CD患者外周血Treg及lncRNA DQ786243作用的相关研究

发布时间：2019-05-27 01:43

【摘要】：目的：以外周血Treg为研究对象，，在克罗恩病患者中研究Treg功能、表型的变化，以及LncRNA DQ786243对Treg控制基因Foxp3表达的影响。分析Treg数量与CD患者特征的关系。 方法：使用磁珠对外周血CD4+CD25+Treg及CD4+CD25-Teff进行分选，用CFSE稀释法研究患者与健康对照Treg与Teff共培养时Treg抑制功能的差异。采用流式荧光抗体标记法，分别检测患者与健康对照者的CD4+Foxp3+Treg细胞及CD4+CD45RA-Foxp3+Treg细胞的数量差异。通过qRT-PCR检测患者与健康对照者之间lncRNA DQ786243、CREB及Foxp3的表达，并与结合临床数据进行相关性分析。通过在Jurkat细胞中转染lncRNADQ786243观察CREB、Foxp3的表达变化，以及CREB磷酸化水平的变化。扩大样本，通过检测CD患者与健康对照者外周血Foxp3TSDR去甲基化水平，了解Treg在CD患者与健康对照组中的数量差异，以及Treg在CD不同治疗及不同临床特征下的差异。 结果：Treg与Teff共培养时，CD患者中Teff在24小时内的平均增殖率为与对照组相比，两者在统计学上无显著差异。CD4+CD45RA-Foxp3hiaTreg的比例在活动期CD患者减少（P 0.01）。定量RT-PCR结果显示，DQ786243的表达在活动期CD患者中显著升高(P=0.004)。CRP与DQ786243存在良好的相关性（r=0.489, P=0.034）。DQ786243与Foxp3的表达存在一定的线性相关性（r=0.435,P=0.021）。CREB和Foxp3之间无明显的相关性。在DQ786243转染48小时之后，Foxp3的mRNA表达水平在Jurkat细胞中显著升高(P=0.046)，p-CREB/t-CREB在转染后24小时及48小时出现增加（P=0.0043）。缓解期患者TSDR去甲基化的比例与活动期患者及健康对照者之间比较未发现显著差异。TSDR去甲基化的比例肛周病变患者中升高（T-Test，P=0.0036）。在DQ786243转染后TSDR去甲基化水平出现了升高，且具有统计学意义（ANOVA, P=0.0010）。 结论：正常对照与CD患者外周血Treg的抑制功能无显著差异。采用CD4+CD45RA-Foxp3hi作为流式标志发现CD患者aTreg数量低于正常对照。DQ786243与CD患者的疾病活动性有关并可以影响CREB及Foxp3的表达。CREB本身并不介导DQ786243上调Foxp3的表达，这一过程可能通过CREB的磷酸化来完成。目前尚无足够的证据显示CD患者外周血Treg数量与正常人之间存在差异。Treg的数量在具有肛周病变的CD患者中出现升高。LncRNADQ786243在Jurkat体外实验中可以影响Foxp3TSDR去甲基化的水平，这可能是DQ786243调节体内Foxp3表达的另一机制。
[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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