葡萄糖神经酰胺合成酶对白血病耐药细胞Bcl-2表达的影响及机制研究

发布时间：2018-04-22 00:25

本文选题：葡萄糖神经酰胺合成酶 + 神经酰胺　；参考：《南京医科大学》2015年硕士论文

【摘要】：多药耐药(multidrug resistance,MDR)的出现已然成为临床治疗白血病的一大难题,对于其耐药机制的研究,目前葡萄糖神经酰胺合成酶(glucosylceramide synthase,GCS)在肿瘤耐药中的作用越来越受到人们的关注。已知GCS是鞘脂代谢中的糖基化酶,其最直接的功能是将尿核苷二磷酸葡萄糖(UDP-glucose)上的糖基转移至神经酰胺(Ceramide,Cer)形成葡萄糖神经酰胺(Glycosylceramide,Glc Cer),为高级鞘糖脂的合成提供合成前体,同时降低了细胞内的Cer水平。作为信号转导的脂质第二信使,Cer广泛参与细胞生长、分化、衰老及死亡等生理过程。Cer水平降低使细胞分化、凋亡等功能抑制,自然而然也成为GCS诱导肿瘤耐药的原因之一。以往关于GCS诱导肿瘤耐药的研究多集中在GCS对细胞膜上药物转运排出泵的影响。多药耐药基因1(multidrug resistance gene 1,MDR1)编码的P170糖蛋白(P-glycoprotein,P-gp)是研究最多的肿瘤耐药分子。当药物进入细胞,P-gp与药物分子结合并在ATP的驱动下将药物转移至细胞外,使细胞内药物始终维持在较低水平。研究发现,不同系统肿瘤中GCS可通过上调P-gp促进抗肿瘤药物的泵出,进而导致细胞耐药的产生。然而亦有研究报道称,即便抗癌药物突破细胞外排这一屏障到达其作用的靶点,由于细胞凋亡通路受抑,这些抗癌药物仍旧无法消灭肿瘤细胞,说明凋亡通路异常也是肿瘤耐药的重要原因之一。Bcl-2是癌细胞特有的一类基因家族,主要在线粒体膜上发挥对细胞凋亡的调控作用。根据Bcl-2家族各分子的结构以及发挥的生物学效应不同可将Bcl-2家族分为:(1)以Bid为代表的只具有BH3结构域、能对刺激产生反应并被激活的BH3蛋白;(2)以Bax为代表的能使线粒体外膜通透性增加进而激活凋亡级联反应的凋亡执行蛋白;(3)以Bcl-2为代表的抑制BH3和凋亡执行蛋白的抑凋亡蛋白。Bcl-2主要通过阻止线粒体细胞色素C的释放从而抑制细胞凋亡。大量数据显示Bcl-2蛋白增加或Bax蛋白减少是多种肿瘤细胞对化疗不敏感的重要原因。上调Bax或下调Bcl-2的基因水平可使细胞对肿瘤药物的敏感性增强。本课题组前期研究发现,与K562敏感细胞株比较,K562/A02白血病多药耐药细胞株中GCS与Bcl-2的表达明显增高,而Bax的表达无明显差异;下调GCS,Bcl-2的表达水平亦明显下降,而Bax的表达不受影响;此外课题组发现K562/A02的耐药性还与ERK信号通路有关,该通路介导了GCS对P-gp转运蛋白的调控。由于ERK信号通路与细胞凋亡密切相关,ERK介导的信号转导可能是多种信号刺激的共同通路。研究发现ERK通路的激活亦是线粒体去极化、细胞色素C释放、caspase3活化以及细胞凋亡的必要前提。因此,本文就GCS上调Bcl-2表达从而诱导白血病耐药的分子机制进行研究,探讨ERK信号通路是否参与GCS对凋亡的抑制过程,以期揭示GCS诱导白血病的耐药新机制,为逆转肿瘤耐药提供新的思路。目的:探讨人白血病多药耐药细胞株K562/A02中GCS对凋亡相关基因Bcl-2表达水平的影响,明确GCS在白血病耐药中的作用机制。方法:利用RNA干扰技术靶向抑制K562/A02细胞中的GCS后,real-time PCR和western blotting检测Bcl-2 m RNA与蛋白、MAPK成员蛋白表达的变化情况;流式细胞术检测细胞凋亡;CCK-8法检测细胞对阿霉素(Adriamycin,ADM)的敏感性以及高效液相色谱—质谱联用分析细胞中的Cer水平。20μM U0126处理K562/A02细胞24 h后,CCK-8法检测细胞对ADM的敏感性;流式细胞术观察U0126和ADM单独或联合使用时细胞凋亡水平的变化;real-time PCR和western blotting分别检测U0126以及Cer对细胞Bcl-2 m RNA与蛋白、ERK信号通路活化的影响。结果:GCS si RNA下调K562/A02细胞中GCS表达后,细胞对ADM的凋亡增加、药物敏感性增强,Bcl-2 m RNA与蛋白水平下降,Cer含量增加;western blotting结果显示p-ERK表达减少,但总ERK、总的以及磷酸化JNK和p38 MAPK的表达无明显变化。MEK特异性抑制剂U0126作用K562/A02后,细胞对ADM凋亡增加、药物敏感性增强,Bcl-2 m RNA与蛋白以及p-ERK水平呈浓度依赖性和时间依赖性下降;外源C6-神经酰胺(C6-Ceramide,C6-Cer)也可显著抑制K562/A02细胞中ERK的磷酸化激活和Bcl-2抑凋亡蛋白的表达。结论:ERK信号通路介导GCS上调抑凋亡蛋白Bcl-2的表达从而抑制K562/A02细胞对ADM的敏感性,诱导白血病细胞多药耐药。Cer作为GCS的酶催化底物,在GCS高表达时水平下降是激活ERK通路的上游信号。
[Abstract]:The emergence of multidrug resistance (multidrug resistance, MDR) has become a major problem in the clinical treatment of leukemia. For the study of its drug resistance mechanism, the role of glucosylceramide synthase (GCS) in the drug resistance is becoming more and more concerned. It is known that GCS is a glycosylase in sphingolipid metabolism, and it is known as a glycosylase in sphingolipid metabolism. The most direct function is to transfer the glycosyl group on the urine nucleoside two phosphate glucose (UDP-glucose) to Ceramide (Cer) to form Glycosylceramide (Glycosylceramide, Glc Cer), to provide synthetic precursors for the synthesis of advanced sheath glycolipid and reduce the level of Cer in the cell. As a signal transduction lipid second messenger, Cer is widely used as a signal transduction The decrease of.Cer level in cell growth, differentiation, senescence and death and other physiological processes, such as cell differentiation and apoptosis, is one of the reasons for GCS to induce tumor resistance. Previous studies on GCS induced drug resistance mostly focus on the effect of GCS on the drug delivery pump on the cell membrane. Multidrug resistance gene 1 (multidru G resistance gene 1, MDR1) encoded P170 glycoprotein (P-glycoprotein, P-gp) is the most studied drug resistant molecule. When the drug enters the cell, P-gp and drug molecules are combined and the drug is transferred to the extracellular by ATP. The intracellular drug is maintained at a lower level. The study found that GCS can be up regulated in different system tumors by up regulation. P-gp promotes the production of antitumor drugs and leads to the production of cell resistance. However, it is reported that even if the anticancer drug breaks through the target of the outer row of the cell line, the anticancer drugs are still unable to eliminate the tumor cells because of the inhibition of the apoptosis pathway, indicating that the apoptosis pathway is abnormal and the tumor resistance is heavy. One of the causes of.Bcl-2 is a kind of gene family specific to cancer cells, which mainly regulate the apoptosis in the mitochondrial membrane. According to the structure of the Bcl-2 family and the different biological effects, the Bcl-2 family can be divided into: (1) the only BH3 domain, represented by Bid, can respond to and activate the stimulation. BH3 protein; (2) the apoptotic executive protein, represented by Bax, can increase the permeability of the mitochondrial membrane and activate the cascade reaction of apoptosis; (3) the anti apoptotic protein.Bcl-2, which is represented by Bcl-2, inhibits the release of mitochondrial cytochrome C and inhibits apoptosis by inhibiting the release of mitochondrial cytochrome C. A large amount of data shows Bcl-2 protein. Increasing or decreasing Bax protein is an important cause of chemotherapy insensitivity to a variety of tumor cells. Up regulation of Bax or down regulation of Bcl-2 can enhance the sensitivity of cells to tumor drugs. Earlier studies in our group have found that the expression of GCS and Bcl-2 in K562/A02 leukemia multidrug resistant cell lines is significantly higher than that of K562 sensitive cell lines. There was no significant difference in the expression of Bax; the expression level of GCS and Bcl-2 decreased significantly, and the expression of Bax was not affected. In addition, the group found that the drug resistance of K562/A02 was also related to the ERK signaling pathway, which mediates the regulation of P-gp transporters by GCS. The ERK signaling pathway is closely related to apoptosis, and ERK mediated signal transduction The study found that the activation of ERK pathway is also the prerequisite for mitochondrial depolarization, cytochrome C release, Caspase3 activation and cell apoptosis. Therefore, this paper studies the molecular mechanism of GCS up regulation of Bcl-2 expression to induce leukemia resistance, and explores whether ERK signaling pathway participates in GCS to wither. The inhibitory process of death in order to reveal the new mechanism of GCS induced resistance to leukemia and to provide new ideas for reversing the drug resistance of tumor. Objective: To explore the effect of GCS on the expression of apoptosis related gene Bcl-2 in human leukemia multidrug resistant cell line K562/A02 and to clarify the mechanism of GCS in the drug resistance of leukemia. Methods: using RNA interference technique to target inhibition. After making GCS in K562/A02 cells, real-time PCR and Western blotting were used to detect the changes of Bcl-2 m RNA and protein, the expression of MAPK member proteins, flow cytometry to detect cell apoptosis, and CCK-8 method to detect the sensitivity of cells to adriamycin and the level of high performance liquid chromatography-mass spectrometry After treatment of K562/A02 cells 24 h, CCK-8 assay was used to detect the sensitivity of cells to ADM; flow cytometry was used to observe the changes in the level of cell apoptosis when U0126 and ADM were used alone or in combination; real-time PCR and Western blotting detected U0126 and Cer against cells and proteins. After the expression of GCS in 02 cells, the apoptosis of ADM increased, the drug sensitivity was enhanced, the level of Bcl-2 m RNA and protein decreased, and the content of Cer increased, and the Western blotting results showed that the p-ERK expression decreased, but the total ERK, the total phosphorylation JNK and p38 expressions had no obvious changes. Increase in death, increased drug sensitivity, Bcl-2 m RNA and protein and p-ERK levels in a concentration dependent and time dependent decline; exogenous C6- ceramide (C6-Ceramide, C6-Cer) also significantly inhibited the phosphorylation of ERK in K562/A02 cells and the expression of Bcl-2 inhibitor protein. Conclusion: ERK signaling pathway mediates GCS to regulate apoptosis protein The expression of.Cer inhibits the sensitivity of K562/A02 cells to ADM, and induces multidrug resistance of leukemic cells as a substrate for GCS enzyme catalysis. The decrease in level of GCS is the upstream signal of the ERK pathway.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R733.7

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