【摘要】：目的：以外周血Treg為研究對象，，在克羅恩病患者中研究Treg功能、表型的變化，以及LncRNA DQ786243對Treg控制基因Foxp3表達的影響。分析Treg數(shù)量與CD患者特征的關(guān)系。 方法：使用磁珠對外周血CD4+CD25+Treg及CD4+CD25-Teff進行分選，用CFSE稀釋法研究患者與健康對照Treg與Teff共培養(yǎng)時Treg抑制功能的差異。采用流式熒光抗體標(biāo)記法，分別檢測患者與健康對照者的CD4+Foxp3+Treg細胞及CD4+CD45RA-Foxp3+Treg細胞的數(shù)量差異。通過qRT-PCR檢測患者與健康對照者之間lncRNA DQ786243、CREB及Foxp3的表達，并與結(jié)合臨床數(shù)據(jù)進行相關(guān)性分析。通過在Jurkat細胞中轉(zhuǎn)染lncRNADQ786243觀察CREB、Foxp3的表達變化，以及CREB磷酸化水平的變化。擴大樣本，通過檢測CD患者與健康對照者外周血Foxp3TSDR去甲基化水平，了解Treg在CD患者與健康對照組中的數(shù)量差異，以及Treg在CD不同治療及不同臨床特征下的差異。 結(jié)果：Treg與Teff共培養(yǎng)時，CD患者中Teff在24小時內(nèi)的平均增殖率為與對照組相比，兩者在統(tǒng)計學(xué)上無顯著差異。CD4+CD45RA-Foxp3hiaTreg的比例在活動期CD患者減少（P 0.01）。定量RT-PCR結(jié)果顯示，DQ786243的表達在活動期CD患者中顯著升高(P=0.004)。CRP與DQ786243存在良好的相關(guān)性（r=0.489, P=0.034）。DQ786243與Foxp3的表達存在一定的線性相關(guān)性（r=0.435,P=0.021）。CREB和Foxp3之間無明顯的相關(guān)性。在DQ786243轉(zhuǎn)染48小時之后，F(xiàn)oxp3的mRNA表達水平在Jurkat細胞中顯著升高(P=0.046)，p-CREB/t-CREB在轉(zhuǎn)染后24小時及48小時出現(xiàn)增加（P=0.0043）。緩解期患者TSDR去甲基化的比例與活動期患者及健康對照者之間比較未發(fā)現(xiàn)顯著差異。TSDR去甲基化的比例肛周病變患者中升高（T-Test，P=0.0036）。在DQ786243轉(zhuǎn)染后TSDR去甲基化水平出現(xiàn)了升高，且具有統(tǒng)計學(xué)意義（ANOVA, P=0.0010）。 結(jié)論：正常對照與CD患者外周血Treg的抑制功能無顯著差異。采用CD4+CD45RA-Foxp3hi作為流式標(biāo)志發(fā)現(xiàn)CD患者aTreg數(shù)量低于正常對照。DQ786243與CD患者的疾病活動性有關(guān)并可以影響CREB及Foxp3的表達。CREB本身并不介導(dǎo)DQ786243上調(diào)Foxp3的表達，這一過程可能通過CREB的磷酸化來完成。目前尚無足夠的證據(jù)顯示CD患者外周血Treg數(shù)量與正常人之間存在差異。Treg的數(shù)量在具有肛周病變的CD患者中出現(xiàn)升高。LncRNADQ786243在Jurkat體外實驗中可以影響Foxp3TSDR去甲基化的水平，這可能是DQ786243調(diào)節(jié)體內(nèi)Foxp3表達的另一機制。
[Abstract]:Aim: to study the function and phenotypic changes of Treg in peripheral blood Treg patients with Crohn's disease, and the effect of LncRNA DQ786243 on the expression of Treg control gene Foxp3. The relationship between the number of Treg and the characteristics of CD patients was analyzed. Methods: magnetic beads were used to isolate CD4 CD25 Treg and CD4 CD25-Teff in peripheral blood. CFSE dilution method was used to study the difference of Treg inhibitory function between patients and healthy controls in co-culture of Treg and Teff. Flow fluorescence antibody labeling was used to detect the number of CD4 Foxp3 Treg cells and CD4 CD45RA-Foxp3 Treg cells between patients and healthy controls. The expression of lncRNA DQ786243,CREB and Foxp3 between patients and healthy controls was detected by qRT-PCR, and the correlation between them and clinical data was analyzed. The expression of CREB,Foxp3 and the level of CREB phosphorylation were observed by transfection of lncRNADQ786243 into Jurkat cells. The Foxp3TSDR demethylation level of peripheral blood between CD patients and healthy controls was measured to investigate the quantitative difference of Treg in CD patients and healthy controls, and the difference of Treg in different treatments and clinical features of CD. Results: when Treg and Teff were co-cultured, the average proliferation rate of Teff in CD patients within 24 hours was not significantly different from that in the control group, but the proportion of CD4 CD45RA-Foxp3hiaTreg decreased in active CD patients. The results of quantitative RT-PCR showed that the expression of DQ786243 was significantly increased in patients with active CD (P 鈮

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