核内微管相关蛋白1轻链3在急性髓系白血病细胞自噬中的作用以及机制研究

发布时间：2018-05-29 20:53

本文选题：急性髓系细胞白血病 + 核内LC3　；参考：《浙江大学》2017年博士论文

【摘要】：背景:急性髓系白血病(acute myeloid leukemia,AML)是恶性血液系统疾病之一,特征为白血病细胞恶性克隆性增生,我国总体发病率为3-4/10万。该类疾病存在死亡率高、复发率高的特点,并且存在较高的治疗相关死亡,是一种凶险的恶性血液疾病。化疗目前仍是治疗AML的主要手段,但耐药以及治疗相关毒性等问题依然是困扰医务人员,如何有效克服上述问题仍然是目前治疗急性髓系白血病的热点。近年来,随着研究人员对肿瘤细胞自噬的认识不断加深,越来越多的证据表明自噬现象在急性髓系白血病治疗过程中扮演了重要角色。因此进一步探索自噬相关机制将有助于在更有效的治疗急性髓系白血病治疗中取得突破。微管相关蛋白1轻链3(LC3)是自噬标志物,自噬形成时,胞浆型LC3(即LC3-I)会酶解掉一小段多肽,转变为(自噬体)膜型(即LC3-II),结合在自噬体膜上。目前有文献已报道Hela细胞自噬发生时,于形成LC3I前,存在核内LC3外移的过程,然而在急性髓系白血病细胞中是否亦存在这样的表现以及其相关机制目前尚不清楚。目的:用化疗药物阿糖胞苷(Cytarabine,Ara-c)以及靶向药物索拉非尼(Sorafenib)分别作用急性髓系白血病细胞株(THP-1,MV4-11),观察其自噬发生时是核内LC3分布的改变,同时观察其调控机制中蛋白去乙酰化酶家族成员Sirt1 以及核转移蛋白(Diabetes and Obesity Regμlated gene,DOR)表达改变,并探讨其在自噬信号通路中作用以及对细胞增殖生长的影响。在此理论基础上,探讨小剂量西达本胺是否可通过抑制Sirt1表达从而抑制自噬来影响急性髓系白血病细胞株的增殖。方法:(1)Ara-c处理THP-1,Ara-c以及索拉非尼分别处理MV4-11细胞株后,使用Western-Blot技术检测细胞自噬蛋白LC3以及P62表达情况,雷帕霉素以及饥饿处理组为阳性对照组,无药物处理为阴性对照组。使用免疫荧光技术观察药物处理THP-1以及MV4-11后LC3的分布变化,以及DOR分布的改变,雷帕霉素以及饥饿处理组为阳性对照组,无药物处理为阴性对照组。利用荧光定量PCR手段检测药物处理THP-1以及MV4-11后的Sirt1,DORmmRNA的表达水平,以饥饿处理组为阳性对照组,无药物处理为阴性对照组。自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3-MA)联合药物处理THP-1以及MV4-11细胞,使用免疫荧光技术检测LC3的分布变化,以及DOR分布的改变,使用荧光定量PCR手段检测Sirt1,DORmRNA的表达水平。利用荧光定量PCR手段检测药物处理急性髓系白血病患者的原代细胞后的Sirt1,DORmRNA的表达水平。(2)利用小分子RNA干扰技术下调THP-1,MV4-11细胞中的Sirt1,DORmRNA表达水平,使用Western-Blot技术检测细胞自噬蛋白LC3以及P62表达情况,使用免疫荧光技术检测LC3的分布变化,以及DOR分布的改变。同时分别利用Western-Blot技术以及荧光定量PCR检测自噬信号通路中关键调控分子的蛋白和mRNA表达的改变。(3)通过MTS,流式细胞分析方法以及Western-Blot技术分析抑制Sirt1以及DOR后对细胞增殖存活的影响。(4)在上述理论基础上,使用小剂量西达本胺处理THP-1,MV4-11细胞后,通过MTS,流式细胞分析方法,荧光定量PCR以及Western-Blot技术探讨小剂量西达本胺是否可通过影响Sirt1表达来调控自噬,从而影响对急性髓系白血病细胞株的增殖存活。结果:(1)化疗药物Ara-c以及靶向药物索拉非尼在抑制急性髓系白血病细胞生长的同时可以诱导细胞自噬增强,在自噬增强时,出现核内LC3转移到核外的改变,同时核内DOR亦出现外移的改变。其中与核内LC3外移相关的分子Sirt1以及DORmmRNA均有不同程度的上调,原代细胞经化疗药物处理后也出现Sirt1以及DORmRNA的表达上调。经3-MA抑制自噬后,核外LC3分布有减少的趋势,同时Sirt1以及DORmRNA出现下调。(2)经下调Sirt1以及DORmRNA的水平后,急性髓系白血病细胞在药物处理后的自噬有所减弱,且核内LC3外移减弱。(3)Sirt1以及DOR表达的改变可以影响自噬通路中的关键分子Beclin1,高迁移率族蛋白 1(high mobility group box-1 protein,HMGB1)以及一磷酸腺苷活化的蛋白激酶(Adenosine monophosphate-activated protein kinase,AMPK)的表达。(4)下调细胞的Sirt1以及DOR的水平后,可增强Ara-c,索拉非尼的细胞毒性作用,促进其对白血病细胞的凋亡作用。(5)小剂量西达本胺可以通过下调白血病细胞的Sirt1抑制细胞自噬来增加细胞对Ara-c与索拉非尼的敏感性,促进药物对细胞的凋亡作用。结论:(1)核内LC3外移参与急性髓系白血病细胞化疗药物处理后产生的自噬现象,且Sirt1,DOR是核内LC3外移的调控分子。(2)核内LC3外移在自噬信号通路可影响Beclin1通路,HMGB1通路与AMPK通路。(3)通过抑制核内LC3外移达到抑制急性髓系白血病细胞的自噬可增强化疗药物的细胞毒性作用。(4)小剂量西达本胺可通过下调Sirt1水平抑制急性髓系白血病细胞自噬,从而增强相关药物的细胞毒性作用。
[Abstract]:Background: acute myeloid leukemia (AML) is one of the malignant hematological diseases. It is characterized by malignant clonogenic proliferation of leukemia cells. The overall incidence of the disease is 3-4/10 million in our country. The disease has high mortality, high recurrence rate, and high treatment related death. It is a dangerous and malignant blood disease. Chemotherapy is still the main means to treat AML, but drug resistance and treatment related toxicity are still plagued by medical personnel. How to effectively overcome these problems is still a hot spot in the treatment of acute myeloid leukemia. In recent years, more and more evidence has been shown as the researchers have deepened the understanding of autophagy in tumor cells. Phagocytosis plays an important role in the treatment of acute myeloid leukemia. Therefore, further exploration of autophagy related mechanisms will help to make a breakthrough in more effective treatment of acute myeloid leukemia. Microtubule related protein 1 light chain 3 (LC3) is a autophagy marker. When autophagy is formed, cytoplasmic LC3 (LC3-I) can hydrolyse a small segment. Peptide (autophagic) membrane type (autophagic) (autophagic) membrane (LC3-II), combined with autophagic membrane. There are reports that Hela cell autophagy occurs at the time of autophagy, before the formation of LC3I, there is a process of LC3 migration in the nucleus. However, it is not clear in the acute myeloid leukemia cells and the related mechanisms are still unclear. Cytarabine (Ara-c) and the targeting drug Sola Fini (Sorafenib) act on the acute myeloid leukemia cell line (THP-1, MV4-11), and observe the changes in the LC3 distribution in the nucleus when the autophagy occurs, and observe the Sirt1 of the protein deacetylase family and the Diabetes and Obesity Reg micron (Diabetes and Obesity). Gene, DOR) expression changes and its effect on autophagic signaling pathway and cell proliferation growth. On this basis, it is explored whether small doses of Western diamine can inhibit autophagy by inhibiting Sirt1 expression to inhibit the proliferation of acute myeloid leukemia cell lines. Method: (1) Ara-c treatment of THP-1, Ara-c, and sorafy After treatment of MV4-11 cell lines, the expression of autophagic protein LC3 and P62 was detected by Western-Blot technique. Rapamycin and starvation treatment group were positive control group, without drug treatment as negative control group. Immunofluorescence technique was used to observe the distribution of THP-1 and MV4-11 after THP-1 and MV4-11, and the change of DOR distribution. The positive control group of rapamycin and starvation treatment group, without drug treatment as negative control group, the expression level of Sirt1, DORmmRNA after THP-1 and MV4-11 was detected by fluorescence quantitative PCR, the positive control group was treated with starvation treatment group, no drug treatment was negative control group, and the autophagy inhibitor 3- methyl adenine (3-Methylade). Nine, 3-MA) combined with drug treatment of THP-1 and MV4-11 cells, using immunofluorescence technique to detect the distribution of LC3, and the change of DOR distribution. The expression level of Sirt1 and DORmRNA was detected by fluorescence quantitative PCR. Fluorescence quantitative PCR was used to detect the Sirt1, DORmRNA, in the treatment of the primary cells of acute myeloid leukemia patients. Expression level. (2) use small molecule RNA interference technique to reduce Sirt1, DORmRNA expression level in THP-1, MV4-11 cells, use Western-Blot technique to detect the expression of autophagic protein LC3 and P62, use immunofluorescence technique to detect the distribution of LC3, and the change of DOR distribution. Meanwhile, Western-Blot technology and fluorescence are used respectively. Quantitative PCR detection of the changes in the protein and mRNA expression of key regulatory molecules in autophagic signaling pathway. (3) the effects of inhibition of Sirt1 and DOR on cell proliferation and survival were analyzed by MTS, flow cytometry and Western-Blot technique. (4) on the basis of the above theory, small doses of Western diamine were used to treat THP-1, MV4-11 cells, through MTS. Flow cytometry, fluorescence quantitative PCR and Western-Blot techniques are used to investigate whether small doses of Western diamine can regulate autophagy by affecting the expression of Sirt1, thereby affecting the proliferation and survival of acute myeloid leukemia cells. Results: (1) chemotherapeutic drug Ara-c and the target drug sorafeni in the suppression of acute myeloid leukemia cells At the same time, it can induce autophagy to enhance the autophagy. At the time of autophagy, the metastasis of LC3 to the nucleus appears in the nucleus, and the DOR also changes in the nucleus. The molecules Sirt1 and DORmmRNA related to the LC3 migration in the nucleus are up to varying degrees. After the treatment of the primary cells, the Sirt1 and the DORmRNA are also appeared. After the inhibition of autophagy by 3-MA, the distribution of LC3 in the nucleus was reduced and the Sirt1 and DORmRNA decreased. (2) the autophagy of acute myeloid leukemia cells weakened after the treatment of Sirt1 and DORmRNA, and the LC3 migration decreased in the nucleus. (3) the changes in the expression of Sirt1 and DOR could affect the autophagic pathway. Key molecules Beclin1, high mobility group protein 1 (high mobility group box-1 protein, HMGB1) and the expression of protein kinase (kinase, AMPK) activated by adenosine monophosphate (Adenosine monophosphate-activated protein kinase, AMPK). (4) down regulation of cells and the levels of cells can enhance the cytotoxicity of Sola Fini and promote its cytotoxicity. The apoptosis of leukemic cells. (5) small dose of DDA can increase the sensitivity of cell to Ara-c and sorafenib by decreasing the autophagy of Sirt1 cells in leukemic cells to promote the effect of drug apoptosis on cells. Conclusion: (1) the LC3 migration in the nucleus participates in the autophagy produced by the chemotherapy of acute myeloid leukemia cells. Phenomenon, and Sirt1, DOR is a regulator of the LC3 migration in the nucleus. (2) the LC3 migration in the nucleus can affect the Beclin1 pathway, the HMGB1 pathway and the AMPK pathway. (3) the cytotoxicity of the chemotherapeutic drugs can be enhanced by inhibiting the migration of LC3 in the nucleus to inhibit the autophagy of acute myeloid leukemia cells. (4) small dose of Western diamine can be passed down The level of Sirt1 inhibits autophagy in acute myeloid leukemia cells, thereby enhancing the cytotoxicity of related drugs.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R733.71

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