【摘要】：目的：課題組前期研究發(fā)現(xiàn)β-arrestin1在兒童急性淋巴細(xì)胞白血�。ˋLL）白血病起始細(xì)胞（LIC）的異常高表達(dá)，本課題進(jìn)一步研究β-arrestin1在體內(nèi)外對(duì)LIC的增殖能力，及對(duì)白血病發(fā)生發(fā)展的影響；研究β-arrestin1對(duì)LICs細(xì)胞甲基化轉(zhuǎn)移酶（DNMTs）活性與表達(dá)的影響；研究β-arrestin1對(duì)HOXa9、PTEN、P15等基因的甲基化與表達(dá)的影響；研究β-arrestin1與甲基化轉(zhuǎn)移酶抑制劑對(duì)LIC增殖與白血病生存的作用，探索β-arrestin1影響LIC甲基化的表觀(guān)遺傳學(xué)機(jī)制，為兒童ALL診治新靶點(diǎn)提供實(shí)驗(yàn)基礎(chǔ)。 方法：從兒童ALL患者骨髓中分選與鑒定具有CD34+CD38-CD19+標(biāo)記的LIC細(xì)胞，運(yùn)用si-βarrestin1慢病毒重組載體抑制ALL-LIC細(xì)胞β-arrestin1表達(dá)，建立ALL-LIC細(xì)胞和小鼠的β-arrestin1敲除模型；利用克隆形成實(shí)驗(yàn)檢測(cè)si-βarrestin1對(duì)ALL-LIC細(xì)胞增殖能力的影響；LIC細(xì)胞異種移植到si-βarrestin1NOD/SCID小鼠體內(nèi)，檢測(cè)ALL-LIC細(xì)胞體內(nèi)復(fù)制能力，利用小鼠的白血病進(jìn)程與生存周期來(lái)監(jiān)測(cè)β-arrestin1對(duì)白血病進(jìn)程的影響；利用ELISA實(shí)驗(yàn)檢測(cè)不同組細(xì)胞中甲基化轉(zhuǎn)移酶(DNMT)活性；利用熒光定量RT-PCR和Western blot實(shí)驗(yàn)檢測(cè)多基因的mRNA和蛋白表達(dá)差異；利用甲基化特異PCR和熒光定量RT-PCR檢測(cè)相關(guān)基因甲基化水平和mRNA表達(dá)水平；利用甲基化轉(zhuǎn)移酶抑制劑5-Aza作用si-βarrestin1細(xì)胞與老鼠模型。 結(jié)果：成功建立抑制β-arrestin1表達(dá)的ALL-LIC細(xì)胞和小鼠模型，抑制β-arrestin1后ALL-LIC細(xì)胞的克隆形成能力明顯減弱，降低ALL-LIC細(xì)胞移植到NOD/SCID小鼠的白血病發(fā)生和組織浸潤(rùn)程度；經(jīng)抑制β-arrestin1表達(dá)HOXa9、PTEN、P15等多基因的表達(dá)和甲基化存在變化，抑制β-arrestin1表達(dá)可以顯著降低DNMT的活性和表達(dá)，抑制PTEN，P15基因的甲基化水平，使其mRNA表達(dá)水平增高；使用5-Aza處理敲除β-arrestin1的LIC細(xì)胞和白血病小鼠發(fā)現(xiàn)，使用抑制劑能協(xié)同降低敲除β-arrestin1細(xì)胞克隆形成能力，推遲敲除β-arrestin1小鼠白血病發(fā)生發(fā)展進(jìn)程，降低骨髓、肝脾組織白血病細(xì)胞浸潤(rùn)程度，DNMT活性與表達(dá)降低。 結(jié)論：β-arrestin1在LIC細(xì)胞中顯著增高，抑制β-arrestin1可降低ALL-LIC細(xì)胞的增殖和在NOD/SCID小鼠體內(nèi)白血病發(fā)生發(fā)展的能力，降低DNMT的活性和表達(dá)，從而影響PTEN的甲基化水平和表達(dá)，并協(xié)同甲基化抑制劑減緩ALL-LIC增殖能力，延緩白血病進(jìn)程，提示甲基化抑制劑可潛在用于A(yíng)LL的臨床治療，，與β-arrestin1協(xié)同作用有關(guān)。
[Abstract]:Objective: to investigate the expression of 尾 -arrestin1 in leukemia initiation cells (LIC) in children with acute lymphoblastic leukemia (all) in vitro and in vivo, and to investigate the effect of 尾 -arrestin1 on the proliferation of LIC in vitro and in vivo. To study the effect of 尾 -arrestin1 on the activity and expression of methylation transferase (DNMTs) in LICs cells; to study the effect of 尾 -arrestin1 on the methylation and expression of Hoxa9 PTENN P15 and other genes; and to study the effects of 尾 -arrestin1 and methyltransferase inhibitor on the proliferation and survival of LIC. To explore the epigenetic mechanism of 尾 -arrestin1 affecting the methylation of LIC, and to provide experimental basis for the diagnosis and treatment of all in children. Methods: LICs labeled with CD34 CD38-CD19 were isolated and identified from the bone marrow of all children. The 尾 -arrestin1 expression of ALL-LIC cells was inhibited by si- 尾 arrestin1 lentivirus recombinant vector, and the 尾 -arrestin1 knockout model of ALL-LIC cells and mice was established. The effect of si- 尾 arrestin1 on the proliferation of ALL-LIC cells was detected by clone formation assay. The xenotransplantation of si- 尾 arretin 1 NOD / SCID cells was used to detect the ability of ALL-LIC cells to replicate in vivo. The effect of 尾 -arrestin1 on leukemia process was monitored by using the leukemia progression and survival cycle of mice, and the activity of methylated transferase (DNMT) in different groups of cells was detected by Elisa. The difference of mRNA and protein expression of multigene was detected by fluorescence quantitative RT-PCR and Western blot assay, and the methylation level and mRNA expression level of related gene were detected by methylation specific PCR and fluorescence quantitative RT-PCR. Effect of methyltransferase inhibitor 5-Aza on si- 尾 arrestin1 cells and mouse model. Results: the ALL-LIC cells and mouse models were successfully established to inhibit the expression of 尾 -arrestinin-1. After 尾 -arrestinin-1, the cloning ability of ALL-LIC cells was significantly decreased, and the leukaemia and tissue infiltration degree of ALL-LIC cells transplanted to nod / SCID mice were decreased. After inhibiting the expression and methylation of 尾 -arrestin1, the activity and expression of DNMT and the methylation of PTEN15 were significantly decreased, and the mRNA expression of PTENn15 was increased by inhibiting the expression of 尾 -arrestinin-1. 5-Aza was used to treat the LIC cells and leukemia mice with knockout 尾 -arrestin1. It was found that the use of inhibitor could reduce the clone forming ability of 尾 -arrestin1 cells, delay the development of leukemia and lower the bone marrow of knockout 尾 -arrestin1 mice. The activity and expression of DNMT decreased in the infiltrating degree of hepatosplenic leukemia cells. Conclusion: 尾 -arrestinin-1 is significantly increased in LIC cells. Inhibiting 尾 -arrestin1 can reduce the proliferation of ALL-LIC cells and the ability to develop leukemia in nod / SCID mice, and decrease the activity and expression of DNMT, thus affecting the methylation level and expression of PTEN. The synergistic effect of methylation inhibitor on the proliferation of ALL-LIC and the delay of leukaemia progression suggests that methylation inhibitor can potentially be used in clinical therapy of all, which is related to the synergistic effect of 尾 -arrestin1.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R733.7

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