血液ー胸腺関門の分子機構と免疫学的機能の解明

(1)异常条件下Cld 5基因敲除小鼠胸腺细胞数量和T细胞发育的分析Cld 5基因敲除小鼠出生后很快死亡。除了在研究的第一年对Cld 5 KO小鼠进行分析外，我还比较了暴露于LPS后Cld 5 KO和对照野生型胎儿的胸腺细胞表型，以检查BTB是否在异常条件下保护胸腺微环境中发挥作用。我还将一种应激激素地塞米松注射到具有Cld 5 KO胎儿的Cld 5 +/-× Cld 5 +/-妊娠小鼠中，然而，Cld 5 KO小鼠和对照小鼠的胸腺细胞发育均被地塞米松损害。(2)用OT-I-TCR Tg Cld 5 KO小鼠检测BTB破坏对T细胞选择的影响虽然我发现静脉注射卵蛋白肽（257-264）可以影响OT-I-TCR Tg胎儿的胸腺细胞发育，但很难收集足够的OT-I-TCR Cld 5 KO胎儿以得到稳定可靠的结论。因此，该测定系统不适合于检查BTB破坏的影响。(3)流式细胞术检测胸腺血管内皮细胞（VECs）的表型在建立流式细胞术检测Cld 5表达的方法后，发现Cld 5+胸腺VECs的比例随着年龄的增长而降低。为了了解Cld 5+和Cld 5- VECs的差异，我将Cld 5与各种血管内皮标志物共染色，发现Ly 6c的表达与胸腺VECs上Cld 5的表达有很好的相关性，尽管其生理意义仍有待验证。

(1) Analysis of the thymocyte number and T-cell development in Cld5 KO mice in abnormal conditionsCld5 KO mice die soon after birth. In addition to the analysis of Cld5 KO mice in the first year of the research, I compared thymocyte phenotypes of Cld5 KO and control wild type fetuses after the exposure of LPS to examine whether BTB plays a role in protecting thymic microenvironment in an abnormal condition. I also injected dexamethasone, a kind of stress hormones, into the pregnant Cld5+/-× Cld5+/- mice with Cld5 KO fetuses, however, thymocyte development of both Cld5 KO and control mice were impaired by dexamethasone.(2) Examine the effects of BTB disruption on T-cell selection using OT-I-TCR Tg Cld5 KO miceAlthough I found that intravenous injection of ova-peptide (257-264) into pregnant mice could affect the thymocyte development of OT-I-TCR Tg fetuses, it is hard to collect enough OT-I-TCR Cld5 KO fetuses to get stable and reliable conclusion. Thus, this assay system was not suitable to examine the effects of BTB disruption.(3) Phenotypic analysis of thymic vascular endothelial cells (VECs) by flow cytometryAfter establishing the method to examine Cld5 expression by FACS, I found that the proportion of Cld5+ thymic VECs decreases with age. To understand the differences of Cld5+ and Cld5- VECs, I co-stained Cld5 with various vascular endothelial markers and found that the expression of Ly6c is well correlated to Cld5 expression on thymic VECs, although its physiological significance remains to be verified.