【作者】 熊芳；

【导师】 黄士昂；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 硕士

【摘要】 目的:急性髓细胞白血病外周血中CD4+CD25high调节性T细胞比例的升高是其免疫功能受损的一个重要机制。TLR8激动剂可有效的阻断调节性T细胞的免疫抑制作用而加强抗肿瘤免疫,提示利用TLR8激动剂阻断调节性T细胞的功能可能是急性髓细胞白血病免疫治疗的一个重要靶点。本实验通过研究人急性髓系白血病细胞U937细胞Toll样受体(TLR1-TLR9)的表达,以及TLR8受体激动剂ssRNA40/LyoVec对其增殖、凋亡、细胞周期和表面分子(CD80、CD86、CD95)的影响,探讨以TLR8为急性髓细胞白血病免疫治疗靶点的可能性。方法:以人急性髓系白血病细胞U937细胞为研究对象,利用逆转录聚合酶联反应(RT-PCR)法检测其TLR1-TLR9 mRNA的表达,用流式细胞术检测其胞内TLR8的表达。采用CCK-8试剂盒检测了不同浓度的TLR8激动剂ssRNA40/LyoVec(0μg/ml、0.2μg/ml、1μg/ml)在不同的作用时间(24h、48h、72h)处理后U937细胞的增殖活性;1μg/mlssRNA40/LyoVec处理U937细胞72h后,经AnnexinV-FITC/PI双标流式细胞仪检测细胞凋亡,经PI单标流式细胞仪检测细胞周期,流式细胞仪检测其表面分子(CD80、CD86、CD95)的变化。结果:RT-PCR示U937细胞有TLR1-TLR9的表达,FCM证明了细胞胞内TLR8的表达;CCK-8法示TLR8激动剂ssRNA40/LyoVec对U937细胞的生长有明显的抑制作用,呈时间和浓度依赖性,抑制率可达70%(P<0.05);1μg/ml TLR8激动剂ssRNA40/LyoVec作用于U937细胞72h后,处于G0/G1期细胞比例由(44.67±1.05)%增高到(54.08±1.19)%,但凋亡细胞的比例、表面分子(CD80、CD86、CD95)的表达无明显变化。结论:TLR1-TLR9可表达在U937细胞,TLR8激动剂ssRNA40/LyoVec具有抑制U937细胞增殖的作用,使细胞阻滞在G0/G1期,但无明显的促凋亡作用,对U937细胞表面分子(CD80、CD86、CD95)的表达无影响。更多还原

【Abstract】 Objective: Acute myeloid leukemia (AML) is a malignant hematopoietic disorder with considerable impairment of the immune system. We have previously reported that the T-cell-mediated immunity in AML patients could be in part damaged by the increased prevalence of CD4+CD25high regulatory T cells (Tregs). Recent studies demonstrated that synthetic and natural ligands for human TLR8 could completely reverse the suppressive function of Tregs and enhance antitumor immunity, which provides a possible application of TLR8 ligands in immunotherapy for a patient with AML. To explore the possibility, the expression of Toll-like receptors and the effect of TLR8 agonist ssRNA40/LyoVec on the proliferation, apoptosis, cell cycle and expression of CD80, CD86 and CD95 of U937 cells were studied.Methods: The expression of TLR1-TLR9 in U937 was studied by using reverse transcription polymerase chain reaction (RT-PCR) and the expression of TLR8 was assayed by flow cytometry (FCM). U937 cells were treated with various concentrations ssRNA40/LyoVec (0μg/ml, 0.2μg/ml, 1μg/ml), and the proliferation was determined by CCK-8 kit at different time points (24h, 48h, 72h). To U937 cells treated by 1μg/ml ssRNA40/LyoVec with 72h, apoptosis was analyzed with AnnexinV-FITC/PI staining by FCM, cell cycle was investigated with PI staining by FCM, expression of CD80, CD86 and CD95 was assayed by FCM. Results: U937 cells expressed TLR1-TLR9. TLR8 agonist ssRNA40/LyoVec could inhibit the growth of U937 cells in time and dose dependent manner and the inhibitory rate could reach 70%(P<0.05). It could also increase the percentage of cells in G0/G1 period. There was no significant difference of percentage of apoptosis cells and the expression of CD80, CD86 and CD95 between control and treated group.Conclusion: TLRs including TLR1-TLR9 were expressed on U937 cells and TLR8 agonist ssRNA40/LyoVec could inhibit the growth of U937 cells, arrest the cells in G0/G1 period, but have no effect on promoting apoptosis and the expression of CD80, CD86 and CD95.更多还原