本文選題：葡萄糖神經(jīng)酰胺合成酶 + 神經(jīng)酰胺　； 參考：《南京醫(yī)科大學(xué)》2015年碩士論文

【摘要】：多藥耐藥(multidrug resistance,MDR)的出現(xiàn)已然成為臨床治療白血病的一大難題,對于其耐藥機(jī)制的研究,目前葡萄糖神經(jīng)酰胺合成酶(glucosylceramide synthase,GCS)在腫瘤耐藥中的作用越來越受到人們的關(guān)注。已知GCS是鞘脂代謝中的糖基化酶,其最直接的功能是將尿核苷二磷酸葡萄糖(UDP-glucose)上的糖基轉(zhuǎn)移至神經(jīng)酰胺(Ceramide,Cer)形成葡萄糖神經(jīng)酰胺(Glycosylceramide,Glc Cer),為高級鞘糖脂的合成提供合成前體,同時(shí)降低了細(xì)胞內(nèi)的Cer水平。作為信號(hào)轉(zhuǎn)導(dǎo)的脂質(zhì)第二信使,Cer廣泛參與細(xì)胞生長、分化、衰老及死亡等生理過程。Cer水平降低使細(xì)胞分化、凋亡等功能抑制,自然而然也成為GCS誘導(dǎo)腫瘤耐藥的原因之一。以往關(guān)于GCS誘導(dǎo)腫瘤耐藥的研究多集中在GCS對細(xì)胞膜上藥物轉(zhuǎn)運(yùn)排出泵的影響。多藥耐藥基因1(multidrug resistance gene 1,MDR1)編碼的P170糖蛋白(P-glycoprotein,P-gp)是研究最多的腫瘤耐藥分子。當(dāng)藥物進(jìn)入細(xì)胞,P-gp與藥物分子結(jié)合并在ATP的驅(qū)動(dòng)下將藥物轉(zhuǎn)移至細(xì)胞外,使細(xì)胞內(nèi)藥物始終維持在較低水平。研究發(fā)現(xiàn),不同系統(tǒng)腫瘤中GCS可通過上調(diào)P-gp促進(jìn)抗腫瘤藥物的泵出,進(jìn)而導(dǎo)致細(xì)胞耐藥的產(chǎn)生。然而亦有研究報(bào)道稱,即便抗癌藥物突破細(xì)胞外排這一屏障到達(dá)其作用的靶點(diǎn),由于細(xì)胞凋亡通路受抑,這些抗癌藥物仍舊無法消滅腫瘤細(xì)胞,說明凋亡通路異常也是腫瘤耐藥的重要原因之一。Bcl-2是癌細(xì)胞特有的一類基因家族,主要在線粒體膜上發(fā)揮對細(xì)胞凋亡的調(diào)控作用。根據(jù)Bcl-2家族各分子的結(jié)構(gòu)以及發(fā)揮的生物學(xué)效應(yīng)不同可將Bcl-2家族分為:(1)以Bid為代表的只具有BH3結(jié)構(gòu)域、能對刺激產(chǎn)生反應(yīng)并被激活的BH3蛋白;(2)以Bax為代表的能使線粒體外膜通透性增加進(jìn)而激活凋亡級聯(lián)反應(yīng)的凋亡執(zhí)行蛋白;(3)以Bcl-2為代表的抑制BH3和凋亡執(zhí)行蛋白的抑凋亡蛋白。Bcl-2主要通過阻止線粒體細(xì)胞色素C的釋放從而抑制細(xì)胞凋亡。大量數(shù)據(jù)顯示Bcl-2蛋白增加或Bax蛋白減少是多種腫瘤細(xì)胞對化療不敏感的重要原因。上調(diào)Bax或下調(diào)Bcl-2的基因水平可使細(xì)胞對腫瘤藥物的敏感性增強(qiáng)。本課題組前期研究發(fā)現(xiàn),與K562敏感細(xì)胞株比較,K562/A02白血病多藥耐藥細(xì)胞株中GCS與Bcl-2的表達(dá)明顯增高,而Bax的表達(dá)無明顯差異;下調(diào)GCS,Bcl-2的表達(dá)水平亦明顯下降,而Bax的表達(dá)不受影響;此外課題組發(fā)現(xiàn)K562/A02的耐藥性還與ERK信號(hào)通路有關(guān),該通路介導(dǎo)了GCS對P-gp轉(zhuǎn)運(yùn)蛋白的調(diào)控。由于ERK信號(hào)通路與細(xì)胞凋亡密切相關(guān),ERK介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)可能是多種信號(hào)刺激的共同通路。研究發(fā)現(xiàn)ERK通路的激活亦是線粒體去極化、細(xì)胞色素C釋放、caspase3活化以及細(xì)胞凋亡的必要前提。因此,本文就GCS上調(diào)Bcl-2表達(dá)從而誘導(dǎo)白血病耐藥的分子機(jī)制進(jìn)行研究,探討ERK信號(hào)通路是否參與GCS對凋亡的抑制過程,以期揭示GCS誘導(dǎo)白血病的耐藥新機(jī)制,為逆轉(zhuǎn)腫瘤耐藥提供新的思路。目的:探討人白血病多藥耐藥細(xì)胞株K562/A02中GCS對凋亡相關(guān)基因Bcl-2表達(dá)水平的影響,明確GCS在白血病耐藥中的作用機(jī)制。方法:利用RNA干擾技術(shù)靶向抑制K562/A02細(xì)胞中的GCS后,real-time PCR和western blotting檢測Bcl-2 m RNA與蛋白、MAPK成員蛋白表達(dá)的變化情況;流式細(xì)胞術(shù)檢測細(xì)胞凋亡;CCK-8法檢測細(xì)胞對阿霉素(Adriamycin,ADM)的敏感性以及高效液相色譜—質(zhì)譜聯(lián)用分析細(xì)胞中的Cer水平。20μM U0126處理K562/A02細(xì)胞24 h后,CCK-8法檢測細(xì)胞對ADM的敏感性;流式細(xì)胞術(shù)觀察U0126和ADM單獨(dú)或聯(lián)合使用時(shí)細(xì)胞凋亡水平的變化;real-time PCR和western blotting分別檢測U0126以及Cer對細(xì)胞Bcl-2 m RNA與蛋白、ERK信號(hào)通路活化的影響。結(jié)果:GCS si RNA下調(diào)K562/A02細(xì)胞中GCS表達(dá)后,細(xì)胞對ADM的凋亡增加、藥物敏感性增強(qiáng),Bcl-2 m RNA與蛋白水平下降,Cer含量增加;western blotting結(jié)果顯示p-ERK表達(dá)減少,但總ERK、總的以及磷酸化JNK和p38 MAPK的表達(dá)無明顯變化。MEK特異性抑制劑U0126作用K562/A02后,細(xì)胞對ADM凋亡增加、藥物敏感性增強(qiáng),Bcl-2 m RNA與蛋白以及p-ERK水平呈濃度依賴性和時(shí)間依賴性下降;外源C6-神經(jīng)酰胺(C6-Ceramide,C6-Cer)也可顯著抑制K562/A02細(xì)胞中ERK的磷酸化激活和Bcl-2抑凋亡蛋白的表達(dá)。結(jié)論:ERK信號(hào)通路介導(dǎo)GCS上調(diào)抑凋亡蛋白Bcl-2的表達(dá)從而抑制K562/A02細(xì)胞對ADM的敏感性,誘導(dǎo)白血病細(xì)胞多藥耐藥。Cer作為GCS的酶催化底物,在GCS高表達(dá)時(shí)水平下降是激活ERK通路的上游信號(hào)。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R733.7

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