【作者】 李超；

【导师】 周平；

【作者基本信息】 华中科技大学 ， 外科学， 2013， 博士

【摘要】 第一部分MyD88抑制剂TJ-M2010对树突状细胞生物学功能的影响[目的]探究新型MyD88抑制剂TJ-M2010对体外培养的小鼠骨髓来源树突状细胞共刺激分子表达、吞噬功能、迁移能力、凋亡、刺激T细胞增殖等生物学功能方面的影响。[方法]体外诱导培养得到BALB/c小鼠不成熟骨髓源性DC (bone marrow-derived dendritic cells, BMDCs),分为四组：空白组(不做任何处理),CpG刺激组,CPG刺激+低浓度TJ-M2010组(20μM),CpG刺激+高浓度TJ-M2010组(40μM)。孵育24小时后流式检测DC共刺激分子CD80、CD86及MHCⅡ和趋化因子受体CCR7的表达；westblot的方法检测NF-κB在细胞核内的表达；收集上述经不同浓度TJ-M2010处理的BALB/c BMDC,与CFSE标记的C57小鼠T细胞体外在CpG刺激下行混合淋巴细胞培养,3天后流式检测T细胞增殖情况。BALB/c BMDC中加入不同浓度TJ-M2010,利用FITC-Dextran法检测DC吞噬功能,利用Annexin-V-PI双染法检测DC凋亡及坏死情况。[结果]流式结果显示：DC经CpG刺激后高表达CD80/CD86/MHCⅡ (53.1±0.3%/55.6±3.5%/64.6±4.3%)。20μM、40μM TJ-M2010分别下降上述分子水平至(CD80:38.2%±2.2%,20.7%±1.1%; CD86:40.4%±1.6%,26.3±1.5%; MHCⅡ45.1%士2.4%，28.1士3.4%,p<0.05,vs CpG对照组)。DC经CpG刺激后CCR7表达率由25.43%上升至46.86%,20μM.40μM TJ-M2010分别使CCR7下降至36.07%和27.20%。TJ-M2010能够剂量依赖性的抑制由CpG引起的NF-κB p65的核转位。TJ-M2010能够剂量依赖性地抑制BMDC与同种反应性T细胞混合淋巴细胞培养中CD3+CFSE+T细胞比例。空白组DC吞噬FITC-Dextran阳性率为63.4%±3.4%,20μM、40μM TJ-M2010与DC共孵育后,其FITC-Dextran阳性率未见明显改变,分别为65.7%±2.6%和62.6%±2.6%(p>0.05,vs空白组)。空白组DC培养24小时后Annexin-V-PI双阳细胞比例为：6.6%士0.9%,20μM、40μM TJ-M2010与DC共孵育后,Annexin-V-PI双阳细胞比例未见明显改变,分别为：6.5%±1.1%和6.6%±1.1%(p>0.05,νs空白组)。[结论]TJ-M2010能够剂量依赖性地抑制CpG引起的DC的成熟、高迁移能力及NF-κB活化,并能够剂量依赖性地间接抑制同种反应性T淋巴细胞增殖；TJ-M2010不影响DC吞噬功能,对DC也为见明显细胞毒性作用。第二部分MyD88抑制剂TJ-M2010促进小鼠同种异基因心脏移植耐受及机制[目的]探讨MyD88抑制剂TJ-M2010在诱导小鼠同种异基因心脏移植耐受中的作用及其可能的机制。【方法】(1)建立BALB/c (H-2d)→C57BL/6(H-2b)的小鼠腹部心脏移植模型。实验分为三组：空白对照组,CMC溶媒对照组及TJ-M2010用药组。TJ-M2010用0.5%羧甲基纤维素钠(Carboxymethyl cellulose, CMC)混悬,腹腔注射给药；对照组使用相同剂量的0.5%CMC腹腔注射。手术当天记为Day0, TJ-M2010给药方案为：Day-2～Day7连续每天给药,剂量50mg/kg/天。术后每天扪诊移植心脏,观察移植物存活时间。(2)术后7天获取移植心,HE染色检测各组移植物病理改变；利用II型胶原酶消化移植物获得移植物中单个核细胞,流式检测CD11c+CD80+双阳细胞比例；实时定量荧光PCR检测移植心中IL-1、IL-6、TNF-α mRNA水平。(3)流式细胞术检测移植心长期存活小鼠脾脏中CD4+CD25+Foxp3+Treg细胞比例；分离各组脾脏细胞,INF-y ELISPOT检测各组脾脏细胞抗原反应性。(4)二次心脏移植：对移植物长期存活小鼠行供者来源二次颈部心脏移植,观察二次移植物存活时间。【结果】空白对照组(n=5)移植心存活时间MST为7.4±0.5天,CMC溶媒对照组(n=5)移植物MST为7.6±0.5天,无统计学差异(p>0.05)。而TJ-M2010组可使50%的移植心存活时间超过100天。对移植物长期存活的受者行二次心脏移植,受者可持续不排斥BALB/c心脏(>100天)。术后7天移植物病理显示TJ-M2010组淋巴细胞浸润、组织破坏等排斥反应明显轻于CMC对照组。实时定量荧光PCR示：与CMC溶媒对照组相比,TJ-M2010组移植心中IL-1、IL-6、TNF-α mRNA水平明显降低(p<0.05)。同时TJ-M2010组移植心中单个核细胞CD11c+CD80+双阳细胞比例明显低于CMC对照组。脾脏中Treg比例：CMC对照组Treg比例为6.43%,TJ-M2010组Treg比例升高至12.92%。INF-y ELISPOT显示：TJ-M2010组移植物长期存活小鼠脾脏细胞对C3H抗原反应正常(p>0.05,VS未做心脏移植的B6脾脏细胞),而对BALB/c抗原反应性明显降低(p<0.05,VS未做心脏移植的B6脾脏细胞)。[结论]单独短期应用MyD88抑制剂TJ-M2010能够诱导供者特异性小鼠心脏移植耐受,机制上可能与减轻炎性因子分泌、诱导调节性T细胞等有关。第三部分MyD88抑制剂TJ-M2010联合anti-CD154诱导小鼠同种异基因皮肤移植耐受[目的]探讨MyD88抑制剂TJ-M2010联合anti-CD154诱导小鼠同种异基因皮肤移植耐受的可能性及其机制。[方法]建立BALB/c (H-2d)→C57BL/6(H-2b)的小鼠皮肤移植模型。实验分为三组：TJ-M2010用药组,anti-CD154组,TJ-M2010+anti-CD154组。手术当天记为Day0, TJ-M2010给药方案为：Day-2～Day7, Day9, Day11, Day13, Day15,腹腔注射,剂量50mg/kg/天。Anti-CD154(MR1clone)给药方案：Day0～Day3, Day7, Day14,腹腔注射,剂量200ug/只/次。以移植皮片完全坏死判断为排斥时间点,观察各组移植物存活时间。对移植物长期存活小鼠行供者(BALB/c)及第三方(C3H)来源二次皮肤移植或心脏移植,观察二次移植物存活时间。分离移植物长期存活小鼠脾脏中单个核细胞,流式检测CD4+CD25+Foxp3+Treg细胞比例；INF-yELISPOT检测各组脾脏细胞对供者(BALB/c)及第三方(C3H)抗原反应性。[结果]TJ-M2010用药组及anti-CD154组皮肤移植物存活时间分别为(9.0±0.71)天与(9.6±0.55)天,两组之间无显著统计学差异(p>0.05),而TJ-M2010+anti-CD154组可使22%的皮肤移植物长期存活(>100天)。对移植物长期存活的小鼠行二次心脏移植,心脏移植物全部长期存活(>100天)；二次皮肤移植,BALB/c来源皮肤移植物存活时间为(16±1.4)天,C3H来源皮肤移植物存活时间为(13.5±0.7)天：行二次皮肤移植受体同时短期给予TJ-M2010,则BALB/c皮肤移植物存活时间可延长至41天、72天(n＝2)。脾脏中Treg比例：与对照组相比,TJ-M2010+anti-CD154组Treg比例明显升高。TJ-M2010+anti-CD154组移植物长期存活小鼠脾脏对C3H抗原反应正常(p>0.05,VS未做皮肤移植的t36脾脏细胞),而对BALB/c抗原反应性明显降低(p<0.05,VS未做皮肤移植的B6脾脏细胞)。[结论]MyD88抑制剂TJ-M2010联合anti-CD154可以诱导小鼠同种异基因皮肤移植耐受,此种耐受是一种不稳定的耐受状态,而MyD88分子在此种耐受的维持中发挥重要作用。更多还原

【Abstract】 Part I The effect of MyD88inhibitor TJ-M2010on the biological functions of dendritic cells.[Objective] To the effect of TJ-M2010on the biological functions of mice bone marrow-derived dentritic cells including the activation of NF-κB, the expression of co-stimulatory molecules, phagocytic function, migration ability, apoptosis, the ability to activate T cells proliferation.[Methods] Mice immature bone marrow-derived dendritic cells were induced and cultivated in vitro and the cells were divided to4groups:Normal control group (cells without any treatment), CpG group, CpG+TJ-M2010(low concentration,20μM) group, CpG+TJ-M2010(high concentration,40μM) group. After incubation for24hours, the activation of NF-κB was detected by western blot. The expression of CD80, CD86, MHCⅡand CCR7on DCs were detected by flow cytometry. C57T cells stained with CSFE were co-cultured with BALB/c DCs treated with TJ-M2010of different concentrations in the presence of CpG for3days, and CD3+CFSE+T cells were analyzed by flow cytometry. BMDCs were cultured with TJ-M2010at different concentrations in the presence of FITC-Dextran and the phagocytic function was detected by flow cytometry. Apoptosis and necrosis were detected with the Annexin-V/PI apoptosis kit.[Results] The flow cytometry results demonstrated that CpG could induce high level expression of CD80/CD86/MHCⅡ on DCs (53.1±0.3%/55.6±3.5%/64.6±4.3%). TJ-M2010could reduce the up-regulation of these molecules dose-dependently (40μM:20.7%±.1%/26.3±1.5%/28.1±3.4%VS20μM:38.2%±2.2%/40.4%±1.6%/45.1%±2.4%, p<0.05). TJ-M2010could reduce the up-regulation of CCR7upon stimulation of CpG. TJ-M2010could also reduce the frequency of reactive CD3+CFSE+T cells during the mix lymphocyte culture with BMDCs. There was no significant difference in the phagocytic rate of FITC-Dextran between the normal control group and TJ-M2010groups (63.4%±3.4%vs65.7%±2.6%vs62.6%±2.6%, p>0.05); The frequency of Annexin-V/PI double positive DCs in TJ-M2010groups also showed no significant difference when compared with the normal control group (6.6%±0.9%vs6.5%±1.1%vs6.6±1.1%p>0.05).[Conclusion] TJ-M2010could dose-dependently inhibit DCs maturation, migration ability and activation of NF-κB, as well as the proliferation of reactive T cells indirectly. TJ-M2010did not influence DCs phagocytic function and it also showed no obvious toxicity to DCs. Part Ⅱ MyD88inhibitor TJ-M2010promotes tolerance to cardiac allografts in mice and its mechanism[Objective] To investigate the role of MyD88inhibitor TJ-M2010in the induction of tolerance for mice cardiac transplantation and the possible mechanism.[Methods](1) A fully allogenic BALB/c (H-2d) to C57BL/6(H-2b) mice abdominal cardiac transplantation model was established. Cardiac recipients were left untreated or given either TJ-M2010or control vehicle (0.5%carboxymethyl cellulose, CMC). The day of operation was recorded as day0. TJ-M2010(50mg/kg/day) suspended in0.5%CMC was administrated to experimental recipients by intraperitoneal injection (i.p.) from Day-2to Day7. Graft beating was monitored by daily palpation. The survival of cardiac grafts was observed.(2) Cardiac grafts were obtained7days after the transplantation. HE staining was performed to estimate the severity of rejection. Mononuclear cells in grafts were obtained by grafts digestion with collagenase Ⅱ and CD11c+CD80+double positive cells were detected by flow cytometry. The mRNA expression of IL-1, IL-6and TNF-a in allografts were analyzed by real-time PCR.(3) The frequency of CD4+CD25+Foxp3+Tregs in recipients was detected by flow cytometry. The allo-response of splenocytes were detected by INF-y ELISPOT.(4) For long-term-survived recipients, second cervical cardiac transplantation was carried out and the allografts survival time was observed.[Results] The grafts of normal control group and CMC group were promptly rejected at early period (MST=7.4±0.5days vs MST=7.6±0.5days, p>0.05). However, TJ-M2010monotherapy (50mg/kg/day, from Day-2to Day7) led to%of allografts surviving for more than100days. For second cardiac transplantation, long-term-survival recipients did not reject the BALB/c hearts for more than100days. Pathological examination demonstrated that TJ-M2010attenuated the rejection responses in allografts in the aspect of lymphocytes infiltration and tissue damage. Real-time PCR results showed that the mRNA expression of IL-1, IL-6and TNF-a in allografts of TJ-M2010group was significantly reduced when compared with that of CMC group (p<0.05). The frequency of CD11c+CD80+double positive cells in allografts of TJ-M2010group was also reduced when compared with that of CMC group. The frequency of CD4+CD25+Foxp3+Treg in recipients treated with CMC was6.43%while12.92%in long-term-survived recipients treated with TJ-M2010. INF-y ELISPOT results showed that splenocytes in long-term-survived recipients had a normal response to C3H antigen in vitro (p>0.05) but an attenuated response to BALB/c antigen (p<0.05).[Conclusion] Short-term monotherapy of MyD88inhibitor TJ-M2010could induce donor-specific tolerance for mice cardiac transplantation. Reduced inflammatory cytokines secretion and Treg induction may involved in the mechanism. Part III MyD88inhibitor combined with anti-CD154lead to tolerance in mice skin transplantation[Objective] To investigate the possibility that MyD88inhibitor TJ-M2010combined with anti-CD154can induce tolerance in mice skin transplantation and the mechanisms. [Methods] A fully allogenic BALB/c (H-2d) to C57BL/6(H-2b) mice skin transplantation model was established. Generally, full thickness skin grafts (-1cm2) from tails of donor mice were transplanted to dorsal flank of recipient mice. The experiment was grouped as follows:TJ-M2010treatment group; anti-CD154group; TJ-M2010+anti-CD154group. The day of operation was recorded as day0. TJ-M2010(50mg/kg/day) suspended in0.5%CMC was administrated to experimental recipients by intraperitoneal injection (i.p.) on day-2-day7, day9, day11, day13, day15. Anti-CD154(MR1clone,200μg/dose) was administrated by i.p. on day0to day3, day7and day14. Rejection was defined as necrosis of the entire graft surface by daily inspection. The survival of cardiac grafts was observed. For long-term-survived recipients, second skin or cardiac transplantation were carried out and the survival time was observed. The frequency of CD4+CD25+Foxp3+Tregs in recipients was detected by flow cytometry. The allo-response of splenocytes were detected by INF-γ ELISPOT.[Results] There were no significant differences between TJ-M2010group and anti-CD154group in allografts survival time (9.0±0.71days VS9.6±0.55days, p>0.05) while TJ-M2010combined with anti-CD154led to22%of allografts surviving for more than100days. For second cardiac transplantation, long-term-survived recipients did not reject the BALB/c hearts for more than100days. For second skin transplantation, long-term-survived recipients rejected both the BALB/c and C3H skin allografts at early stage. However, when the recipients were treated with TJ-M2010at the time of second skin transplantation, the allografts survival time prolonged significantly. The frequency of CD4+CD25+Foxp3+Treg in recipients treated with TJ-M2010+anti-CD154was significantly rised. INF-γELISPOT results showed that splenocytes in long-term-survived recipients had a normal response to C3H antigen in vitro (p>0.05) but an attenuated response to BALB/c antigen (p<0.05).[Conclusion] MyD88combined with anti-CD154induced tolerance in mice allogenic skin transplantation. The tolerance was unstable. MyD88played an essential role in the maintenance of this tolerance.更多还原