camosic acid inhibited cell growth and decreased cell viability in a dose- and time- dependent manner,and induced G1 arrest and apoptosis.Carnosic acid also augmented these effects when they were induced by a low（physiological） concentration of arsenic trioxide,which was associated with upregulation of p27 and activation of caspace-9.These effects were apparently mediated by the induction of PTEN expression and by blocking Akt pathway.In vivo:（1）Transplantation was always successful,and all animals died generally in 24-32 d.（2）After 4 w treatment,the average survival time was as follows: 200mg/Kg.34.9±3.28 days,300mg/Kg:42.6±5.82 days,500mg/Kg:47.0±6.17 days. These results showed camosic acid treatment increased the average survival time in a dose- dependent manner（P＜0.05）.（3）After 4 w treatment,the average survival time was as follows:control group:27.0±2.49 days,CA grpup:50.8±3.12 days,As₂O₃ group:62.2±2.66 days,CA+As₂O₃ group:80.1±5.02 days.The findings demonstrated that carnosic acid could particularly promote the average survival time induced by As₂O₃（P＜0.05）.The cells in G1 phase（%）were as follows:control group: 36.0±4.53,CA group:46.4±6.47,As₂O₃ group:51.0±3.54,CA+As₂O₃ group: 75.2±8.53.These data showed that carnosic acid could particularly promote cell cycle arrest of HL-60 cells induced by As₂O₃（P＜0.05）.The apoptotic cells（%）were as follows:control group:4.8±1.79,CA group:13.2±3.19,As₂O₃ group:58.2±5.97, CA+ As₂O₃ group:76.2±4.87.These observations showed that carnosic acid could enhance As₂O₃-induced apoptosis（P＜0.05）.Microscopy indicated that in the tissue of untreated animals,leukemic blasts infiltrated the parenchyme as very large perivascular masses associated with smaller aggregates of leukemic cells that obstructed sinusoids.In the tissue of treated animals,some cells with a condensed nucleus had apoptotic-like features.After drugs treatments,immunohistochemistry showed that cleaved caspase-3、p27、PTEN level was increased.Conclusions:These findings indicate that PTEN/Akt pathway has an important role in the cooperated induction of apoptosis and G1 arrest by carnosic acid and As₂O₃. Carnosic acid may have potential as an adjuvant in As₂O₃ -induced apoptosis therapy for its anticipated safety and its great potency in enhancing apoptosis-inducing action of low concentration of As₂O₃.更多还原