發(fā)布時(shí)間：2018-05-29 20:53

  本文選題：急性髓系細(xì)胞白血病 + 核內(nèi)LC3�。� 參考：《浙江大學(xué)》2017年博士論文

【摘要】：背景:急性髓系白血病(acute myeloid leukemia,AML)是惡性血液系統(tǒng)疾病之一,特征為白血病細(xì)胞惡性克隆性增生,我國(guó)總體發(fā)病率為3-4/10萬(wàn)。該類(lèi)疾病存在死亡率高、復(fù)發(fā)率高的特點(diǎn),并且存在較高的治療相關(guān)死亡,是一種兇險(xiǎn)的惡性血液疾病�；熌壳叭允侵委烝ML的主要手段,但耐藥以及治療相關(guān)毒性等問(wèn)題依然是困擾醫(yī)務(wù)人員,如何有效克服上述問(wèn)題仍然是目前治療急性髓系白血病的熱點(diǎn)。近年來(lái),隨著研究人員對(duì)腫瘤細(xì)胞自噬的認(rèn)識(shí)不斷加深,越來(lái)越多的證據(jù)表明自噬現(xiàn)象在急性髓系白血病治療過(guò)程中扮演了重要角色。因此進(jìn)一步探索自噬相關(guān)機(jī)制將有助于在更有效的治療急性髓系白血病治療中取得突破。微管相關(guān)蛋白1輕鏈3(LC3)是自噬標(biāo)志物,自噬形成時(shí),胞漿型LC3(即LC3-I)會(huì)酶解掉一小段多肽,轉(zhuǎn)變?yōu)?自噬體)膜型(即LC3-II),結(jié)合在自噬體膜上。目前有文獻(xiàn)已報(bào)道Hela細(xì)胞自噬發(fā)生時(shí),于形成LC3I前,存在核內(nèi)LC3外移的過(guò)程,然而在急性髓系白血病細(xì)胞中是否亦存在這樣的表現(xiàn)以及其相關(guān)機(jī)制目前尚不清楚。目的:用化療藥物阿糖胞苷(Cytarabine,Ara-c)以及靶向藥物索拉非尼(Sorafenib)分別作用急性髓系白血病細(xì)胞株(THP-1,MV4-11),觀(guān)察其自噬發(fā)生時(shí)是核內(nèi)LC3分布的改變,同時(shí)觀(guān)察其調(diào)控機(jī)制中蛋白去乙�；讣易宄蓡TSirt1 以及核轉(zhuǎn)移蛋白(Diabetes and Obesity Regμlated gene,DOR)表達(dá)改變,并探討其在自噬信號(hào)通路中作用以及對(duì)細(xì)胞增殖生長(zhǎng)的影響。在此理論基礎(chǔ)上,探討小劑量西達(dá)本胺是否可通過(guò)抑制Sirt1表達(dá)從而抑制自噬來(lái)影響急性髓系白血病細(xì)胞株的增殖。方法:(1)Ara-c處理THP-1,Ara-c以及索拉非尼分別處理MV4-11細(xì)胞株后,使用Western-Blot技術(shù)檢測(cè)細(xì)胞自噬蛋白LC3以及P62表達(dá)情況,雷帕霉素以及饑餓處理組為陽(yáng)性對(duì)照組,無(wú)藥物處理為陰性對(duì)照組。使用免疫熒光技術(shù)觀(guān)察藥物處理THP-1以及MV4-11后LC3的分布變化,以及DOR分布的改變,雷帕霉素以及饑餓處理組為陽(yáng)性對(duì)照組,無(wú)藥物處理為陰性對(duì)照組。利用熒光定量PCR手段檢測(cè)藥物處理THP-1以及MV4-11后的Sirt1,DORmmRNA的表達(dá)水平,以饑餓處理組為陽(yáng)性對(duì)照組,無(wú)藥物處理為陰性對(duì)照組。自噬抑制劑3-甲基腺嘌呤(3-Methyladenine,3-MA)聯(lián)合藥物處理THP-1以及MV4-11細(xì)胞,使用免疫熒光技術(shù)檢測(cè)LC3的分布變化,以及DOR分布的改變,使用熒光定量PCR手段檢測(cè)Sirt1,DORmRNA的表達(dá)水平。利用熒光定量PCR手段檢測(cè)藥物處理急性髓系白血病患者的原代細(xì)胞后的Sirt1,DORmRNA的表達(dá)水平。(2)利用小分子RNA干擾技術(shù)下調(diào)THP-1,MV4-11細(xì)胞中的Sirt1,DORmRNA表達(dá)水平,使用Western-Blot技術(shù)檢測(cè)細(xì)胞自噬蛋白LC3以及P62表達(dá)情況,使用免疫熒光技術(shù)檢測(cè)LC3的分布變化,以及DOR分布的改變。同時(shí)分別利用Western-Blot技術(shù)以及熒光定量PCR檢測(cè)自噬信號(hào)通路中關(guān)鍵調(diào)控分子的蛋白和mRNA表達(dá)的改變。(3)通過(guò)MTS,流式細(xì)胞分析方法以及Western-Blot技術(shù)分析抑制Sirt1以及DOR后對(duì)細(xì)胞增殖存活的影響。(4)在上述理論基礎(chǔ)上,使用小劑量西達(dá)本胺處理THP-1,MV4-11細(xì)胞后,通過(guò)MTS,流式細(xì)胞分析方法,熒光定量PCR以及Western-Blot技術(shù)探討小劑量西達(dá)本胺是否可通過(guò)影響Sirt1表達(dá)來(lái)調(diào)控自噬,從而影響對(duì)急性髓系白血病細(xì)胞株的增殖存活。結(jié)果:(1)化療藥物Ara-c以及靶向藥物索拉非尼在抑制急性髓系白血病細(xì)胞生長(zhǎng)的同時(shí)可以誘導(dǎo)細(xì)胞自噬增強(qiáng),在自噬增強(qiáng)時(shí),出現(xiàn)核內(nèi)LC3轉(zhuǎn)移到核外的改變,同時(shí)核內(nèi)DOR亦出現(xiàn)外移的改變。其中與核內(nèi)LC3外移相關(guān)的分子Sirt1以及DORmmRNA均有不同程度的上調(diào),原代細(xì)胞經(jīng)化療藥物處理后也出現(xiàn)Sirt1以及DORmRNA的表達(dá)上調(diào)。經(jīng)3-MA抑制自噬后,核外LC3分布有減少的趨勢(shì),同時(shí)Sirt1以及DORmRNA出現(xiàn)下調(diào)。(2)經(jīng)下調(diào)Sirt1以及DORmRNA的水平后,急性髓系白血病細(xì)胞在藥物處理后的自噬有所減弱,且核內(nèi)LC3外移減弱。(3)Sirt1以及DOR表達(dá)的改變可以影響自噬通路中的關(guān)鍵分子Beclin1,高遷移率族蛋白 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)的表達(dá)。(4)下調(diào)細(xì)胞的Sirt1以及DOR的水平后,可增強(qiáng)Ara-c,索拉非尼的細(xì)胞毒性作用,促進(jìn)其對(duì)白血病細(xì)胞的凋亡作用。(5)小劑量西達(dá)本胺可以通過(guò)下調(diào)白血病細(xì)胞的Sirt1抑制細(xì)胞自噬來(lái)增加細(xì)胞對(duì)Ara-c與索拉非尼的敏感性,促進(jìn)藥物對(duì)細(xì)胞的凋亡作用。結(jié)論:(1)核內(nèi)LC3外移參與急性髓系白血病細(xì)胞化療藥物處理后產(chǎn)生的自噬現(xiàn)象,且Sirt1,DOR是核內(nèi)LC3外移的調(diào)控分子。(2)核內(nèi)LC3外移在自噬信號(hào)通路可影響B(tài)eclin1通路,HMGB1通路與AMPK通路。(3)通過(guò)抑制核內(nèi)LC3外移達(dá)到抑制急性髓系白血病細(xì)胞的自噬可增強(qiáng)化療藥物的細(xì)胞毒性作用。(4)小劑量西達(dá)本胺可通過(guò)下調(diào)Sirt1水平抑制急性髓系白血病細(xì)胞自噬,從而增強(qiáng)相關(guān)藥物的細(xì)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R733.71

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