不同剂量地西他滨对调节性T细胞的影响

发布时间：2018-05-29 16:12

本文选题：地西他滨 + 5-aza-2’-deoxycytidine　；参考：《天津医科大学》2012年硕士论文

【摘要】：目的：探讨不同剂量甲基转移酶抑制剂地西他滨(decitabine, Aza)通过调节Foxp3基因表达在体内外实验中对CD4+CD25+Treg细胞变化的影响,寻找体内外Aza影响CD4+CD25+Treg变化的合适剂量。方法：(1)体外实验部分,利用密度梯度离心法分离纯化C57BL/6(H-2b)小鼠脾脏单个核细胞,联合使用抗小鼠CD3单克隆抗体、抗小鼠CD28单克隆抗体、IL-2、TGF-β及不同剂量的Aza,进行淋巴细胞培养,观察不同剂量的Aza对培养体系的影响,以流式细胞技术检测培养体系CD4+CD25+Foxp3+Treg比例,实时荧光定量PCR检测培养体系Foxp3mRNA表达水平。(2)体内实验部分,按不同分组经C57BL/6(H-2b)小鼠阴茎背静脉注射不同剂量的Aza,观察不同剂量的Aza对小鼠机体的影响,以流式细胞技术检测外周血CD4+CD25+Foxp3+Treg比例,实时荧光定量PCR检测脾脏Foxp3mRNA表达水平。结果：(1)体外实验中发现,①联合使用抗小鼠CD3单克隆抗体、抗小鼠CD28单克隆抗体、IL-2. TGF-β及Aza能够使小鼠脾脏淋巴细胞培养体系中初始CD4+CD25-T细胞分化为CD4+CD25+Treg细胞。同时Foxp3mRNA表达水平上升。②不同剂量Aza诱导初始CD4+CD25-T细胞分化为CD4+CD25+Foxp3+Treg细胞的数量不同。Foxp3mRNA表达水平也不同。③浓度为5uM Aza组淋巴细胞培养体系中CD4+CD25+Foxp3+Treg比例和Foxp3mRNA表达水平与其他浓度组相比升高最明显,且差异有统计学意义(P0.05)。(2)体内实验结果显示,①Aza使经阴茎背静脉注射给药后第7天小鼠外周血CD4+CD25+Treg细胞数量增加,脾脏中Foxp3mRNA表达水平上升。②不同剂量Aza对小鼠外周血CD4+CD25+Treg细胞数量和脾脏中Foxp3mRNA表达水平影响不同。③按4ug/g.体重/day剂量给予Aza组对小鼠外周血CD4+CD25+Foxp3+Treg比例和脾脏Foxp3mRNA表达水平的影响与其他实验组相比升高最明显,且均有显著性差异(P0.05)。结论：在本实验中,无论是体内还是体外,甲基转移酶抑制剂5-氮杂-2’-脱氧胞苷(5-aza-2'-deoxycytidine, Aza)即地西他滨(Decitabine)均可诱导初始CD4+CD25T细胞分化为CD4+CD25+Treg且Foxp3mRNA表达水平上调,但是不同剂量Aza对CD4+CD25+Foxp3+Treg细胞的影响效果不同。在体内,小鼠按4ug/g/d剂量给予Aza,在体外,浓度为5uM的Aza诱导CD4+CD25+Treg细胞的增殖和分化最明显。
[Abstract]:Objective: To investigate the effect of decitabine (Aza) on the changes of CD4+CD25+Treg cells in vitro and in vivo by regulating the expression of Foxp3 gene in different doses of methyltransferase inhibitor, and to find the appropriate dosage of Aza in vitro and in vivo to affect the change of CD4+CD25+Treg.
Methods: (1) in vitro, the splenic mononuclear cells of C57BL/6 (H-2b) mice were separated and purified by density gradient centrifugation. The monoclonal antibodies against mouse CD3, anti mouse CD28 monoclonal antibody, IL-2, TGF- beta and Aza in different doses were used for lymphocyte culture, and the effects of Aza on the culture system of different doses were observed by flow cytometry. CD4+CD25+Foxp3+Treg ratio of cell technology detection culture system and real-time fluorescence quantitative PCR were used to detect the expression level of Foxp3mRNA. (2) in the experimental part of the body, different doses of Aza were injected into the dorsal vein of the penis in C57BL/6 (H-2b) mice by different groups, and the effects of different doses of Aza on the mice body were observed, and the peripheral blood C was detected by flow cytometry. D4+CD25+Foxp3+Treg ratio and real-time fluorescence quantitative PCR were used to detect the expression level of Foxp3mRNA in spleen.
Results: (1) in the experiment in vitro, the combined use of anti mouse CD3 monoclonal antibody, anti mouse CD28 monoclonal antibody, IL-2. TGF- beta and Aza can make the initial CD4+CD25-T cells in the mouse spleen lymphocyte culture system differentiate into CD4+CD25+Treg cells. Meanwhile, the level of Foxp3mRNA expression rises. 2. Aza induced initial CD4+CD25-T in different doses of Aza. The number of.Foxp3mRNA cells differentiated into CD4+CD25+Foxp3+Treg cells was also different. (3) the CD4+CD25+Foxp3+Treg ratio and Foxp3mRNA expression level in the lymphocyte culture system of 5uM Aza group were most significantly higher than those of other concentration groups, and the difference was statistically significant (P0.05). (2) in vivo experimental results showed that (1) Aza made Seventh days after the injection of the penis dorsal vein, the number of CD4+CD25+Treg cells in the peripheral blood of mice increased and the expression level of Foxp3mRNA in the spleen increased. (2) different doses of Aza had different effects on the number of CD4+CD25+Treg cells in peripheral blood and the expression level of Foxp3mRNA in the spleen of mice. (3) CD4+CD25+F in Aza group was given to the peripheral blood of mice in Aza group according to the 4ug/g. weight /day dose. The ratio of oxp3+Treg and spleen Foxp3mRNA expression increased most significantly compared with other experimental groups, and there was a significant difference (P0.05).
Conclusion: in this experiment, both in vivo and in vitro, the methyltransferase inhibitor 5- nitrogen -2 '- deoxycytidine (5-aza-2'-deoxycytidine, Aza), namely, Decitabine, can induce the initial CD4+CD25T cells to differentiate into CD4+CD25+Treg and the level of Foxp3mRNA expression, but Aza to CD4+CD25+Foxp3+Treg cells at different doses. In vivo, mice were given Aza according to the dose of 4ug/g/d, and the proliferation and differentiation of CD4+CD25+Treg cells were most obvious in vitro, with the concentration of 5uM Aza.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.4

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