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  本文關鍵詞：前列腺素E2和白三烯B4對調(diào)節(jié)性T細胞分化的影響　出處：《河北醫(yī)科大學》2011年碩士論文　論文類型：學位論文

  更多相關文章： CD4~+T細胞 調(diào)節(jié)性T細胞 FOXP3 前列腺素E2 白三烯B4

【摘要】：目的:初始CD4~+T細胞接受抗原刺激后,首先分化為Th0細胞,在不同的細胞因子作用下,可分化為Th1、Th2、Th17及Treg(regulatory T cells, Treg)細胞,發(fā)揮不同的生物學作用。Treg細胞具有免疫抑制作用,主要功能是維持免疫耐受和免疫穩(wěn)態(tài),并與類風濕關節(jié)炎(rheumatoid arthritis, RA)密切相關。Treg細胞是由CD4~+T細胞在TGF-β的作用下分化而來,而叉狀/翼狀螺旋轉(zhuǎn)錄因子(forkhead/wingedhelix transcription factor, FOXP3)是檢測CD4~+T細胞向Treg細胞分化程度的特異性轉(zhuǎn)錄因子。 前列腺素E2(Prostaglandin E2, PGE2)和白三烯B4(leukotriene B4, LTB4)均為花生四烯酸的代謝產(chǎn)物,是RA病程中的重要炎癥介質(zhì)。目前,臨床上多用非甾體類抗炎藥治療RA,其作用機制正是通過抑制環(huán)氧化酶進而抑制PGE2的生成。有研究表明,應用LTB4受體阻斷劑可以治療RA。本室以往研究證實,PGE2通過前列腺素受體EP2和EP4抑制Th0細胞向Treg細胞分化,參與機體的免疫調(diào)節(jié)。LTB4可抑制Th0分化為Treg細胞,且抑制膠原誘導的關節(jié)炎(collagen induced arthritis,CIA)小鼠Th0細胞分化為Treg細胞,參與機體免疫調(diào)節(jié)。那么,PGE2和LTB4是否可以調(diào)節(jié)人CD4~+T細胞向Treg細胞的分化,目前尚無文獻報道,因此本課題以人外周血CD4~+T細胞為研究對象,觀察PGE2和LTB4對Treg細胞分化的影響,進而確定PGE2和LTB4是否通過調(diào)節(jié)Treg細胞的分化,參與人體的免疫調(diào)節(jié),以期進一步揭示二者在RA中的免疫調(diào)節(jié)作用。 方法: 1 PGE2對Treg細胞分化的調(diào)節(jié) (1)免疫磁珠法分選人外周血CD4~+T細胞,流式細胞術檢測分選細胞的純度;收集分選后的細胞,接種于anti-CD3預先包被的24孔板,加入anti-CD28和TGF-β1,誘導CD4~+T細胞向Treg細胞分化,于不同時間收集細胞,觀察FOXP3 mRNA的時間依從性表達;收集分選后的細胞,接種于anti-CD3預先包板的24孔板,加入anti-CD28、TGF-β1和IL-2,7d后收集細胞,流式細胞術檢測CD~4+CD25~+FOXP3~+細胞數(shù)量。 (2)不同濃度PGE2對Treg細胞分化的影響。PGE2對Treg細胞FOXP3 mRNA表達的影響:分為四組,對照組和PGE2(0.1μM、1μM、10μM)組,孵育36h,收集細胞,觀察FOXP3 mRNA的表達情況;不同時間10μM PGE2對Treg細胞分化的影響:分為對照組和PGE2 10μM組,于不同時間收集細胞,觀察FOXP3 mRNA的表達情況;PGE2對CD~4+CD25~+ FOXP3~+細胞數(shù)量的影響:分為四組,對照組和PGE2 (0.1μM、1μM、10μM)組,培養(yǎng)7d,收集細胞,流式細胞術觀察CD4~+CD25~+FOXP3~+細胞的數(shù)量。 2 LTB4對Treg細胞分化的調(diào)節(jié) LTB4對Treg細胞FOXP3 mRNA表達的影響:分為四組,對照組和LTB4 (0.01μM、0.1μM、1μM)組,孵育36h,收集細胞,觀察FOXP3 mRNA的表達情況;LTB4對CD~4+CD25~+FOXP3~+細胞數(shù)量的影響:分為四組,對照組和LTB4 (0.01μM、0.1μM、1μM)組,培養(yǎng)7d,流式細胞術檢測CD4~+ CD25~+FOXP3~+細胞的數(shù)量。 應用SPSS13.0軟件進行統(tǒng)計學分析,數(shù)據(jù)用均數(shù)±標準差(x±s)表示,各組均數(shù)的比較行單因素方差分析(one-way ANOVA),用最小顯著差法(least significant difference, LSD)作兩兩比較,P0.05為有顯著性差異。 結(jié)果: 1 PGE2對Treg細胞分化的調(diào)節(jié) (1)經(jīng)磁珠分選后,流式細胞儀檢測CD4~+T細胞的純度達97%以上;在anti-CD3和anti-CD28,TGF-β1作用下Treg細胞關鍵轉(zhuǎn)錄因子FOXP3 mRNA在36h達表達高峰;在anti-CD3和anti-CD28存在的條件下,TGF-β1和IL-2作用7d后,可使(72.9533±17.2270)%的細胞同時表達CD4、CD25和FOXP3,證明體外成功誘導CD4~+T細胞分化為Treg細胞。 (2)不同濃度的PGE2在CD4~+T細胞誘導分化為Treg細胞36h時均對FOXP3 mRNA的表達有抑制作用,且呈劑量依賴性,PGE2(0.1μM、1μM、10μM)組(0.8544±0.0745、0.6972±0.1047、0.2220±0.0377)與對照組(1.0000±0.0000)相比差異均有統(tǒng)計學意義(P0.05);不同時間10μM PGE2對Treg細胞FOXP3 mRNA的表達均有抑制作用,且在7d時抑制作用最明顯,5d和7d時,PGE2 10μM組(4.1127±0.5213、3.6510±0.5057)與對照組(10.0970±1.7363、21.0310±4.1888)相比,有顯著差異(P0.05);PGE2作用7d,流式細胞術檢測CD4~+CD25~+FOXP3~+細胞的數(shù)量:發(fā)現(xiàn)PGE2(0.1、1、10μM)組均可減少CD4~+CD25~+FOXP3~+細胞的數(shù)量,且呈劑量依賴性,PGE2 1μM和10μM組(41.4933±9.5664、23.9367±5.3066)%與對照組(72.9533±17.2270)%相比差異有統(tǒng)計學意義(P0.05)。 上述結(jié)果顯示PGE2抑制CD4~+T細胞向Treg細胞的分化。2 LTB4對Treg細胞分化的調(diào)節(jié) 不同濃度的LTB4在CD4~+T細胞誘導分化為Treg細胞36h時,LTB4 (0.01μM、0.1μM、1μM)組(0.9757±0.2335、1.0058±0.1900、1.0779±0.2348)與對照組(1.0000±0.0000)相比,差異無統(tǒng)計學意義(P0.05);LTB4作用7d后,應用流式細胞術檢測CD4~+CD25~+FOXP3~+細胞的數(shù)量,發(fā)現(xiàn)LTB4 (0.01μM、0.1μM、1μM)各組(78.7000±12.9752、76.5200±9.9611、77.8133±13.8570)%與對照組(72.9533±17.2270)%相比,無顯著差異(P 0.05)。 上述結(jié)果顯示LTB4對CD4~+T細胞向Treg細胞的分化無明顯影響。 結(jié)論: (1)通過磁珠分選人外周血CD4~+T細胞,成功誘導其向Treg細胞分化,首次證實PGE2對Treg細胞的分化有抑制作用,且呈劑量依賴性。 (2)本實驗未檢測到LTB4對Treg細胞的分化有調(diào)節(jié)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.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

【共引文獻】

相關博士學位論文 前1條

1 張綱;bFGF、rhBMP-2聚乳酸納米微球促進下頜骨骨折愈合的實驗研究[D];第三軍醫(yī)大學;2007年

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