本文選題：胞內(nèi)染色 + galectin-9��； 參考：《華中科技大學》2010年博士論文

【摘要】： [目的]探討galectin-9蛋白抗完全異基因小鼠心臟移植排斥的機制。 [方法]構(gòu)建含galectin-9基因的腺病毒,感染CHO細胞使其過表達galectin-9,免疫細胞化學和流式細胞染色確定其真核表達定位。以PMA+Ionomycin+BFA激活脾臟淋巴細胞,以流式細胞染色探討活化前后CD4+淋巴細胞和CD8+T淋巴細胞亞群表達galectin-9的平均熒光強度。原核表達并純化基因重組人galectin-9蛋白。流式分選CD4+CD25-T細胞,體外誘導其向Th17細胞分化,加或不加galectin-9蛋白,檢測誘導的CD4+IL-17+T細胞比例。膠原酶消化法分離完全異基因小鼠移植心內(nèi)浸潤淋巴細胞,分析CD4+Tim-3+T細胞和CD8+Tim-3+T細胞的比例以及天然免疫細胞Tim-3+的比例。構(gòu)建小鼠BALB/c→C57BL/6心臟移植模型,予galectin-9蛋白短期干預,觀察移植物存活情況,探討不同組別移植物引流淋巴結(jié)和小鼠外周血內(nèi)CD4+IFN-γ+、CD4+IL-17+、CD8+IFN-γ+, CD8+IL-17+, CD4+Tim-3+、CD4+Tim-1+淋巴細胞的比例。對比各組別移植受者脾臟內(nèi)CD4+CD25+Foxp3+T細胞(Treg)的比例。 [結(jié)果]galectin-9表達于CHO細胞的胞漿,galectin-9在活化的CD4+和CD8+淋巴細胞內(nèi)胞漿有表達,可能以分泌形式發(fā)揮作用。在體外,galectin-9可抑制Th17細胞的生成。完全異基因移植心臟物浸潤細胞內(nèi)的Tim-3+淋巴細胞以CD8+細胞為主,CD8+Tim-3+淋巴細胞占CD8+淋巴細胞的比例達到40.5%±5.2%。給予galectin-9短期治療可顯著延長完全異基因小鼠移植心的存活時間(MST=23.0±1.0天vs對照組7.2±0.4天),移植物存活延長的同時伴隨引流淋巴結(jié)內(nèi)CD8+IFN-γ+、CD8+IL-17+和外周血中CD4+Tim-3+淋巴細胞比例下調(diào),而CD4+Tim-1+比例上調(diào),但galectin-9治療組小鼠體內(nèi)的Treg比例并無明顯上調(diào)。 [結(jié)論]Galectin-9在活化的CD4+T和CD8+T細胞胞漿內(nèi)可檢測到,可能通過分泌形式發(fā)揮免疫負反饋抑制作用。在體外,galectin-9可抑制Th17細胞的生成。galectin-9延長小鼠移植心的存活與CD8+T細胞免疫下調(diào),Th1和Th17免疫受抑制(CD4+Tim-3+)而Th2免疫(CD4+Tim-1+)占優(yōu)勢有一定關(guān)聯(lián)。 [目的]探討galectin-9促進DC成熟的效應,用Rapamycin在體外能否抑制galectin-9的促成熟效應。探索利用小劑量rapamycin和galectin-9能否誘導小鼠移植心耐受。 [方法]分離BALB/c→C56BL/6排斥心臟內(nèi)浸潤細胞,流式檢測圈定單核細胞/DC細胞亞群進行Tim-3的表達水平分析,Western Blot檢測移植心內(nèi)Tim-3L(galectin-9)的表達。體外培養(yǎng)BALB/c小鼠的BMDC,檢測Tim-3的表達情況。以一定濃度梯度的galectin-9處理DC,或聯(lián)合一定濃度梯度的Rapamycin檢測DC表面共刺激分子CD80/CD86的表達。 建立BALB/c→C56BL/6小鼠移植模型,按照以下處理進行分組：A.同基因?qū)φ战M(C56BL/6→C56BL/6);B.異基因移植galectin-9單獨治療組；C.異基因移植Rapamycin治療組；D. galectin-9+Rapamycin聯(lián)合治療組；E.異基因移植PBS治療組。觀察小鼠移植心存活情況。ELISPOT檢測長期存活小鼠對供者抗原或非相關(guān)第三方抗原反應分泌IL-4和IFN-γ的情況。 [結(jié)果]移植心內(nèi)單核細胞/DC細胞Tim-3的陽性率高達47.3%±5.6%。移植后第3天galectin-9的表達水平顯著上調(diào),而第7天時表達減少。培養(yǎng)的不成熟BMDC組成性表達Tim-3,陽性率約為6.7%左右。使用galectin-9可促進DC細胞上調(diào)共刺激分子Cd80/CD86,而在聯(lián)合使用Rapamycin后DC表達的共刺激分子水平明顯減少。體內(nèi)聯(lián)合galectin-9和Rapamycin可促進全部移植物長期接受(180天,n=6)；而單用galectin-9治療,僅短期延長移植物存活(MST=23.5±2.2天)；單用rapamycin的治療組,4例移植心在分別在移植后不同時間點被排斥,2例存活大于180天,與galectin-9聯(lián)合Rapamycin使用組差異有統(tǒng)計學意義(p0.05)。聯(lián)合治療組移植物長期受者對供者抗原刺激分泌IFN-γ和IL-4明顯弱于第三方,獲得針對供者抗原特異的免疫低反應。 [結(jié)論]移植物內(nèi)天然免疫細胞高表達Tim-3和移植心早期上調(diào)galectin-9,兩者可能參與了移植排斥的發(fā)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,兩者聯(lián)合使用可誘導移植心獲得耐受。聯(lián)合治療的受者針對供者抗原產(chǎn)生顯著減弱的Th1和Th2免疫反應。 [目的]探討ST2825作用于TLR信號關(guān)鍵接頭蛋白MyD88以減輕小鼠異基因心臟移植排斥的機制。 [方法]①GM-SCF+IL-4誘導BALB/c小鼠骨髓造血干細胞分化為DC,使用多種TLR配體及壞死心肌勻漿刺激其活化。檢測ST2825抑制MyD88對DC表達共刺激分子CD80/CD86的影響。②分離C57BL/6小鼠淋巴結(jié)細胞(以T細胞為主),以BALB/c來源的DC刺激其增殖。加或不加CpG(TLR9配體),檢測ST2825對混培體系中T細胞激活的影響。③行BALB/c→C57BL/6心臟移植,術(shù)前兩小時給予250mg/kgST2825,以后每日給藥1次直到術(shù)后第6天,第7日獲取不同組別的移植心和脾臟,并留取7例觀察移植心完全停跳時間。實時定量PCR檢測移植心內(nèi)促炎因子IL-1β,IL-6和TNF-α的mRNA水平,HE病理分析淋巴細胞浸潤和心肌完整性,流式細胞儀檢測移植受者脾臟內(nèi)CD4+IL-17+和CD4+IFN-y+淋巴細胞以及CD4+CD25+Foxp3+調(diào)節(jié)性T細胞比例。 [結(jié)果]體外使用ST2825可顯著抑制各種TLR配體及壞死心肌勻漿刺激DC引起的CD80/CD86上調(diào)(除TLR3配體外),ST2825呈劑量依賴性抑制DC刺激的T細胞活化。單獨使用ST2825顯著延長小鼠心移植物存活時間(MST=19.4±0.98天vs 7.2±0.4天)。ST2825治療組心肌結(jié)構(gòu)較為完整,淋巴細胞浸潤減少,移植物內(nèi)的IL-1β和IL-6轉(zhuǎn)錄顯著下調(diào),CD4+IL-17+和CD4+IFN-γ+淋巴細胞比例亦顯著下調(diào),而CD4+CD25+Foxp3+調(diào)節(jié)性T細胞比例略上調(diào)。 [結(jié)論] ST2825可顯著顯著延長小鼠同種異基因心移植物存活時間,與ST2825抑制DC活化,進而抑制T細胞活化并與CD4+CD25+Foxp3+調(diào)節(jié)性T細胞的生成有關(guān)。 [目的]探討利用MyD88阻斷劑ST2825阻斷TLR信號,聯(lián)合共刺激分子阻斷劑anti-CD 154誘導皮膚移植耐受。 [方法]建立BALB/c→C57BL/6小鼠皮膚移植模型,摸索ST2825阻斷劑聯(lián)合anti-CD 154體內(nèi)給予的方案,觀察移植皮片的存活時間。對移植皮膚長期存活的小鼠行ELISPOT檢測對供者抗原的反應性：取受者脾細胞與供者脾細胞(BALB/c)或非相關(guān)第三方脾細胞(C3H)共孵育,檢測IFN-γ和IL-4的斑點數(shù)。 [結(jié)果]單獨anti-CD 154干預組,單獨ST2825干預組,CMC對照干預組異基因移植皮膚均不能獲得保護,短期內(nèi)即排斥。而聯(lián)合使用anti-CD154+ST2825的治療組,大多數(shù)皮膚移植物獲得長期存活(150天)。ELISPOT檢測提示移植受者體內(nèi)針對供者抗原呈現(xiàn)特異性低反應,脾細胞分泌IFN-γ的反應明顯減弱,但分泌IL-4的反應保留。 [結(jié)論]利用我們建立的anti-CD154+ST2825誘導方案可誘導完全異基因小鼠皮膚移植物長期接受。移植受者對供者抗原呈現(xiàn)特異性免疫低反應,可能與Th1免疫向Th2免疫偏移有關(guān)。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R392

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7 婁域峰;施新萍;張華;;半乳糖凝集素-3在心臟疾病診斷中的應用[A];中華醫(yī)學會第九次全國檢驗醫(yī)學學術(shù)會議暨中國醫(yī)院協(xié)會臨床檢驗管理專業(yè)委員會第六屆全國臨床檢驗實驗室管理學術(shù)會議論文匯編[C];2011年

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相關(guān)博士學位論文 前10條

1 湯東;胰腺星狀細胞高表達Galectin-1促進胰腺癌免疫抑制及進展的研究[D];南京醫(yī)科大學;2012年

2 安U，

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