前列腺素E2和白三烯B4对调节性T细胞分化的影响

发布时间：2018-01-05 16:27

本文关键词：前列腺素E2和白三烯B4对调节性T细胞分化的影响　出处：《河北医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:初始CD4~+T细胞接受抗原刺激后,首先分化为Th0细胞,在不同的细胞因子作用下,可分化为Th1、Th2、Th17及Treg(regulatory T cells, Treg)细胞,发挥不同的生物学作用。Treg细胞具有免疫抑制作用,主要功能是维持免疫耐受和免疫稳态,并与类风湿关节炎(rheumatoid arthritis, RA)密切相关。Treg细胞是由CD4~+T细胞在TGF-β的作用下分化而来,而叉状/翼状螺旋转录因子(forkhead/wingedhelix transcription factor, FOXP3)是检测CD4~+T细胞向Treg细胞分化程度的特异性转录因子。前列腺素E2(Prostaglandin E2, PGE2)和白三烯B4(leukotriene B4, LTB4)均为花生四烯酸的代谢产物,是RA病程中的重要炎症介质。目前,临床上多用非甾体类抗炎药治疗RA,其作用机制正是通过抑制环氧化酶进而抑制PGE2的生成。有研究表明,应用LTB4受体阻断剂可以治疗RA。本室以往研究证实,PGE2通过前列腺素受体EP2和EP4抑制Th0细胞向Treg细胞分化,参与机体的免疫调节。LTB4可抑制Th0分化为Treg细胞,且抑制胶原诱导的关节炎(collagen induced arthritis,CIA)小鼠Th0细胞分化为Treg细胞,参与机体免疫调节。那么,PGE2和LTB4是否可以调节人CD4~+T细胞向Treg细胞的分化,目前尚无文献报道,因此本课题以人外周血CD4~+T细胞为研究对象,观察PGE2和LTB4对Treg细胞分化的影响,进而确定PGE2和LTB4是否通过调节Treg细胞的分化,参与人体的免疫调节,以期进一步揭示二者在RA中的免疫调节作用。方法: 1 PGE2对Treg细胞分化的调节 (1)免疫磁珠法分选人外周血CD4~+T细胞,流式细胞术检测分选细胞的纯度;收集分选后的细胞,接种于anti-CD3预先包被的24孔板,加入anti-CD28和TGF-β1,诱导CD4~+T细胞向Treg细胞分化,于不同时间收集细胞,观察FOXP3 mRNA的时间依从性表达;收集分选后的细胞,接种于anti-CD3预先包板的24孔板,加入anti-CD28、TGF-β1和IL-2,7d后收集细胞,流式细胞术检测CD~4+CD25~+FOXP3~+细胞数量。 (2)不同浓度PGE2对Treg细胞分化的影响。PGE2对Treg细胞FOXP3 mRNA表达的影响:分为四组,对照组和PGE2(0.1μM、1μM、10μM)组,孵育36h,收集细胞,观察FOXP3 mRNA的表达情况;不同时间10μM PGE2对Treg细胞分化的影响:分为对照组和PGE2 10μM组,于不同时间收集细胞,观察FOXP3 mRNA的表达情况;PGE2对CD~4+CD25~+ FOXP3~+细胞数量的影响:分为四组,对照组和PGE2 (0.1μM、1μM、10μM)组,培养7d,收集细胞,流式细胞术观察CD4~+CD25~+FOXP3~+细胞的数量。 2 LTB4对Treg细胞分化的调节 LTB4对Treg细胞FOXP3 mRNA表达的影响:分为四组,对照组和LTB4 (0.01μM、0.1μM、1μM)组,孵育36h,收集细胞,观察FOXP3 mRNA的表达情况;LTB4对CD~4+CD25~+FOXP3~+细胞数量的影响:分为四组,对照组和LTB4 (0.01μM、0.1μM、1μM)组,培养7d,流式细胞术检测CD4~+ CD25~+FOXP3~+细胞的数量。应用SPSS13.0软件进行统计学分析,数据用均数±标准差(x±s)表示,各组均数的比较行单因素方差分析(one-way ANOVA),用最小显著差法(least significant difference, LSD)作两两比较,P0.05为有显著性差异。结果: 1 PGE2对Treg细胞分化的调节 (1)经磁珠分选后,流式细胞仪检测CD4~+T细胞的纯度达97%以上;在anti-CD3和anti-CD28,TGF-β1作用下Treg细胞关键转录因子FOXP3 mRNA在36h达表达高峰;在anti-CD3和anti-CD28存在的条件下,TGF-β1和IL-2作用7d后,可使(72.9533±17.2270)%的细胞同时表达CD4、CD25和FOXP3,证明体外成功诱导CD4~+T细胞分化为Treg细胞。 (2)不同浓度的PGE2在CD4~+T细胞诱导分化为Treg细胞36h时均对FOXP3 mRNA的表达有抑制作用,且呈剂量依赖性,PGE2(0.1μM、1μM、10μM)组(0.8544±0.0745、0.6972±0.1047、0.2220±0.0377)与对照组(1.0000±0.0000)相比差异均有统计学意义(P0.05);不同时间10μM PGE2对Treg细胞FOXP3 mRNA的表达均有抑制作用,且在7d时抑制作用最明显,5d和7d时,PGE2 10μM组(4.1127±0.5213、3.6510±0.5057)与对照组(10.0970±1.7363、21.0310±4.1888)相比,有显著差异(P0.05);PGE2作用7d,流式细胞术检测CD4~+CD25~+FOXP3~+细胞的数量:发现PGE2(0.1、1、10μM)组均可减少CD4~+CD25~+FOXP3~+细胞的数量,且呈剂量依赖性,PGE2 1μM和10μM组(41.4933±9.5664、23.9367±5.3066)%与对照组(72.9533±17.2270)%相比差异有统计学意义(P0.05)。上述结果显示PGE2抑制CD4~+T细胞向Treg细胞的分化。2 LTB4对Treg细胞分化的调节不同浓度的LTB4在CD4~+T细胞诱导分化为Treg细胞36h时,LTB4 (0.01μM、0.1μM、1μM)组(0.9757±0.2335、1.0058±0.1900、1.0779±0.2348)与对照组(1.0000±0.0000)相比,差异无统计学意义(P0.05);LTB4作用7d后,应用流式细胞术检测CD4~+CD25~+FOXP3~+细胞的数量,发现LTB4 (0.01μM、0.1μM、1μM)各组(78.7000±12.9752、76.5200±9.9611、77.8133±13.8570)%与对照组(72.9533±17.2270)%相比,无显著差异(P 0.05)。上述结果显示LTB4对CD4~+T细胞向Treg细胞的分化无明显影响。结论: (1)通过磁珠分选人外周血CD4~+T细胞,成功诱导其向Treg细胞分化,首次证实PGE2对Treg细胞的分化有抑制作用,且呈剂量依赖性。 (2)本实验未检测到LTB4对Treg细胞的分化有调节作用。
[Abstract]:Objective: the initial CD4~+T cells after antigen stimulation, the differentiation of Th0 cells, the cell factor under different can be divided into Th1, Th2, Th17 and Treg (regulatory T cells, Treg cells,.Treg cells) play different biological effects have immunosuppressive effects, the main function is to maintain the immune tolerance and immune homeostasis and, in patients with rheumatoid arthritis (rheumatoid arthritis, RA) is closely related to.Treg cells by CD4~+T cells in the TGF- beta differentiation, and fork / winged helix transcription factor (forkhead/wingedhelix transcription, factor, FOXP3) is to detect CD4~+T cell specific transcription factor of Treg cell differentiation.
Prostaglandin E2 (Prostaglandin E2 PGE2) and B4 (leukotriene B4 LTB4, leukotrienes) are four arachidonic acid metabolites, are important mediators of inflammation in the course of the RA. At present, most of the clinical use of non steroidal anti-inflammatory drugs for the treatment of RA, its mechanism is generated by inhibiting cyclooxygenase inhibiting PGE2. Studies have shown that the application of LTB4 receptor antagonist RA. can treat our previous studies confirmed that PGE2 receptor EP2 and EP4 inhibition by prostaglandin Th0 cells to differentiate into Treg cells, involved in immune regulation of.LTB4 can inhibit the differentiation of Th0 into Treg cells, and the inhibition of collagen induced arthritis (collagen induced, arthritis, CIA) differentiation mouse Th0 cells into Treg cells involved in immune regulation. Then, whether PGE2 and LTB4 can regulate the differentiation of CD4~+T cells into Treg cells, there is no literature reports, so this topic in human peripheral blood CD4~+T Cell as the research object, we observed the effect of PGE2 and LTB4 on the differentiation of Treg cells, and further determined whether PGE2 and LTB4 participated in the regulation of Treg cells by regulating the differentiation of Treg cells, in order to further reveal the two immunoregulatory functions in RA.
Method:
Regulation of 1 PGE2 on Treg cell differentiation
(1) immunomagnetic beads human peripheral blood CD4~+T cells, detect the purity of sorted cells by flow cytometry; collected sorted cells were inoculated on anti-CD3 pre coated 24 well plate, adding anti-CD28 and TGF- beta 1 induced CD4~+T cells to differentiate into Treg cells, cells were collected at different time, time dependence to observe the expression of FOXP3 of mRNA; collect the sorted cells, 24 hole plate, inoculated on anti-CD3 pre coated plate add anti-CD28, TGF- beta 1 and IL-2,7d cells were collected, the number of CD~4+CD25~+FOXP3~+ cells was detected by flow cytometry.
(2) effects of different concentrations of PGE2 on the differentiation of Treg cells and.PGE2 cells on the expression of Treg FOXP3 mRNA were divided into four groups, control group and PGE2 (0.1 M, 1 M, 10 M) group, 36h incubation, cells were collected to observe the expression of FOXP3 mRNA; effect of different time of 10 M PGE2 on the Treg cell differentiation: divided into PGE2 control group and 10 M group at different time were collected and the expression of FOXP3 was observed in mRNA; the effect of PGE2 on the number of CD~4+CD25~+ FOXP3~+ cells were divided into four groups, control group and PGE2 (0.1 M, 1 M, 10 M) group, cultured 7d cells, the collection, the number of CD4~+CD25~+FOXP3~+ cells were observed by flow cytometry.
Regulation of 2 LTB4 on Treg cell differentiation
Effect of LTB4 on the expression of Treg FOXP3 mRNA cells were divided into four groups, control group and LTB4 (0.01 M, 0.1 M, 1 M) group, 36h incubation, cells were collected to observe the expression of FOXP3 mRNA; the effect of LTB4 on CD~4+CD25~+FOXP3~+ cells were divided into four groups, control group and LTB4 (0.01 M, 0.1 M, 1 M) group, 7d medium, flow cytometry was used to detect the number of CD4~+ CD25~+FOXP3~+ cells.
SPSS13.0 software was used for statistical analysis. The data were expressed by mean + standard deviation (x + s). The mean number of each group was compared with one-way ANOVA (one-way ANOVA), and the 22 difference was compared with the smallest significant difference method (least significant difference, LSD). P0.05 showed significant difference.
Result:
Regulation of 1 PGE2 on Treg cell differentiation
(1) by magnetic separation, the purity of CD4~+T cells were detected by flow cytometry for more than 97%; in anti-CD3 and anti-CD28, TGF- beta 1 Treg under the action of the key transcription factor FOXP3 of mRNA cells in 36h reached the peak of expression; in the presence of anti-CD3 and anti-CD28, TGF- beta 1 and IL-2 7d, can make (72.9533 + 17.2270)% of the cells expressed CD4, CD25 and FOXP3, proved successful in vitro differentiation of CD4~+T cells induced by Treg cells.
(2) different concentrations of PGE2 in the differentiation of CD4~+T cells into Treg cells at 36h of FOXP3 mRNA expression was inhibited, in a dose-dependent manner, PGE2 (0.1 M, 1 M, 10 M) group (0.8544 + 0.0745,0.6972 + 0.1047,0.2220 + 0.0377) and control group (1 + 0) had a significant difference (P0.05); inhibition of expression in different time of 10 M PGE2 of Treg FOXP3 mRNA cells, and the most obvious inhibition in 7d, 5D and 7d, PGE2 10 M group (4.1127 + 0.5213,3.6510 + 0.5057) and control group (10.0970 + 1.7363,21.0310 + 4.1888) compared, there was significant difference (P0.05); PGE2 7d, flow cytometry was used to detect the number of CD4~+CD25~+FOXP3~+ cells: PGE2 (0.1,1,10 M) group can reduce the number of CD4~+CD25~+FOXP3~+ cells in a dose-dependent manner, PGE2 1 M and 10 M group (41.4933 + 9.5664,23.9367 + 5.3066) and control group (% 72.9533 + 17. 2270)% of the difference was statistically significant (P0.05).
These results suggest that PGE2 inhibits the differentiation of CD4~+T cells from Treg cells to the differentiation of.2 LTB4 to Treg cells
Different concentrations of LTB4 in the differentiation of CD4~+T cells into Treg cells 36h, LTB4 (0.01 M, 0.1 M, 1 M) group (0.9757 + 0.2335,1.0058 + 0.1900,1.0779 + 0.2348) and control group (1 + 0) compared to the difference was not statistically significant (P0.05); LTB4 7d, should be used flow cytometry was used to detect the number of CD4~+CD25~+FOXP3~+ cells and found that LTB4 (0.01 M, 0.1 M, 1 M) groups (78.7000 + 12.9752,76.5200 + 9.9611,77.8133 + 13.8570)% and the control group (72.9533 + 17.2270)% compared with no significant difference (P 0.05).
The above results showed that LTB4 had no significant effect on the differentiation of CD4~+T cells to Treg cells.
Conclusion:
(1) CD4~+T cells from peripheral blood were successfully induced by magnetic beads to differentiate into Treg cells. For the first time, PGE2 was found to inhibit the differentiation of Treg cells in a dose-dependent manner.
(2) the effect of LTB4 on the differentiation of Treg cells was not detected in this experiment.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【共引文献】