【作者】 何畏；

【导师】 黄峥兰；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2023， 硕士

【摘要】 目的:慢性髓细胞白血病(chronic myleoid leukemia,CML)是一种起源于造血干细胞的骨髓增殖性肿瘤,主要累及粒系。CML疾病的特点是在受累的细胞中,可检测到特征性的BCR-ABL融合基因。随着伊马替尼(imatinib,IM)的问世,慢性髓细胞白血病的治疗取得了显著进展,但在进一步治疗的过程中部分患者出现了对伊马替尼的耐药或不耐受。在此之前,有研究者通过开发具有更高效的靶向BCR-ABL的酪氨酸激酶药物,BCR-ABL依赖性的伊马替尼耐药问题已经得到了部分解决,但BCR-ABL非依赖性耐药逐渐成为伊马替尼耐药的主要途径,并且尚未找到良好的解决方案。因此我们将研究重点聚焦在BCR-ABL非依赖性途径诱导的耐药相关靶点及其靶向药物。哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)是细胞生长和增殖的重要调节分子,其在肿瘤细胞的增殖中发挥着重要的作用。我们发现,mTOR信号通路在伊马替尼耐药的CML细胞系K562/G01以及我们收集的临床患者标本的外周血单个核细胞中表达明显上调。同时,AZD2014是一种新型的mTOR抑制剂,具有良好的水溶性和生物安全性,其有效性在包含白血病在内的多种肿瘤疾病中得到验证。故本课题拟研究AZD2014对伊马替尼耐药的CML细胞株K562/G01的生物学效应,并探究AZD2014杀伤K562/G01细胞、克服IM耐药的具体机制,为AZD2014应用于临床上BCR-ABL非依赖性耐药患者的治疗提供理论基础。方法:1.利用生物信息学分析mTOR信号通路在伊马替尼敏感的CML细胞系K562、伊马替尼耐药的CML细胞系K562/G01中的表达情况;Western blot和Q-PCR对生信结果进行验证;收集临床标本,分离外周血单个核细胞后通过Q-PCR检测mTOR的表达;CCK-8和克隆形成实验验证AZD2014对CML细胞增殖的作用。2.流式细胞术检测AZD2014对CML细胞周期及凋亡的影响;Western blot检测细胞周期和凋亡相关蛋白的表达;DAPI染色观察凋亡细胞的细胞核形态学的改变;AZD2014联合IM作用于CML细胞,CCK-8检测细胞活性,使用Compu Syn软件分析两药对CML细胞是否具有协同效应。Western blot检测IM处理后mTOR信号通路以及AZD2014作用后BCR-ABL下游相关分子的表达,探究mTOR与BCR-ABL的关系。3.Western blot检测AZD2014处理后细胞自噬相关蛋白的表达;细胞核细胞浆蛋白分提实验检测药物处理后TFEB在细胞中亚定位的改变,并利用免疫荧光实验进行验证;使用自噬抑制剂3-MA共处理CML耐药细胞观察其回复作用。4.最后通过尾静脉注射K562/G01细胞构建CML耐药小鼠模型,在体内验证AZD2014对CML耐药小鼠模型的抗白血病效应。结果:1.生信分析以及在临床标本中进行验证后,我们发现相比K562细胞,mTOR信号通路在K562/G01细胞中上调,AZD2014能够抑制CML细胞的增殖,上调p27、下调Cyclin D1的表达进而导致CML细胞周期阻滞在G1期。2.AZD2014没有诱导CML细胞凋亡的能力,但是与IM联用后能够有效促进CML耐药细胞的凋亡,提高CML耐药细胞对于IM的敏感性,Compu Syn软件分析AZD2014和IM在CML细胞上表现出协同作用,且在对IM耐受的CML细胞系K562/G01上的协同作用更强。IM对mTOR信号通路没有明显的抑制作用,并且AZD2014对BCR-ABL相关的信号通路也不存在影响,证实mTOR是以BCR-ABL非依赖性的方式参与IM耐药。3.AZD2014处理后TFEB的定位由细胞质进入细胞核,入核后自噬相关分子的转录增强并提高了细胞的自噬水平。自噬抑制剂3-MA的回复实验证实AZD2014发挥生物学效应与细胞自噬水平的升高有关。4.AZD2014单药在K562/G01小鼠模型中具有有效的抗肿瘤作用,且联用IM后发挥出更强的效应。结论:本研究证明了AZD2014通过增强细胞自噬对慢性髓细胞白血病细胞,尤其是IM耐药的CML细胞发挥抗白血病作用,这为BCR-ABL非依赖性耐药的CML患者的治疗提供了一种新的治疗策略,具有一定的应用前景。更多还原

【Abstract】 Objective: Chronic myleoid leukemia(CML)is a proliferative malignancy neoplasm originated from hematopoietic stem cell,which primarily involves the granulocytic lineage.CML is characterized by the detection of BCR-ABL fusion genes in the affected cells.With the application of imatinib,significant progress has been made in the treatment of chronic myeloid leukemia,but resistance or intolerance to imatinib has emerged during further treatment.Previously,the problem of BCR-ABL dependent imatinib resistance has been partially solved by some investigators through the development of with more efficient tyrosine kinase inhibitors(TKIs),but BCR-ABL independent resistance is gradually becoming the major challenge and there is still no ideal solution.Therefore,we focused on the relevant molecules mediated IM resistance via BCR-ABL independent pathway and their targeted drugs of resistance via BCR-ABL independent pathway.The mammalian target of rapamycin(mTOR)is an important regulatory molecule for cell growth and proliferation,and it executes a particularly important role in tumor cells.We found that the mTOR signaling pathway was up-regulated in imatinib-resistant CML cell line K562/G01 as well as in peripheral blood mononuclear cells from CML patients.Meanwhile,AZD2014 is a novel mTOR inhibitor with good water solubility and bio-safety,and its effectiveness has been verified in a variety of tumor including leukemia.Therefore,we intended to investigate the biological effect of AZD2014 on imatinib resistant CML cell line K562/G01,and explore the potential mechanism how AZD2014 overcoming IM resistance,so as to provide a theoretical and experimental basis for the application of AZD2014 in the clinical treatment of BCR-ABL independent drug-resistant patients.Methods: 1.Bioinformatics was used to analyze the expression of mTOR signaling pathway in imatinib-sensitive CML cell line K562 and imatinib-resistant CML cell line K562/G01.Western blot and Q-PCR were used to validate the results above;Clinical samples were collected and peripheral blood mononuclear cells(PBMC)were separated,then mTOR expression was detected by Q-PCR.CCK-8 and colony formation assay were performed to verify the effect of AZD2014 on CML cell proliferation.2.Flow cytometry was used to detect the effect of AZD2014 on CML cell cycle and apoptosis.Western blot was used to detect the expression of cell cycle and apoptosis-related proteins.DAPI staining was used to observe the changes of nuclear morphology of apoptotic cells.CML cells was treated with AZD2014 and IM,CCK-8 was used to detect the cell viability,and Compu Syn software was used to analyze the combination effect.Western blot was performed to detect the expression of mTOR signaling pathway and BCR-ABL downstream related molecules after IM and AZD2014 treatment accordingly to investigate the relationship between mTOR and BCR-ABL.3.The expression of autophagy-related proteins after AZD2014 treatment was detected by Western blot.The localization of TFEB in cells after AZD2014 treatment was detected by analyzing the portion of TFEB in the nuclear and cytoplasmic protein and verified by immunofluorescence assay.CML-resistant cells were co-treated with the autophagy inhibitor 3-MA to observe its rescue effects.4.Finally,CML-resistant mouse model was constructed by injection of K562/G01 cells through tail vein,and the anti-leukemic effect of AZD2014 was verified in the CML-resistant mouse model.Results: 1.Through bioinformatics analysis and validation in clinical samples,we found that the mTOR signaling pathway was up-regulated in K562/G01 cells compared to K562 cells,and AZD2014 was able to inhibit the proliferation of CML cells by up-regulating p27 and down-regulating Cyclin D1,which in turn led to cycle arrest of CML cells in G1 phase.2.AZD2014 could not induce apoptosis individually in CML cells,but could effectively promote apoptosis in CML-resistant cells when in combination with IM and improve the sensitivity of CML-resistant cells to IM.Our result suggested there was a synergistic effect between AZD2014 and IM on CML cells,and the synergistic effect was stronger on IM-resistant CML cell line K562/G01.IM had no inhibitory effect on mTOR signaling pathway and AZD2014 had no effect on BCR-ABL downstream signaling,indicating that mTOR was involved in IM resistance via BCR-ABL independent pathway.3.The TFEB translocated from cytoplasm to the nucleus after AZD2014 treatment,then the transcription of autophagy-related molecules was enhanced and the cell autophagy increased after TFEB entering the nucleus.The results of rescue experiments induced by autophagy inhibitor 3-MA confirmed that AZD2014 exerted biological effects through the increase of cellular autophagy.4.AZD2014 had effective anti-tumor effect on K562/G01 mouse model and exerted stronger effect with IM.更多还原