Investigation of novel therapeutic approach through regulatory T cells (Tr) and dendritic cells (DCs) for the patients with autoimmune hepatitis (AIH)

(1)自身免疫性肝炎（AIH）患者树突状细胞（dc）上toll样受体（TLR）家族的表达。从分离的T-reg和非T-reg部分提取总RNA，并通过实时荧光定量PCR检测TLR2、3、4、6、7、8、9的mRNA含量。TLR3、6、7、9在对照组的循环髓样DC （MDC）和浆细胞样DC （PDC）上表达，而AIH患者中只有tlr6表达。因此，TLRs在dc中的不同表达模式可能对AIH的发病机制有一定影响。(2) AIH患者dc上凋亡相关分子的表达及dc对TNF-R1诱导的凋亡的易感性。提取循环髓细胞DC （MDC）和浆细胞样DC （PDC）的总RNA，采用实时荧光定量PCR检测细胞中TNF-R1、TNF-RS18、Fas、Bcl-2、Bax、Bad、FAST、PKR、Apaf-1和c-FLIP的mRNA表达量。进一步，在TNFα存在下孵育MDC+PDC后，通过计数凋亡dc的数量来评估TNF-R1诱导的凋亡敏感性。在AIH和对照组中，新鲜分离的MDC和PDC上均表达TNF-RS18、TNF-R1、Bad和FAST。然而，与对照组相比，AIH中TNF-R1和Bad的表达明显受到抑制。TNFα作用72 h后，对照组细胞中凋亡的MDC和PDC数量从30.0%增加到45.8%，AIH细胞中凋亡的MDC和PDC数量从33.3%增加到34.5%，提示AIH细胞中TNF-R1信号诱导的MDC和PDC凋亡可能受到抑制。

(1) Expression of toll-like receptor (TLR) family on Dendritic cells (DCs) in patients with autoimmune hepatitis (AIH). Total RNA was extracted from isolated T-reg and non-T-reg fractions and the amount of mRNA for TLR2, 3, 4, 6, 7, 8, 9were evaluated by real time PCR. TLR3, 6, 7, 9were expressed on circulating myeloid DC (MDC) and plasmacytoid DC (PDC) in controls, while only TLR6was expressed on those in patients with AIH. Thus different expression pattern of TLRs on DCs might have some influence on the pathogenesis of AIH.(2) Expression of apoptosis-related molecules on DCs, and susceptibility of DCs to apoptosis induced through TNF-R1 in patients with AIH.Total RNA was extracted from circulating myeloid DC (MDC) and plasmacytoid DC (PDC), the amount of mRNA for TNF-R1, TNF-RS18, Fas, Bcl-2, Bax, Bad, FAST, PKR, Apaf-1, and c-FLIP were evaluated by real time PCR. Further, after incubation of MDC+PDC in the presence of TNFα, susceptibility to apoptosis induced through TNF-R1 was evaluated by counting the number of apoptotic DCs. TNF-RS18, TNF-R1, Bad, and FAST were expressed on freshly isolated MDC and PDC both in AIH and control. However, expression of TNF-R1 and Bad was significantly suppressed in AIH compared with controls. After 72 hour incubation in the presence of TNFα, the amount of apoptotic MDC and PDC in controls increased from 30.0% to 45.8%, whereas that in AIH changed from 33.3% to 34.5%, suggesting the possibility that apoptosis of MDC and PDC induced by signals through TNF-R1 is suppressed in AIH.