β-arrestin1调控儿童急性淋巴细胞白血病起始细胞的增殖与甲基化研究

发布时间：2018-07-18 12:37

【摘要】：目的：课题组前期研究发现β-arrestin1在儿童急性淋巴细胞白血病（ALL）白血病起始细胞（LIC）的异常高表达，本课题进一步研究β-arrestin1在体内外对LIC的增殖能力，及对白血病发生发展的影响；研究β-arrestin1对LICs细胞甲基化转移酶（DNMTs）活性与表达的影响；研究β-arrestin1对HOXa9、PTEN、P15等基因的甲基化与表达的影响；研究β-arrestin1与甲基化转移酶抑制剂对LIC增殖与白血病生存的作用，探索β-arrestin1影响LIC甲基化的表观遗传学机制，为儿童ALL诊治新靶点提供实验基础。方法：从儿童ALL患者骨髓中分选与鉴定具有CD34+CD38-CD19+标记的LIC细胞，运用si-βarrestin1慢病毒重组载体抑制ALL-LIC细胞β-arrestin1表达，建立ALL-LIC细胞和小鼠的β-arrestin1敲除模型；利用克隆形成实验检测si-βarrestin1对ALL-LIC细胞增殖能力的影响；LIC细胞异种移植到si-βarrestin1NOD/SCID小鼠体内，检测ALL-LIC细胞体内复制能力，利用小鼠的白血病进程与生存周期来监测β-arrestin1对白血病进程的影响；利用ELISA实验检测不同组细胞中甲基化转移酶(DNMT)活性；利用荧光定量RT-PCR和Western blot实验检测多基因的mRNA和蛋白表达差异；利用甲基化特异PCR和荧光定量RT-PCR检测相关基因甲基化水平和mRNA表达水平；利用甲基化转移酶抑制剂5-Aza作用si-βarrestin1细胞与老鼠模型。结果：成功建立抑制β-arrestin1表达的ALL-LIC细胞和小鼠模型，抑制β-arrestin1后ALL-LIC细胞的克隆形成能力明显减弱，降低ALL-LIC细胞移植到NOD/SCID小鼠的白血病发生和组织浸润程度；经抑制β-arrestin1表达HOXa9、PTEN、P15等多基因的表达和甲基化存在变化，抑制β-arrestin1表达可以显著降低DNMT的活性和表达，抑制PTEN，P15基因的甲基化水平，使其mRNA表达水平增高；使用5-Aza处理敲除β-arrestin1的LIC细胞和白血病小鼠发现，使用抑制剂能协同降低敲除β-arrestin1细胞克隆形成能力，推迟敲除β-arrestin1小鼠白血病发生发展进程，降低骨髓、肝脾组织白血病细胞浸润程度，DNMT活性与表达降低。结论：β-arrestin1在LIC细胞中显著增高，抑制β-arrestin1可降低ALL-LIC细胞的增殖和在NOD/SCID小鼠体内白血病发生发展的能力，降低DNMT的活性和表达，从而影响PTEN的甲基化水平和表达，并协同甲基化抑制剂减缓ALL-LIC增殖能力，延缓白血病进程，提示甲基化抑制剂可潜在用于ALL的临床治疗，，与β-arrestin1协同作用有关。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R733.7

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