【作者】 李杰；

【导师】 罗建民；

【作者基本信息】 河北医科大学 ， 内科学， 2012， 博士

【摘要】 慢性粒细胞白血病（chronic myelogenous leukemia，CML）是我国慢性白血病中常见的一种类型，可发生于各年龄人群。虽然传统治疗药物羟基脲、干扰素等在一定时期内可以改善患者症状，但不能治愈本病，也不能阻断疾病进展。绝大多数患者在一段时期的慢性期后，进入加速期或急变期，最终因造血衰竭或感染等并发症而死亡。融合基因bcr/abl是慢性粒细胞白血病的遗传分子生物学特征，其产物Bcr/Abl激酶是发病的关键因素。因Bcr/Abl激酶具有强烈的酪氨酸激酶活性，目前酪氨酸激酶抑制剂（tyrosine kinase inhibitors, TKIs）如伊马替尼等已作为白血病一线治疗药物应用于临床。在治疗中发现，伊马替尼对慢性粒细胞白血病慢性期有较好的治疗效果，但仍有部分患者发生耐药。研究表明，Bcr/Abl激酶可以激活细胞内Ras/MAPK、Jun/STAT5、PI3K/Akt/mTOR等多种信号通路，进而影响细胞周期分布以及细胞凋亡等，导致CML发病。其中，mTOR信号通路在CML对TKIs耐药机制中发挥重要作用，抑制mTOR信号通路的异常激活可能增强Bcr/Abl激酶阳性细胞对TKIs的敏感性，在慢性粒细胞白血病治疗中发挥作用。哺乳动物雷帕霉素靶点（mammalian target of rapamycin, mTOR）是一种丝氨酸/苏氨酸激酶，在调节蛋白质合成、细胞周期分布、细胞增殖及凋亡等多个方面发挥重要作用。4E-BP1和p70S6K是mTOR下游两个重要的效应分子，是mTOR信号通路功能的主要执行者。mTOR激活可以导致4E-BP1和p70S6K磷酸化水平升高，在转录及翻译水平调节多种蛋白质的合成。研究发现，mTOR信号通路在胃癌、肾癌、小细胞肺癌、宫颈癌等多种人类肿瘤细胞中表达异常，与肿瘤的发生密切相关。此外，mTOR通路的激活可能参与了急性髓系白血病、淋巴瘤等血液系统恶性疾病的发生发展，且是Bcr/Abl阳性细胞恶性转化及早期生存过程中的关键环节。雷帕霉素（rapamycin，RAPA）是一种小分子mTOR活性抑制剂，属于大环内酯类化合物，最早作为免疫抑制剂应用于临床。有研究表明，雷帕霉素对体外培养的多种肿瘤细胞有明显的抑制作用。目前，雷帕霉素在白血病治疗中的作用机制以及mTOR能否成为慢性粒细胞白血病治疗中的靶点等问题尚不清楚，已引起学者们的广泛关注。因此，为了深入探讨mTOR信号通路在慢性粒细胞白血病发生中的作用以及mTOR抑制剂雷帕霉素对白血病的治疗作用，本实验从以下几个方面进行了研究：1.慢性粒细胞白血病患者骨髓细胞中mTOR通路的表达情况；2. mTOR抑制剂雷帕霉素对慢性粒细胞白血病细胞株K562细胞周期分布以及细胞凋亡的影响；3.雷帕霉素对K562细胞mTOR通路相关分子表达的影响；4.雷帕霉素联合塞来昔布对慢性粒细胞白血病K562细胞的抑制作用。本文拟通过以上研究，深入分析mTOR信号通路在CML发生发展中的作用，为mTOR成为CML治疗新靶点提供理论依据。本研究论文分为四个部分：第一部分：mTOR、4E-BP1和p70S6K在慢性粒细胞白血病发病中的作用目的：通过检测mTOR及其效应分子4E-BP1、p70S6K在CML患者骨髓细胞中的表达，探讨mTOR信号通路在CML发病中的作用。方法：选取2009-2010年河北医科大学第二医院及河北省人民医院初治慢性期CML患者34例。运用Western Blot方法检测CML患者骨髓细胞中mTOR及其下游分子4E-BP1、p70S6K的蛋白表达及磷酸化情况。结果：1.1CML患者骨髓细胞mTOR及p-mTOR蛋白表达情况Western blot检测结果表明，mTOR在CML患者骨髓细胞中的阳性表达率为82.4%（28/34），对照组骨髓中的阳性表达率为60.0%（6/10），二者比较无明显差异（P>0.05）。进一步检测mTOR磷酸化水平，发现CML患者骨髓细胞中p-mTOR阳性表达率明显高于对照组（70.6%vs30.0%，P<0.05）。mTOR蛋白表达及其磷酸化水平与患者年龄、性别均无明显相关（P>0.05）。1.24E-BP1及p-4E-BP1在CML患者骨髓细胞中的表达检测结果表明，CML患者和对照组骨髓细胞中4E-BP1蛋白阳性表达率分别为88.2%（30/34）、80.0%(8/10)，二者比较无统计学意义（P>0.05）。然而，CML患者骨髓细胞中4E-BP1磷酸化水平却明显高于对照组（76.5%vs40.0%，P<0.05）。进一步分析4E-BP1蛋白表达及其磷酸化水平与患者临床病理资料的关系，结果表明4E-BP1、p-4E-BP1的表达与患者性别、年龄无明显相关（P>0.05）。1.3CML患者骨髓细胞中p70S6K及其磷酸化水平的检测结果Western blot检测结果表明，p70S6K在CML患者骨髓细胞中的阳性表达率为85.3%（29/34），在正常人群中的阳性表达率为70.0%（7/10），二者比较无明显差异（P>0.05）。进一步检测p70S6K磷酸化水平，发现CML患者骨髓细胞中p-p70S6K阳性表达率明显高于对照组（73.5%vs20.0%，P<0.05）。CML患者中，p70S6K及p-p70S6K表达与患者年龄、性别均无明显相关（P>0.05）。结论：慢性粒细胞白血病骨髓细胞中mTOR、4E-BP1和p70S6K的磷酸化水平明显增高，mTOR途径被激活，提示mTOR途径在慢性粒细胞白血病的发病中发挥重要作用。第二部分：mTOR抑制剂雷帕霉素对慢性粒细胞白血病K562细胞周期分布和凋亡的影响目的：通过检测mTOR抑制剂雷帕霉素对K562细胞周期分布、细胞增殖及细胞凋亡的影响，探讨雷帕霉素对K562细胞株的抑制作用。方法：不同浓度雷帕霉素处理K562细胞株后，采用MTT法检测细胞增殖情况，运用流式细胞分析技术、RT-PCR以及Western Blot检测细胞周期分布的变化及细胞凋亡情况。结果：2.1雷帕霉素对K562细胞存活率的影响MTT检测结果发现，雷帕霉素对K562细胞生长有明显抑制作用。经雷帕霉素处理24h后，20、40、80、100、200、500、1000nmol/L处理组K562细胞存活率均明显低于溶剂对照组（P<0.05）。在20～1000nmol/L浓度范围内，随着雷帕霉素浓度的升高K562细胞存活率均明显降低。而1、5、10nmol/L浓度雷帕霉素处理组对K562细胞存活率无明显影响（P>0.05）。2.2雷帕霉素对K562细胞周期分布的影响FCM结果表明，雷帕霉素处理24h、48h后，处理组G0/G1期细胞比对照组显著增多，而S期细胞比例则明显降低（P<0.05）。各处理组G2/M期细胞所占比例无明显变化。提示雷帕霉素可显著影响K562细胞周期分布，诱导细胞G0/G1期阻滞。进一步分析雷帕霉素作用时间对细胞周期分布的影响，发现20nmol/L、40nmol/L、80nmol/L及160nmol/L浓度雷帕霉素作用48h后G0/G1期细胞比例明显高于作用24h（P<0.05），10nmol/L雷帕霉素作用时间对G0/G1期细胞比例无明显影响（P>0.05）。10nmol/L~80nmol/L浓度雷帕霉素作用48h比24h后S期细胞明显减少（P<0.05），160nmol/L浓度组作用24h、48h后S期细胞比例相比无显著差异（P>0.05）。2.3雷帕霉素对细胞周期关键因子CyclinD1、p21以及CyclinB1的影响采用RT-PCR方法检测雷帕霉素处理对K562细胞细胞周期关键分子表达的影响，以GAPDH mRNA表达作为内参照。结果显示，20nmol/L、40nmol/L及80nmol/L雷帕霉素处理组CyclinD1mRNA表达明显低于对照组（P<0.05）。给予不同浓度雷帕霉素作用24h后，40nmol/L和80nmol/L雷帕霉素处理组p21mRNA表达量分别为0.642±0.069和1.094±0.161，明显高于对照组（0.206±0.033，P<0.05），而20nmol/L雷帕霉素处理组p21mRNA表达与对照组相比无明显差异（P>0.05）。雷帕霉素各浓度处理组CyclinB1mRNA表达与对照组相比均无统计学意义（P>0.05）。2.4雷帕霉素对K562细胞凋亡率的影响采用Annexin V-PI双染凋亡试剂盒检测雷帕霉素对K562细胞凋亡率的影响。凋亡细胞分为早期凋亡细胞和晚期凋亡细胞两部分。结果显示，雷帕霉素各浓度处理组K562细胞凋亡率明显高于对照组（P<0.05）。进一步分析早期凋亡细胞和晚期凋亡细胞比率，发现雷帕霉素20nmol/L、40nmol/L和80nmol/L浓度作用24h后，早期凋亡细胞比率分别为9.53±1.10%、11.47±0.90%和15.90±1.67%，均明显高于对照组（P<0.05）。而40nmol/L和80nmol/L浓度处理组晚期凋亡细胞比率明显高于对照组（P<0.05），20nmol/L浓度处理组与对照组晚期凋亡细胞比率无显著差异（P>0.05）。结果提示，雷帕霉素可诱导K562细胞发生凋亡，且主要影响细胞早期凋亡。2.5雷帕霉素对凋亡相关蛋白Caspase-3、Bcl-2的影响Caspase是哺乳动物细胞凋亡中的关键蛋白酶，雷帕霉素作用24h后对照组及各浓度雷帕霉素处理组均在32kD位置（酶原）出现荧光阳性条带，而雷帕霉素20nmol/L、40nmol/L及80nmol/L处理组在17kD的位置出现Caspase-3的活性片段。结果表明雷帕霉素可以激活K562细胞中的Caspase-3，其诱导的细胞凋亡可能与Caspase-3激活有关。采用Western Blot检测Bcl-2蛋白的表达，发现雷帕霉素20nmol/L、40nmol/L、80nmol/L处理组Bcl-2蛋白相对表达量分别为0.977±0.150、0.733±0.123、0.603±0.095，其中40nmol/L、80nmol/L浓度处理组Bcl-2蛋白表达量显著低于对照组（1.080±0.137, P<0.05）。结论：mTOR靶向抑制剂雷帕霉素可影响细胞周期、凋亡关键分子的表达，诱导细胞G0/G1期阻滞以及凋亡。第三部分：雷帕霉素对K562细胞mTOR通路相关分子表达的影响目的：通过检测雷帕霉素对K562细胞中mTOR及其下游分子mRNA、蛋白表达的影响，探讨雷帕霉素抑制K562细胞生长及诱导凋亡的可能机制。方法：采用Western Blot、RT-PCR方法检测雷帕霉素对K562细胞内mTOR、4E-BP1及p70S6K mRNA、蛋白表达及其磷酸化水平。结果：3.1雷帕霉素对K562细胞mTOR及p-mTOR表达的影响mTOR蛋白分子量为289kD，经SDS-PAGE电泳、转膜后，K562细胞对照组、雷帕霉素处理组均在289kD位置出现荧光条带。以GAPDH（38kD）作为参照，经图像分析系统对荧光条带进行定量分析，结果显示，雷帕霉素对mTOR蛋白表达无明显影响（P>0.05），但可以显著降低mTOR蛋白磷酸化水平（P<0.05）。RT-PCR结果显示，各浓度雷帕霉素处理组细胞内mTOR mRNA表达量与对照组相比无明显差异（P>0.05）。3.24E-BP1及p-4E-BP1在K562细胞中表达采用Western Blot技术观察雷帕霉素对K562细胞中4E-BP1蛋白去磷酸化和磷酸化水平的影响。结果显示，雷帕霉素处理组4E-BP1和p-4E-BP1蛋白表达均明显低于对照组（P<0.05）。运用RT-PCR检测4E-BP1在mRNA水平上的表达。结果表明，各浓度雷帕霉素处理组细胞内4E-BP1mRNA表达量比对照组均明显降低（P<0.05）。结果提示，雷帕霉素可降低mTOR下游4E-BP1mRNA以及蛋白的表达，同时可影响其磷酸化。3.3雷帕霉素对K562细胞p70S6K及p-p70S6K表达的影响Western Blot结果显示，雷帕霉素处理组p70S6K蛋白表达及磷酸化水平均明显低于对照组（P<0.05）。RT-PCR结果表明，20nmol/L、40nmol/L、80nmol/L雷帕霉素处理组细胞内p70S6K mRNA表达量分别为0.633±0.067、0.513±0.057、0.403±0.051，比对照组明显降低（0.801±0.077，P<0.05）。结果提示，雷帕霉素可抑制p70S6K的表达及磷酸化水平。结论：雷帕霉素可通过抑制mTOR及其下游底物4E-BP1和p70S6K的活性，诱导细胞G0/G1期阻滞以及细胞凋亡，进而抑制肿瘤细胞的生长。第四部分：雷帕霉素联合塞来昔布对慢性粒细胞白血病的抑制及其机制目的：通过观察雷帕霉素联合塞来昔布对K562细胞增殖、凋亡以及mTOR信号通路的影响，探讨雷帕霉素与塞来昔布对K562细胞的协同抑制作用及可能机制。方法：选用低浓度雷帕霉素（20nmol/L）联合低浓度塞来昔布（10μmol/L）处理K562细胞株，采用流式细胞学技术、RT-PCR及WesternBlot方法检测细胞凋亡、mTOR通路相关分子的表达。结果：4.1塞来昔布对K562细胞存活率的影响采用MTT方法检测不同浓度塞来昔布对K562细胞存活率的影响。结果显示，塞来昔布对K562细胞生长有明显抑制作用。塞来昔布处理24h及48h后，10、20、40、80、160μmol/L处理组K562细胞存活率均明显低于溶剂对照组（P<0.05）。在10～160μmol/L浓度范围内，随着塞来昔布浓度的升高K562细胞存活率明显降低。5μmol/L浓度塞来昔布处理对K562细胞存活率无明显影响（P>0.05）。4.2雷帕霉素联合塞来昔布对K562存活率的影响观察雷帕霉素联合塞来昔布对K562细胞株细胞存活率的影响，结果表明，联合用药组细胞存活率比对照组、雷帕霉素及塞来昔布单药组明显降低（P<0.05）。结果提示联合用药可明显抑制K562细胞的增殖。4.3雷帕霉素联合塞来昔布对K562细胞凋亡率的影响采用Annexin V-PI双染凋亡试剂盒检测雷帕霉素联合塞来昔布对K562细胞凋亡率的影响。结果显示，20nmol/L雷帕霉素处理组、10μmol/L塞来昔布处理组及两药联合处理组细胞凋亡率均明显高于对照组（P<0.05）；而且联合用药组细胞凋亡率为15.87±2.21%，比各单药处理组明显升高（P<0.05）。进一步比较不同处理措施对早期凋亡和晚期凋亡的影响，结果发现雷帕霉素处理组细胞早期凋亡率比对照组明显增多（6.03±0.70%vs4.23±0.35%，P<0.05），而塞来昔布处理组细胞晚期凋亡率明显高于对照组（4.17±0.61%vs2.27±0.25%，P<0.05）。联合用药组细胞早期凋亡率与晚期凋亡率均比对照组明显增高（P<0.05）。结果提示，雷帕霉素及塞来昔布单药均可诱导K562细胞发生凋亡，二者联合用药可增强单药诱导细胞凋亡的作用，在诱导细胞凋亡中可能具有协同作用。4.4雷帕霉素联合塞来昔布对mTOR信号通路相关分子的影响Western Blot检测结果表明，雷帕霉素及塞来昔布联合处理组mTOR蛋白表达及磷酸化水平均明显低于对照组（P<0.05）。雷帕霉素及塞来昔布单药对K562细胞中mTOR蛋白表达无明显影响，但可显著降低mTOR磷酸化水平（P<0.05）。另外，联合用药组mTOR及p-mTOR均明显低于单药处理组（P<0.05）。RT-PCR结果显示，雷帕霉素、塞来昔布单药作用24h后，K562细胞mTOR mRNA表达与对照组无明显差异（P>0.05），而联合用药组mTOR mRNA相对表达量为0.280±0.046，明显低于对照组（0.707±0.059，P<0.05），与单药作用组相比也明显降低（P<0.05）。结果提示，联合用药可降低K562细胞mTOR在mRNA、蛋白水平的表达及其磷酸化水平。运用Western Blot方法检测4E-BP1和p-4E-BP1的表达。结果表明，雷帕霉素、塞来昔布单药及联合用药组4E-BP1蛋白表达及磷酸化水平均明显低于对照组（P<0.05），而且联合用药组4E-BP1和p-4E-BP1比单药处理组降低的更为明显（P<0.05）。RT-PCR检测结果发现，雷帕霉素、塞来昔布单药及联合用药组4E-BP1mRNA相对表达量分别为1.437±0.150、1.373±0.127、0.723±0.084，均比对照组（1.697±0.160）明显减少（P<0.05）。进一步比较单药处理组及联合用药组4E-BP1mRNA表达的变化，结果表明联合用药后K562细胞内4E-BP1mRNA表达明显减少（P<0.05）。雷帕霉素、塞来昔布单药及联合用药作用于K562细胞24h后，药物处理组p70S6K蛋白表达及磷酸化水平均明显低于对照组（P<0.05），而且联合用药其表达水平明显低于单药处理组。RT-PCR结果与蛋白检测结果一致，单药及联合用药组K562细胞内p70S6K mRNA相对表达量比对照组明显减少（P<0.05），联合用药组p70S6K mRNA表达比单药处理明显降低（P<0.05）。结论：雷帕霉素联合塞来昔布可明显抑制K562细胞增殖、诱导细胞凋亡，而且联合用药可增强单药对K562细胞的生长抑制作用。结论：1慢性粒细胞白血病骨髓细胞中mTOR、4E-BP1和p70S6K的磷酸化水平明显增高，mTOR途径被激活，提示mTOR途径在慢性粒细胞白血病的发病中发挥重要作用。2mTOR靶向抑制剂雷帕霉素可影响细胞周期、凋亡关键分子的表达，诱导细胞G0/G1期阻滞以及凋亡。3雷帕霉素可通过抑制mTOR及其下游底物4E-BP1和p70S6K的活性，诱导细胞G0/G1期阻滞以及细胞凋亡，进而抑制肿瘤细胞的生长。4雷帕霉素联合塞来昔布可明显抑制K562细胞增殖、诱导细胞凋亡，而且联合用药可增强单药对K562细胞的生长抑制作用。更多还原

【Abstract】 Chronic myelogenous leukemia (CML) is a type of common chronicleukemia in China, which can occur in various age groups. Traditional drugssuch as hydroxycarbamide and IFN can relieve some of the patients’symptoms, but they can not cure the disease or block its process. Most of thepatients entered accelerated or blastic phases after a period of chronic phase,and died in hematopoietic failure or complications. bcr/abl fusion gene is amain molecular biological characteristic of CML. Its encoding productBcr/Abl kinase is a critical factor in pathogenesis of CML. Inhibitors ofBcr/Abl kinase (such as imatinib) have achieved satisfactory effects in thetreatment of patients with CP CML and become the first line therapeutic drug.However, there are always some patients that resistance to Bcr/Abl kinaseinhibitors. Studies have shown that Bcr/Abl kinase had intensive tyrosinekinase activity. It could activate many important signaling pathways includingRas/MAPK, Jun/STAT5and PI3K/Akt/mTOR, in sequence affect cell cycledistribution and cell apoptosis. Some studies have proved that mTORcontributed to tyrosine kinase inhibitors (TKIs) resistance of CML, andinhibition of mTOR signaling could enhance the sensitivity ofBCR/ABL-positive cells to TKIs.Mammalian target of rapamycin (mTOR) is a kind of serine/threoninekinase and plays a critical role in many physiological functions such as proteintranslation, cell cycle distribution, cell proliferation and apoptosis.4E-BP1and p70S6K are two major molecules in downstream of mTOR. Theyundertake most of the mTOR signaling’s functions. When mTOR signaling isactivated, the phosphorylations of4E-BP1and p70S6K are increased. Asresults, the synthesis of various proteins is dysregulated both at transcriptionand translation level. Abnormal activation of mTOR signaling could be observed in many kinds of tumor cells, such as gastric cancer, small cell lungcancer, cervical cancer, ovarian cancer and hepatocellular carcinoma.Activation of mTOR signaling might also be involved in the tumorigenesisand progress of some hematological malignants, for example acute myeloidleukemia and lymphoma. Furthermore, PI3K/Akt/mTOR signaling wasrequired in the malignant transformation of Bcr/Abl-positive cells and initialcellulars survival. Studies showed that mTOR inhibitors (rapamycin andRD001) significantly inhibited the growth of various tumor cells in vitro. Theprecise mechanism of mTOR in pathogenesis of CML needs to be clarified,and mTOR may become a therapeutic target of CML in the furtuer.In the present study, to study the role of mTOR signaling in pathogenesisof CML and the mechanism of potential therapeutic effects of rapamycin onCML, we will detect four parts in our study:1. Expression of mTOR signalingin CML bone marrow cells;2. Effects of rapamycin on cell cycle distributionand apoptosis in K562cells;3. Mechanism of cell cycle arrest and apoptosisinduced by rapamycin in K562cells;4. Effcets of rapamycin combined withcelecoxib treatment on K562cells. The aim of this study is to explore thefunction of mTOR signaling in pathogenesis and progression of CML, and toprovide theoretical evidence for targeting mTOR signaling in CML therapy.This study includes four parts:Part ⅠExpression of mTOR signaling in CML bone marrow cellsObjective: To explore the possible role of mTOR signaling inpathogenesis of CML by detecting the expression of mTOR and itsdownstream effectors,4E-BP1and p70S6K, in CML bone marrow cells.Methods: Thirty-four patients with chronic phase CML included in thisstudy were collected from the Second Hospital, Hebei Medical University andHebei General Hospital between June2009and December2010. Theexpression of mTOR,4E-BP1and p70S6K and their phosphorylation in CMLbone marrow cells was detected by Western Blot.Results：1.1Expression of mTOR and p-mTOR in CML bone marrow cells Western Blot analysis showed that the positive expression rate of mTORin bone marrow cells with CML showed no statistically significant differenceas compared with that in normal bone marrow cells (P>0.05). However, theexpression of p-mTOR in bone marrow cells with CML was obviouslyincreased, compared with that in normal bone marrow cells (70.6%vs30.0%，P<0.05). There was no relationship between the expression of mTOR and itsphosphorylation and patient’s age and gender (P>0.05).1.2Expression of4E-BP1and p-4E-BP1in CML bone marrow cellsThe positive expression rate of4E-BP1in CML bone marrow cellsshowed no statistically significant difference as compared with that in normalbone marrow cells (P>0.05). The expression of p-4E-BP1in CML bonemarrow cells was significant increased as compared with that in normal bonemarrow cells (76.5%vs40.0%，P<0.05). The expression and phosphorylationof4E-BP1showed no relationship with patient’s age and gender (P>0.05).1.3Expression of p70S6K and p-p70S6K in CML bone marrow cellsWestern Blot analysis showed that the positive expression rate of p70S6Kin CML bone marrow cells showed no statistically significant difference ascompared with that in normal bone marrow cells (P>0.05). The higherexpression of p-p70S6K was detected in in bone marrow cells with CML,compared with that in normal bone marrow cells (73.5%vs20.0%，P<0.05).However, there was no relationship between expression of p70S6K, p-p70S6Kand patient’s age and gender (P>0.05).Conclusion：The phosphorylation level of mTOR,4E-BP1and p70S6Kin CML bone marrow cells was significantly increased, indicating that mTORsignaling was activated and played an important role in pathogenesis of CML.Part Ⅱ Effects of rapamycin on cell cycle, proliferation and apoptosis inK562cellsObjective: To explore Effects of rapamycin on cell cycle, proliferationand apoptosis in K562cells.Methods: The proliferation, cell cycle distribution and apoptosis of K562cell was detected by Flow cytometry (FCM) assay, Western Blot and RT-PCR after treated with different concentrations of rapamycin.Results:2.1Effects of rapamycin on the survival rates in K562cellsMTT result showed obvious inhibition of rapamycin on the proliferationof K562cells. The cell survival rates in groups treated with rapamycindisplayed a significant decrease in a dose-dependent manner ranging from20to1000nmol/L (P<0.05). The cell survival rates in1nmol/L,5nmol/L and10nmol/L rapamycin treatment groups showed no statistically significantdifference as compared with that in control group (P>0.05).2.2Rapamycin arrests K562cells at G0/G1phaseExponentially growing K562cells were treated with rapamycin for24hand48h, and detected by flow cytometry. After treated with rapamycin for24h the proportion of cells in G0/G1phase in treated groups was significantlyincreased, compared with that in solvent control group (29.56±2.89, P<0.05).With regard to S phase cells, the decreased population was accompanied withthe increase of G0/G1phase cells. No difference was found in G2/M cellpopulations after treatment with rapamycin, as compared with the controlgroup. Furthermore, the significant increase of G0/G1phase cells and decreaseof S phase cells were found in groups with rapamycin treatment for48h, aswell as the treated groups for24h. These results indicated that rapamycincould arrest K562cells at G0/G1phase.The FCM analysis also showed that the proportion of cells at G0/G1phasein groups treated with20nmol/L,40nmol/L,80nmol/L, and160nmol/Lrapamycin for48h was significantly increased, compared with that for24h(P<0.05). The proportion of cells at G0/G1phase in10nmol/L rapamycintreatment group showed no obvious change no matter how long the treatmentperiod was (P>0.05). However, the proportion of cells at S phase in treatedgroups with10nmol/L~80nmol/L rapamycin for48h was obviouslydecreased, compared with that treated for24h (P<0.05). As regard to theproportion of cells at S phase in160nmol/L rapamycin treatment groups,there was no difference between treatment for24h and48h (P>0.05). 2.3Effects of rapamycin on CyclinD1, p21, and CyclinB1in K562cellsRT-PCR analysis showed the lower expression of CyclinD1mRNA ingroups with20nmol/L,40nmol/L and80nmol/L rapamycin treatment than incontrol group (P<0.05). On the contrary, higher expression of p21at mRNAlevel was detected in cells with40nmol/L and80nmol/L rapamycin treatment(P<0.05). Expression of CyclinB1show no variety in K562cells afterrapamycin incubation.2.4Rapamycin induced apoptosis in K562cellsTo determine if rapamycin resulted in increased apoptosis of K562cells,we studied apoptosis after treatment of rapamycin with increasingconcentrations for24h. The percentage of apoptotic cells in treated groupswith20,40and80nmol/L was significantly higher than in control group(P<0.05). The result indicated that treatment with20nmol/L or higherconcentrations of rapamycin increased apoptotic cells, including early and lateapoptotic cells, in a concentration dependent manner.Furthermore, the percentage of early apoptotic cells of K562cells ingroups treated with20nmol/L,40nmol/L and80nmol/L rapamycin was9.53±1.10%,11.47±0.90%and15.90±1.67%respectively, significantly higherthan in solvent control group (P<0.05). Meanwhile, the higher percentage oflate apoptotic cells of K562cells was detected in groups with40nmol/L and80nmol/L rapamycin treatment (P<0.05), and no significant difference wasfound in20nmol/L rapamycin treatment group (P>0.05).2.5Effects of rapamycin on Caspase-3and Bcl-2in K562cellsTo study the underlying apoptotic mechanisms, expression of apoptosisregulators, caspase-3and bcl-2, were assessed after treatment with20,40and80nmol/L rapamycin. Caspase-3(32kD) was detected in all groups includingrapamycin treatment groups and solvent control group. However, thecaspase-3activity (17kD) was only detected in K562cells in treated groups.It indicated that rapamycin could activate Caspase-3in K562cells, and mightinduce apoptosis through Caspasse-3activation.The relative expression quantity of Bcl-2protein in treated groups with 40nmol/L and80nmol/L rapamycin was0.733±0.123and0.603±0.095,respectively, significantly lower compared with that in solvent control group(1.080±0.137, P<0.05).Conclusion：Rapamycin, as the inhibitor of mTOR, could arrest K562cells at G0/G1phase by regulationg key factors of the cell cycle and induceapoptosis by decreasing Bcl-2expression and activating Caspase-3.Part Ⅲ Effects of rapamycin on expression of mTOR,4E-BP1and p0S6Kin K562cellsObjective: To explore the possible mechanism of cell growth inhibitionand apoptosis induced by rapamycin in K562cells, the expression of mTORand its downstream molecules was studied.Methods: The expression of mTOR,4E-BP1and p70S6K at mRNA andprotein level was detected by RT-PCR and Western Blot, respectively.Results:3.1Effects of rapamycin on mTOR expression in K562cellsThe expression of mTOR at protein level in rapamycin treatment groupsshowed no statistically significant difference as compared with that in controlgroup (P>0.05). However, the lower expression of p-mTOR was found inrapamycin treatment groups (P<0.05).The expression of mTOR at mRNA level detected by RT-PCR showedthe coincident result with at protein level investigated by Western Blot.3.2Effects of rapamycin on4E-BP1expression in K562cellsWestern Blot showed that the expression of4E-BP1and p-4E-BP1ingroups with rapamycin treatment was significantly decreased, compared withthat in control group (P<0.05). Moreover, RT-PCR also displayed thesignificantly lower expression of4E-BP1mRNA in rapamycin treatmentgroups (P<0.05). It indicated that rapamycin could decrease the expression of4E-BP1in K562cells.3.3Expression of p70S6K and p-p70S6K in K562cells after rapamycintreatmentThe lower expression of p70S6K and p-p70S6K was investigated in rapamycin treatment groups (P<0.05). Accordingly, the mRNA expression ofp70S6K in groups with20nmol/L,40nmol/L and80nmol/L rapamycintreatment was significantly decreased detected by RT-PCR (P<0.05). Theresults indicated that rapamycin could play its role by inhibiting p70S6K.Conclusion：Rapamycin might have an effect on cell cycle and apoptosisby inhibiting mTOR and its downstream substrates (4E-BP1and p70S6K),and then inhibit the growth of tumor cells.PART ⅣEffects of rapamycin combined with celecoxib on mTORsignaling in K562cellsObjective: To explore effects of rapamycin combined with celecoxib onmTOR signaling by detecting proliferation and apoptosis of K562cells.Methods: The treated concentration was determined by MTT, and thenK562cells were incubated with rapamycin (20nmol/L), celecoxib (10μmol/L)and rapamycin combined with celecoxib. The apoptosis of K562cells wasmeasured by flow cytometry, and the expression of mTOR,4E-BP1andp70S6K was detected by RT-PCR and Western Blot as before.Results:4.1Effects of celecoxib on the survival rates in K562cellsMTT assay showed that celecoxib had a potent inhibitory effect on theproliferation of K562cells (P<0.05). The cell survival rates in celecoxibtreated groups displayed a significant decrease in a dose-dependent mannerranging from10to160μmol/L (P<0.05). The cell survival rate in5μmol/Lcelecoxib treatment group showed no statistically significant difference,compared with that in control group (P>0.05).4.2Effects of rapamycin combined with celecoxib on the survival rates inK562cellsMTT study showed the lower survival rate of K562cells was investigatedin the group with two drugs, compared with rapamycin, celecoxib or no drug(P<0.05), indicating that rapamycin combined with celecoxib could inhibitK562cells growth.4.3Rapamycin combined with celecoxib induced K562cells apoptosis Cells Apoptosis induced by rapamycin combined with celecoxib wasdetermined by annexin V/PI apoptosis detection kit. The result showed that theapoptosis rate in groups with rapamycin treatment, celecoxib treatment andrapamycin combined with celecoxib treatment were significantly higher thanthat in solvent control group (P<0.05). And, more apoptosis cells weredetected in the group treated with two drugs than treated with one drug(P<0.05).Furthermore, the early apoptosis of K562cells incubated with rapamycinwas obviously increased as compared with solvent control (P<0.05). On thecontrary, the late apoptosis cells was more in the group with celecoxibtreatment than in solvent control group (P<0.05). The rate of the earlyapoptosis and the late apoptosis in rapamycin combined with celecoxibtreatment group was10.10±1.37%and5.77±0.83%, respectively, significantlyhigher as compared with that in solvent control group (P<0.05). These datasshowed that rapamycin or celecoxib could induce apoptosis in K562cells, andthe rapamycin combined with celecoxib was able to enhance the role of onedrug. It indicated that Rapamycin and celecoxib might have synergistic effectsin inducing apoptosis of K562cells.4.4Effects of rapamycin combined with celecoxib on major molecules ofmTOR signaling in K562cellsWestern Blot showed the expression of mTOR and its phosphorylation inthe group treated with rapamycin and celecoxib was significantly decreased ascompared with that in control group (P<0.05). With regard to expression ofmTOR, there was no significant difference between the K562cells incubatedwith one drug and solvent (P>0.05), however, the lower phosphorylation levelof mTOR was found in two groups treated with one drug (P<0.05). RT-PCRshowed lower mRNA expression of mTOR in K562cells treated by bothrapamycin and celecoxib, as well as the data detected by Western blot. Theresults indicated that rapamycin combined with celecoxib could affect theexpression of mTOR and its phosphorylation level in K562cells.Expression of4E-BP1and its phosphorylation in K562cells incubated with one or both drugs was significantly decreased as compared with that incontrol group (P<0.05). RT-PCR showed the lower expression of4E-BP1mRNA in K562cells, accordance with protein expression.With regard to p70S6K, the lower expression of both at mRNA andprotein level was investigated in the groups treated with rapamycin, celecoxiband rapamycin combined with celecoxib (P<0.05). Interestedly, the expressionof p70S6K in K562cells incubated with both drugs was significantlydecreased as compared with only one drug (P<0.05). Moreover, thephosphorylation level of p70S6K was obviously decreased in the group withrapamycin combined with celecoxib treatment, as well as the expression ofp70S6K studied by Western blot.Conclusion： Rapamycin combined with celecoxib could inhibit cellproliferation and induce apoptosis，and increased the inhibitory effects inK562cells than single agent.Conclusions:1The phosphorylation level of mTOR,4E-BP1and p70S6K in CML bonemarrow cells was significantly increased, indicating that mTOR signalingwas activated and played an important role in pathogenesis of CML.2Rapamycin, as the inhibitor of mTOR, could arrest K562cells at G0/G1phase by regulationg key factors of the cell cycle and induce apoptosis bydecreasing Bcl-2expression and activating Caspase-3.3Rapamycin might have an effect on cell cycle and apoptosis by inhibitingmTOR and its downstream substrates (4E-BP1and p70S6K), and theninhibit the growth of tumor cells.4Rapamycin combined with celecoxib could inhibit cell proliferation andinduce apoptosis，and increased the inhibitory effects in K562cells thansingle agent.更多还原