發(fā)布時(shí)間：2019-03-23 17:45

【摘要】：目的：急性早幼粒細(xì)胞白血病(acute promyelocytic leukemia,APL)是髓系細(xì)胞停止在早幼粒細(xì)胞階段的一類較為特殊的急性髓系白血病。隨著全反式維甲酸(all-trans retinoid acid,ATRA)常規(guī)用于APL的治療后,APL患者的預(yù)后得到了顯著提高。但是,單用ATRA后耐藥問題也日益嚴(yán)重。依維莫司(Everolimus, RAD001)一種新型哺乳動物雷帕霉素受體(mTOR)抑制劑,為雷帕霉素的衍生物,具有免疫抑制、抗腫瘤、抗病毒等多種作用。因此,本研究旨在探討依維莫司協(xié)同ATRA對APL的作用及機(jī)制。方法：用RAD001和ATRA分別或聯(lián)合處理急性早幼粒細(xì)胞白血病細(xì)胞株NB4-R1細(xì)胞。應(yīng)用CDllb染色流式細(xì)胞術(shù)及硝基四唑氮藍(lán)(NBT)還原實(shí)驗(yàn)檢測細(xì)胞分化,運(yùn)用CCK-8法檢測細(xì)胞增殖情況,周期試劑盒流式檢測細(xì)胞周期情況,AnnexinV/PI雙染色檢測細(xì)胞凋亡情況,免疫印跡法檢測自噬相關(guān)蛋白LC3、Beclinl,凋亡相關(guān)蛋白caspase家族,周期相關(guān)蛋白CyclinD1、P27Kip1及其他P70S6K、 P-P70S6K、4E-BP1、P-4E-BP1、PML-RARa等蛋白質(zhì)表達(dá)水平,RT-PCR方法檢測分化相關(guān)基因C/EBPε的mRNA表達(dá)水平。結(jié)果：(1)用1nM、10nM、100nM的RAD001和1μM的ATRA分別或聯(lián)合處理NB4-R1細(xì)胞24h、48h、72h后。流式細(xì)胞術(shù)和NBT還原實(shí)驗(yàn)顯示,聯(lián)合用藥組能夠明顯誘導(dǎo)NB4-R1細(xì)胞的分化,而單獨(dú)使用RAD001或ATRA并不能有效誘導(dǎo)NB4-R1細(xì)胞的分化。其中100nM的RAD001聯(lián)合1μM的ATRA和單獨(dú)1μM的ATRA處理NB4-R1細(xì)胞48h后分化比率分別為(54.47±4.91)%和(17.07±2.65)%(p0.01)。(2)用1nM、10nM、100nM的RAD001和1 μM的ATRA分別或聯(lián)合處理NB4-R1細(xì)胞48h后。CCK-8法檢測藥物對細(xì)胞的增殖情況發(fā)現(xiàn),聯(lián)合用藥明顯抑制細(xì)胞的增殖(p0.01),并且隨著聯(lián)用RAD001的濃度的增加,抑制作用也逐漸增強(qiáng)。而單獨(dú)用藥對細(xì)胞的抑制作用不明顯。(3)用濃度為100nM的RAD001和1μM的ATRA分別或聯(lián)合處理APL細(xì)胞株NB4-R1細(xì)胞48h后,流式細(xì)胞術(shù)周期檢測聯(lián)合用藥組G1期細(xì)胞明顯增多,而S期細(xì)胞明顯減少(p0.05)。免疫印跡法結(jié)果顯示聯(lián)合用藥后細(xì)胞周期相關(guān)蛋白cyclinD1表達(dá)相對單獨(dú)用ATRA組明顯下調(diào),而P27Kip1蛋白表達(dá)水平上調(diào)。(4)用濃度為100nM的RAD001和1μM的ATRA分別或聯(lián)合處理APL細(xì)胞株NB4-R1細(xì)胞48h后,流式細(xì)胞術(shù)凋亡檢測發(fā)現(xiàn)聯(lián)合用藥組和單獨(dú)用藥組細(xì)胞并未發(fā)生明顯凋亡。免疫印跡法檢測凋亡相關(guān)蛋白Caspase家族caspase3、caspase8、 caspase9發(fā)現(xiàn)各組之間并未發(fā)生明顯變化。(5)用濃度為100nM的RAD001和1μM的ATRA分別或聯(lián)合處理APL細(xì)胞株NB4-R1細(xì)胞48h后,RT-PCR檢測分化相關(guān)基因C/EBPε的mRNA水平顯示聯(lián)合用藥組相對與單獨(dú)用藥組明顯上調(diào)(p0.01)。免疫印跡檢測PML-RARa融合蛋白發(fā)現(xiàn),聯(lián)合用藥使融合蛋白表達(dá)下降, RAD001協(xié)同ATRA明顯抑制了mTOR下游底物P70S6K、E-BP1的磷酸化,自噬相關(guān)蛋白LC3-Ⅱ、Beclinl表達(dá)明顯上調(diào)。而加入10mM的自噬抑制劑3-MA處理后,APL細(xì)胞株NB4-R1細(xì)胞分化水平明顯下降(p0.01)并且可逆轉(zhuǎn)聯(lián)合用藥對PML-RARa融合蛋白的部分降解作用。結(jié)論：(1)RAD001協(xié)同ATRA可以誘導(dǎo)耐藥的急性早幼粒細(xì)胞白血病細(xì)胞株NB4-R1細(xì)胞分化,達(dá)到逆轉(zhuǎn)其耐藥的作用�？赡苁峭ㄟ^上調(diào)轉(zhuǎn)錄因子C/EBPε的表達(dá)和抑制mTOR信號通路誘導(dǎo)NB4-R1發(fā)生自噬部分降解PML-RARa融合蛋白等多種途徑來實(shí)現(xiàn)的。(2)RAD001協(xié)同ATRA明顯抑制NB4-R1細(xì)胞的增殖。(3)RAD001協(xié)同ATRA使NB4-R1細(xì)胞的細(xì)胞周期被阻斷在G1期,S期細(xì)胞減少,可能與聯(lián)合用藥下調(diào)CyclinD1蛋白,上調(diào)P27Kip1蛋白有關(guān)。(4)RAD001協(xié)同ATRA作用NB4-R1細(xì)胞后,細(xì)胞不發(fā)生明顯的凋亡,對凋亡相關(guān)蛋白Caspase家族沒有影響,因此聯(lián)合用藥不影響細(xì)胞的生存。
[Abstract]:Objective: Acute promyelocytic leukemia (APL) is a specific type of acute myeloid leukemia in the stage of early promyelocytic leukemia. With all-trans retinoic acid (ATRA) in the treatment of APL, the prognosis of APL patients has been improved significantly. However, the problem of drug resistance after ATRA alone is also increasing. Everolimus (RAD001), a novel mammalian rapamycin receptor (mTOR) inhibitor, is a derivative of rapamycin, and has various effects such as immunosuppression, anti-tumor, and anti-virus. Therefore, the purpose of this study is to explore the effect and mechanism of everolimus-based ATRA on APL. Methods: The cells of acute promyelocytic leukemia cell line NB4-R1 were treated with RAD001 and ATRA, respectively. The cell differentiation was detected by means of CD11b staining flow cytometry and NBT reduction. The cell proliferation was detected by CCK-8 method. The cell cycle and Annexin V/ PI double staining were used to detect the cell cycle, and the autophagy-related proteins LC3 and Beclinl were detected by immunoblotting. The expression levels of apoptosis-related protein caspase-family, cyclin D1, P27Kip1 and other P70S6K, P-P70S6K, 4E-BP1, P-4E-BP1, PML-RARa were detected by RT-PCR. Results: (1) NB4-R1 cells were treated with 1 nM,10 nM,100 nM RAD001 and 1. m u.M ATRA respectively or in combination with NB4-R1 cells for 24 h,48 h, and 72 h. Flow cytometry and NBT reduction experiments show that the combination group can obviously induce the differentiation of NB4-R1 cells, and the use of RAD001 or ATRA alone can not effectively induce the differentiation of NB4-R1 cells. The rate of differentiation was 54.47 (4.91)% and (17.07% 2.65)% (p0.01) in 100 nM of RAD001 in combination with 1.mu. M of ATRA and 1. m (2) NB4-R1 cells were treated with 1 nM,10 nM,100 nM of RAD001 and 1.mu. M of ATRA, respectively, or in combination with NB4-R1 cells for 48 h. The proliferation of the cells was detected by the CCK-8 method, and the combined administration of the drug significantly inhibited the proliferation of the cells (p0.01), and the inhibition was also enhanced with the increase of the concentration of the combination of RAD001. And the inhibition effect of the individual medicine on the cells is not obvious. (3) After 48 h of the APL cell line NB4-R1 cells were treated with ATRA with a concentration of 100 nM and ATRA of 1. m u.M, respectively, the number of G1 cells in the combined administration group was significantly increased by the flow cytometry cycle, and the S-phase cells were significantly reduced (p0.05). The results of immunoblotting showed that the expression of cyclinD1 in the cell cycle after combined administration was significantly reduced with the ATRA group, while the level of P27Kip1 protein was up-regulated. (4) After 48 h of the APL cell line NB4-R1 cells were treated with RAD001 and 1. m u.M of ATRA at a concentration of 100 nM, the cell apoptosis was detected by flow cytometry. The expression of caspase-3, caspase-8 and caspase-9 in the apoptosis-related protein was detected by immunoblotting. (5) After 48 h of the APL cell line NB4-R1 cells were treated with RAD001 and 1. m u.M of ATRA at a concentration of 100 nM, the mRNA level of the related gene C/ EBP was significantly up-regulated by RT-PCR (p0.01). The fusion protein of PML-RARa was detected by immunoblotting. In combination, the expression of fusion protein was decreased, and the co-administration of RAD001 and ATRA significantly inhibited the phosphorylation of P70S6K, E-BP1 in the downstream substrate of mTOR, and the expression of autophagy-related protein LC3-鈪，

本文編號：2446092