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)表面Toll样受体(TLR)家族的表达。从分离的T-reg组分和非T-reg组分中提取总RNA，用实时荧光定量聚合酶链式反应(Real Time PCR)检测TLR2、3、4、6、7、8、9的mRNA含量。对照组循环髓系树突状细胞(MDC)和浆细胞样树突状细胞(PDC)表达TLR3、6、7、9，而AIH患者仅表达TLR6。(2)AIH患者树突状细胞表面TLRs的表达及对肿瘤坏死因子受体1诱导的细胞凋亡的易感性。提取循环髓系树突状细胞(MDC)和浆细胞样树突状细胞(PDC)的总RNA，用实时荧光定量聚合酶链式反应(Real-time PCR)检测肿瘤坏死因子-R1、肿瘤坏死因子Rs18、Fas、Bcl2、Bax、Bad、FAST、PKR、APAF-1和c-fllip的表达。此外，将mDC+pDC与肿瘤坏死因子α共同孵育后，通过计数凋亡树突状细胞的数量来评估其对肿瘤坏死因子-1诱导的细胞凋亡的敏感性。AIH组和对照组新鲜分离的MDC和PDC上均有肿瘤坏死因子受体18、肿瘤坏死因子受体1、Bad和FAST的表达。而AIH组肿瘤坏死因子-R1和Bad的表达明显低于对照组。与肿瘤坏死因子α共同孵育72小时后，对照组MDC和PDC的凋亡率从30.0%增加到45.8%，而AIH组的凋亡率从33.3%增加到34.5%，提示AIH可能通过肿瘤坏死因子-R1信号诱导的MDC和PDC的凋亡受到抑制。

(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.