阻断天然免疫Tim-3信号或TLR信号促进移植耐受诱导

发布时间：2018-06-03 11:11

本文选题：胞内染色 + galectin-9　；参考：《华中科技大学》2010年博士论文

【摘要】： [目的]探讨galectin-9蛋白抗完全异基因小鼠心脏移植排斥的机制。 [方法]构建含galectin-9基因的腺病毒,感染CHO细胞使其过表达galectin-9,免疫细胞化学和流式细胞染色确定其真核表达定位。以PMA+Ionomycin+BFA激活脾脏淋巴细胞,以流式细胞染色探讨活化前后CD4+淋巴细胞和CD8+T淋巴细胞亚群表达galectin-9的平均荧光强度。原核表达并纯化基因重组人galectin-9蛋白。流式分选CD4+CD25-T细胞,体外诱导其向Th17细胞分化,加或不加galectin-9蛋白,检测诱导的CD4+IL-17+T细胞比例。胶原酶消化法分离完全异基因小鼠移植心内浸润淋巴细胞,分析CD4+Tim-3+T细胞和CD8+Tim-3+T细胞的比例以及天然免疫细胞Tim-3+的比例。构建小鼠BALB/c→C57BL/6心脏移植模型,予galectin-9蛋白短期干预,观察移植物存活情况,探讨不同组别移植物引流淋巴结和小鼠外周血内CD4+IFN-γ+、CD4+IL-17+、CD8+IFN-γ+, CD8+IL-17+, CD4+Tim-3+、CD4+Tim-1+淋巴细胞的比例。对比各组别移植受者脾脏内CD4+CD25+Foxp3+T细胞(Treg)的比例。 [结果]galectin-9表达于CHO细胞的胞浆,galectin-9在活化的CD4+和CD8+淋巴细胞内胞浆有表达,可能以分泌形式发挥作用。在体外,galectin-9可抑制Th17细胞的生成。完全异基因移植心脏物浸润细胞内的Tim-3+淋巴细胞以CD8+细胞为主,CD8+Tim-3+淋巴细胞占CD8+淋巴细胞的比例达到40.5%±5.2%。给予galectin-9短期治疗可显著延长完全异基因小鼠移植心的存活时间(MST=23.0±1.0天vs对照组7.2±0.4天),移植物存活延长的同时伴随引流淋巴结内CD8+IFN-γ+、CD8+IL-17+和外周血中CD4+Tim-3+淋巴细胞比例下调,而CD4+Tim-1+比例上调,但galectin-9治疗组小鼠体内的Treg比例并无明显上调。 [结论]Galectin-9在活化的CD4+T和CD8+T细胞胞浆内可检测到,可能通过分泌形式发挥免疫负反馈抑制作用。在体外,galectin-9可抑制Th17细胞的生成。galectin-9延长小鼠移植心的存活与CD8+T细胞免疫下调,Th1和Th17免疫受抑制(CD4+Tim-3+)而Th2免疫(CD4+Tim-1+)占优势有一定关联。 [目的]探讨galectin-9促进DC成熟的效应,用Rapamycin在体外能否抑制galectin-9的促成熟效应。探索利用小剂量rapamycin和galectin-9能否诱导小鼠移植心耐受。 [方法]分离BALB/c→C56BL/6排斥心脏内浸润细胞,流式检测圈定单核细胞/DC细胞亚群进行Tim-3的表达水平分析,Western Blot检测移植心内Tim-3L(galectin-9)的表达。体外培养BALB/c小鼠的BMDC,检测Tim-3的表达情况。以一定浓度梯度的galectin-9处理DC,或联合一定浓度梯度的Rapamycin检测DC表面共刺激分子CD80/CD86的表达。建立BALB/c→C56BL/6小鼠移植模型,按照以下处理进行分组：A.同基因对照组(C56BL/6→C56BL/6);B.异基因移植galectin-9单独治疗组；C.异基因移植Rapamycin治疗组；D. galectin-9+Rapamycin联合治疗组；E.异基因移植PBS治疗组。观察小鼠移植心存活情况。ELISPOT检测长期存活小鼠对供者抗原或非相关第三方抗原反应分泌IL-4和IFN-γ的情况。 [结果]移植心内单核细胞/DC细胞Tim-3的阳性率高达47.3%±5.6%。移植后第3天galectin-9的表达水平显著上调,而第7天时表达减少。培养的不成熟BMDC组成性表达Tim-3,阳性率约为6.7%左右。使用galectin-9可促进DC细胞上调共刺激分子Cd80/CD86,而在联合使用Rapamycin后DC表达的共刺激分子水平明显减少。体内联合galectin-9和Rapamycin可促进全部移植物长期接受(180天,n=6)；而单用galectin-9治疗,仅短期延长移植物存活(MST=23.5±2.2天)；单用rapamycin的治疗组,4例移植心在分别在移植后不同时间点被排斥,2例存活大于180天,与galectin-9联合Rapamycin使用组差异有统计学意义(p0.05)。联合治疗组移植物长期受者对供者抗原刺激分泌IFN-γ和IL-4明显弱于第三方,获得针对供者抗原特异的免疫低反应。 [结论]移植物内天然免疫细胞高表达Tim-3和移植心早期上调galectin-9,两者可能参与了移植排斥的发生。Galectin-9具有刺激DC成熟的作用,而Rapamycin可抑制galectin-9引起的DC成熟,两者联合使用可诱导移植心获得耐受。联合治疗的受者针对供者抗原产生显著减弱的Th1和Th2免疫反应。 [目的]探讨ST2825作用于TLR信号关键接头蛋白MyD88以减轻小鼠异基因心脏移植排斥的机制。 [方法]①GM-SCF+IL-4诱导BALB/c小鼠骨髓造血干细胞分化为DC,使用多种TLR配体及坏死心肌匀浆刺激其活化。检测ST2825抑制MyD88对DC表达共刺激分子CD80/CD86的影响。②分离C57BL/6小鼠淋巴结细胞(以T细胞为主),以BALB/c来源的DC刺激其增殖。加或不加CpG(TLR9配体),检测ST2825对混培体系中T细胞激活的影响。③行BALB/c→C57BL/6心脏移植,术前两小时给予250mg/kgST2825,以后每日给药1次直到术后第6天,第7日获取不同组别的移植心和脾脏,并留取7例观察移植心完全停跳时间。实时定量PCR检测移植心内促炎因子IL-1β,IL-6和TNF-α的mRNA水平,HE病理分析淋巴细胞浸润和心肌完整性,流式细胞仪检测移植受者脾脏内CD4+IL-17+和CD4+IFN-y+淋巴细胞以及CD4+CD25+Foxp3+调节性T细胞比例。 [结果]体外使用ST2825可显著抑制各种TLR配体及坏死心肌匀浆刺激DC引起的CD80/CD86上调(除TLR3配体外),ST2825呈剂量依赖性抑制DC刺激的T细胞活化。单独使用ST2825显著延长小鼠心移植物存活时间(MST=19.4±0.98天vs 7.2±0.4天)。ST2825治疗组心肌结构较为完整,淋巴细胞浸润减少,移植物内的IL-1β和IL-6转录显著下调,CD4+IL-17+和CD4+IFN-γ+淋巴细胞比例亦显著下调,而CD4+CD25+Foxp3+调节性T细胞比例略上调。 [结论] ST2825可显著显著延长小鼠同种异基因心移植物存活时间,与ST2825抑制DC活化,进而抑制T细胞活化并与CD4+CD25+Foxp3+调节性T细胞的生成有关。 [目的]探讨利用MyD88阻断剂ST2825阻断TLR信号,联合共刺激分子阻断剂anti-CD 154诱导皮肤移植耐受。 [方法]建立BALB/c→C57BL/6小鼠皮肤移植模型,摸索ST2825阻断剂联合anti-CD 154体内给予的方案,观察移植皮片的存活时间。对移植皮肤长期存活的小鼠行ELISPOT检测对供者抗原的反应性：取受者脾细胞与供者脾细胞(BALB/c)或非相关第三方脾细胞(C3H)共孵育,检测IFN-γ和IL-4的斑点数。 [结果]单独anti-CD 154干预组,单独ST2825干预组,CMC对照干预组异基因移植皮肤均不能获得保护,短期内即排斥。而联合使用anti-CD154+ST2825的治疗组,大多数皮肤移植物获得长期存活(150天)。ELISPOT检测提示移植受者体内针对供者抗原呈现特异性低反应,脾细胞分泌IFN-γ的反应明显减弱,但分泌IL-4的反应保留。 [结论]利用我们建立的anti-CD154+ST2825诱导方案可诱导完全异基因小鼠皮肤移植物长期接受。移植受者对供者抗原呈现特异性免疫低反应,可能与Th1免疫向Th2免疫偏移有关。
[Abstract]:[Objective] to explore the mechanism of galectin-9 protein against complete allogeneic cardiac allograft rejection in mice.
[Methods] the adenovirus containing galectin-9 gene was constructed, and CHO cells were infected with galectin-9, immunocytochemistry and flow cytometry to determine the eukaryotic expression. The spleen lymphocytes were activated by PMA+Ionomycin+BFA, and the expression of galectin-9 in CD4+ lymphoblastic cells and CD8+T lymphocyte subsets before and after activation was investigated by flow cytometry. The average fluorescence intensity. Prokaryotic expression and purification of recombinant human galectin-9 protein. Flow type CD4+CD25-T cells were selected to differentiate into Th17 cells in vitro, and the ratio of induced CD4+IL-17+T cells was detected by adding or without galectin-9 protein. The collagenase digestion method was used to isolate the infiltrating lymphocytes in the heart of completely allogeneic mice, and the analysis of CD4+Tim-3+T The proportion of cells and CD8+Tim-3+T cells and the proportion of Tim-3+ in natural immune cells. The model of BALB/c to C57BL/6 heart transplantation in mice was constructed, and the short-term intervention of galectin-9 protein was given to observe the survival of the grafts. The lymph nodes of different groups of graft and the peripheral blood of CD4+ IFN- gamma +, CD4+IL-17+, CD8+IFN- gamma +, CD8+IL-17+, CD4+Ti in the peripheral blood of mice were discussed. The ratio of m-3+ to CD4+Tim-1+ lymphocytes was compared with the proportion of CD4+CD25+Foxp3+T cells (Treg) in the spleen of transplant recipients.
[results]galectin-9 is expressed in the cytoplasm of CHO cells, galectin-9 is expressed in the cytoplasm of activated CD4+ and CD8+ lymphocytes, may play a role in secretory form. In vitro, galectin-9 can inhibit the formation of Th17 cells. The Tim-3+ lymphocytes in completely allogeneic transplanted heart infiltrating cells are dominated by CD8+ cells, CD8+Tim-3+ lymphatic cells are fine. The ratio of CD8+ lymphocyte to 40.5% + 5.2%. for galectin-9 short-term treatment could significantly prolong the survival time of complete allogeneic mice (MST=23.0 + 1 days vs control group 7.2 + 0.4 days). The prolongation of graft survival was accompanied by CD8+IFN- gamma + in the drainage lymph nodes, CD8 +IL-17+ and the proportion of CD4+Tim-3+ lymphocytes in peripheral blood. While the CD4+Tim-1+ ratio was up-regulated, the proportion of Treg in galectin-9 treated mice did not increase significantly.
[conclusion]Galectin-9 can be detected in the cytoplasm of activated CD4+T and CD8+T cells, may play an immunosuppressive effect through the secretory form. In vitro, galectin-9 can inhibit the formation of.Galectin-9 in Th17 cells to prolong the survival of the transplanted heart and the down-regulation of CD8+T cell immunity, Th1 and Th17 immunity is inhibited (CD4+Tim-3+) and Th2 immunity (CD4+) Tim-1+) there is a certain link in the dominance.
[Objective] to explore the effect of galectin-9 on promoting the maturation of DC, whether or not Rapamycin can inhibit the maturation effect of galectin-9 in vitro. The effect of small dose of rapamycin and galectin-9 on the induction of heart tolerance in mice is explored.
[Methods] BALB/c - C56BL/6 was separated from the infiltrating cells in the heart, and the expression level of the /DC cell subgroup of mononuclear cells was detected by flow cytometry. The expression of Tim-3L (galectin-9) in the transplanted heart was detected by Western Blot. The BMDC of the BALB/c mice was cultured in vitro, and the Tim-3 was detected. The expression of costimulatory molecule CD80/CD86 on DC surface was detected by Rapamycin with a certain concentration gradient.
BALB/c to C56BL/6 mice transplantation model was set up according to the following treatment: A. identical gene control group (C56BL/6 to C56BL/6); B. allogeneic transplantation galectin-9 alone treatment group; C. allogeneic transplantation Rapamycin treatment group; D. galectin-9+Rapamycin combined treatment group; E. isobase transplantation treatment group. Case.ELISPOT detected IL-4 and IFN- gamma secretion from donor or unrelated third party antigens in long-term survival mice.
[results] the positive rate of /DC cell Tim-3 in the transplanted monocyte was up to 47.3% + 5.6%. after third days of transplantation, and the expression level of galectin-9 was significantly up, but the expression of the immature BMDC was reduced at seventh days. The positive rate of the immature BMDC was about 6.7%. The use of galectin-9 can promote the up-regulation of CO stimulator Cd80/CD86 in DC cells. The levels of CO stimulator molecules expressed by DC after Rapamycin were significantly reduced. The combination of galectin-9 and Rapamycin in the body could promote the long-term acceptance of all grafts (180 days, n=6), while the only short-term prolongation of graft survival (MST=23.5 + 2.2 days) was only used by galectin-9 alone, and 4 cases of transplantation hearts were not simultaneously transplanted at the same time after transplantation. The survival of 2 cases was more than 180 days, and there was a significant difference between the 2 cases and the Rapamycin use group (P0.05). The long-term recipients of the combined treatment group were significantly weaker than third parties to the donor antigen stimulation and secretion of IFN- gamma and IL-4, and the antigen specific immune response to donor antigen was obtained.
[Conclusion] the high expression of Tim-3 in the grafts and the early up-regulation of galectin-9 in the transplanted heart may be involved in the role of.Galectin-9 to stimulate the maturation of DC, while Rapamycin can inhibit the maturation of DC caused by galectin-9, and the combination of the two can induce the tolerance of the transplanted heart. The antigen produced significantly reduced Th1 and Th2 immune responses.
[Objective] to explore the mechanism of ST2825 acting on the key signaling protein MyD88 of TLR signaling to alleviate the rejection of allogeneic heart transplantation in mice.
[method] GM-SCF+IL-4 induced BALB/c mouse bone marrow hematopoietic stem cells to differentiate into DC, using a variety of TLR ligands and necrotic myocardial homogenate to stimulate their activation. The effect of ST2825 inhibition MyD88 on DC expression of CO stimulatory molecule CD80/CD86 was detected. (2) isolated C57BL/6 mouse lymph node cells (T cells as the main), and BALB/c source DC to stimulate their proliferation. No CpG (TLR9 ligand) was used to detect the effect of ST2825 on the activation of T cells in the mixed culture system. (3) BALB/c to C57BL/6 heart transplantation was performed, 250mg/kgST2825 was given two hours before operation, and 1 times daily was given until sixth days after the operation, and the heart and spleen of different groups were obtained on the seventh day, and 7 cases were left to observe the complete stop time of the heart transplantation. Real time quantitative PCR The mRNA levels of IL-1 beta, IL-6 and TNF- alpha in the transplanted heart were detected. Lymphocyte infiltration and myocardial integrity were analyzed by HE pathological analysis. The proportion of CD4+IL-17+ and CD4+IFN-y+ lymphocytes in spleen and CD4+CD25+Foxp3+ regulatory T cells in transplanted recipients were detected by flow cytometry.
[results] the use of ST2825 in vitro could significantly inhibit the up regulation of CD80/CD86 induced by various TLR ligands and necrotic myocardial homogenate (except TLR3 in vitro). ST2825 showed a dose-dependent inhibition of DC stimulated T cells activation. The survival time of cardiac allograft in mice was significantly prolonged by ST2825 (MST =19.4 + 0.98 days vs 7.2 + 0.4 days). The structure was more complete, the lymphocyte infiltration decreased, the IL-1 beta and IL-6 transcription in the grafts decreased significantly, and the proportion of CD4+IL-17+ and CD4+IFN- gamma + lymphocytes also decreased significantly, while the proportion of CD4+CD25+Foxp3+ regulatory T cells was slightly up-regulated.
[Conclusion] ST2825 can significantly prolong the survival time of allogeneic cardiac allograft in mice, and inhibit the activation of DC with ST2825, and then inhibit the activation of T cells, and it is related to the formation of CD4+CD25+Foxp3+ regulatory T cells.
[Objective] to investigate the use of MyD88 blocker ST2825 to block TLR signal and induce skin graft tolerance by CO stimulator blocker anti-CD 154.
[Methods] to establish a BALB/c to C57BL/6 mouse skin transplantation model, explore the scheme given by the ST2825 blocker and anti-CD 154 in vivo, and observe the survival time of the transplanted skin. The responsiveness of the donor antigen to the donor's spleen cells (BALB/c) or the donor spleen cells (BALB/c) or the unrelated spleen in the mice with long-term survival of the transplanted skin was observed. Cells (C3H) were incubated to detect the number of IFN- and IL-4 spots.
[results] the individual anti-CD 154 intervention group, the individual ST2825 intervention group, the CMC control group could not obtain the protection and the rejection in the short term. While the combined use of anti-CD154+ST2825 in the treatment group, most of the skin grafts obtained long-term survival (150 days).ELISPOT test suggested that transplant recipients were specific for donor antigen in the body. When the opposite sex was low, the reaction of splenocytes secreting IFN- gamma was obviously weakened, but the reaction of secreting IL-4 remained.
[Conclusion] the anti-CD154+ST2825 induction scheme established by us can induce long-term acceptance of skin graft in completely allogeneic mice. The transplant recipients have specific immune response to donor antigen, which may be related to the immunization of Th1 to Th2.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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