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  本文選題：移植物　切入點：膠原酶Ⅱ　出處：《華中科技大學(xué)》2010年博士論文

【摘要】： 目的建立簡便而高效的分離小鼠心臟移植物內(nèi)浸潤淋巴細胞的方法,使用流式細胞儀檢測移植心內(nèi)浸潤淋巴細胞的亞型。 方法建立小鼠頸部心臟移植模型,分實驗組(同種異體移植組：Balb/c小鼠為供體,C57BL/6小鼠為受體)和對照組(同系移植組：供受體均為C57BL/6小鼠)。移植術(shù)后3天,5天和7天獲取移植心,將其中一部分移植物進行病理切片觀察排斥反應(yīng)情況,剩余移植物剪碎并采用改進的Ⅱ型膠原酶消化法(250 U/ml,30-40 min)消化心臟,然后用Ficoll密度梯度離心法(800g×20 min)分離單個核細胞,以鈣離子熒光染料Indo-1分析細胞活性,最后以熒光標(biāo)記抗體CD4, CD8, CD44, CD62L, Foxp3進行流式細胞染色,流式細胞儀檢測移植物內(nèi)浸潤淋巴細胞亞群組成。 結(jié)果移植物內(nèi)分離的單個核細胞數(shù)量穩(wěn)定在1×106以上,術(shù)后第7天數(shù)量達到最高峰,分離所得的單個核細胞數(shù)量與排斥反應(yīng)嚴(yán)重程度相關(guān)。淋巴細胞占分離所得單個核細胞的比例為(31.9±2.3)%,活性為(95.1±2.1)%。移植物內(nèi)浸潤的T細胞大部分為效應(yīng)性T細胞,CD4+/CD8+比值隨著排斥反應(yīng)的進行逐漸降低。 結(jié)論本法單用膠原酶消化移植心,采用Ficoll密度梯度離心法,減少了對心臟移植物內(nèi)浸潤淋巴細胞的損傷,獲得的細胞可進一步用于流式分析其表型,為直接檢測浸潤到移植物內(nèi)的淋巴細胞亞型提供一種高效和可靠的方法,是一種值得推廣的免疫學(xué)研究手段。 目的檢測Tim3 mRNA在移植后移植物內(nèi)的表達變化及Tim3在受者體內(nèi)不同部位T淋巴細胞上表達,探討其與急性排斥反應(yīng)的關(guān)系。 方法建立小鼠心臟移植模型,分實驗組(同種異體移植組：Balb/c小鼠為供體,C57BL/6小鼠為受體)和對照組(同系移植組：供受體均為C57BL/6小鼠)。移植術(shù)后3天,5天,7天和9天獲取移植物,采用實時定量RT-PCR檢測Tim3 mRNA在移植物內(nèi)的表達變化。術(shù)后3天和6天分離受者外周血、脾臟、引流淋巴結(jié)、移植物內(nèi)淋巴細胞,流式細胞儀檢測Tim3陽性細胞在CD4+和CD8+T細胞中的比例。 結(jié)果同基因組移植物內(nèi)Tim3表達很低。異基因組移植術(shù)后第3天移植物內(nèi)Tim3mRNA的表達明顯升高,并且Tim3 mRNA的表達隨著排斥反應(yīng)的進行而顯著升高(P0.01),于移植物完全排斥前達到最高峰,隨后表達逐漸降低。移植術(shù)后受者外周血和脾臟內(nèi)Tim3陽性細胞比例無明顯變化(P0.05),引流淋巴結(jié)內(nèi)Tim3+/CD4+比值輕度升高(P＜0.05),移植物內(nèi)Tim3+/CD4+和Tim3+/CD8+比值顯著升高(P0.01)。移植術(shù)后第3天和第6天引流淋巴結(jié)內(nèi)Tim3+/CD4+比值差異無統(tǒng)計學(xué)意義(P0.05),而術(shù)后第6天移植物內(nèi)Tim3+/CD4+和Tim3+/CD8+比值均顯著高于第3天(P0.01)。 結(jié)論移植物內(nèi)Tim3 mRNA的表達與小鼠完全異基因心臟移植排斥反應(yīng)的進展動態(tài)相關(guān)。異基因組受者移植物引流淋巴結(jié)和移植物內(nèi)Tim3+細胞比例升高,以移植物內(nèi)升高更為顯著。 目的觀察Galectin-9對同種異體心臟移植物存活時間的影響,探討Galectin-9激活Tim3-Tim3L通路在小鼠心臟移植模型中的免疫調(diào)節(jié)作用。 方法建立小鼠同種異體心臟移植模型,分實驗組和對照組。實驗組受者術(shù)后連續(xù)7天給予穩(wěn)定的Galectin-9蛋白,對照組給予PBS。觀察心臟移植物存活時間。術(shù)后第7天獲取實驗組和對照組心臟移植物,病理切片觀察排斥情況,免疫組化觀察移植物內(nèi)浸潤CD4+和CD8+T細胞數(shù)量。實時定量RT-PCR檢測移植物內(nèi)Tim3,IFN-γ和IL-17mRNA的表達。流式細胞儀檢測引流淋巴結(jié)和移植物內(nèi)Tim3+細胞比例及外周血中Th1和Th17細胞比例。 結(jié)果Galectin-9蛋白處理組心臟移植物平均存活時間為22.7天,對照組僅為7.2天,Galectin-9治療組移植物存活時間顯著延長(P0.05)。病理切片顯示Galectin-9治療組移植物排斥反應(yīng)明顯減輕,浸潤的CD4+和CD8+T細胞明顯減少。Galectin-9治療后移植物內(nèi)Tim3、IFN-γ和IL-17mRNA表達減少。引流淋巴結(jié)和移植物內(nèi)Tim3+細胞比例降低,外周血中Th1和Th17細胞比例也降低。 結(jié)論短期Galectin-9治療能顯著延長同種異體心臟移植物存活時間,其機制是減少移植物內(nèi)淋巴細胞浸潤,并通過觸發(fā)Tim3+細胞程序性死亡而降低受者體內(nèi)Th1和Th17細胞反應(yīng)。 目的觀察阻斷Tim4對小鼠心臟移植物存活時間的影響,探討Tim4在Tim4-Timl通路誘導(dǎo)免疫耐受中的作用。 方法建立小鼠同種異體心臟移植模型,給予受者抗Tim4抗體,觀察其對移植物存活時間的影響,體外混合淋巴細胞培養(yǎng)檢測抗Tim4抗體對同種反應(yīng)性T細胞增殖的作用。比較Tim4基因缺陷和野生型心臟移植物在受體體內(nèi)的存活時間,病理切片判斷排斥情況,免疫熒光染色觀察移植物內(nèi)浸潤淋巴細胞。實時定量RT-PCR檢測移植物內(nèi)免疫相關(guān)基因表達,流式細胞儀檢測移植物浸潤淋巴細胞亞型的變化。過繼輸注受者體內(nèi)效應(yīng)性T細胞和調(diào)節(jié)性T細胞給Babl/c SCID小鼠,觀察其對皮膚移植物存活時間的影響。給予小劑量雷帕霉素觀察其對Tim4基因缺陷的移植物存活時間的影響,并通過術(shù)前給予抗CD25抗體清除受者體內(nèi)Treg明確Treg在本實驗中的作用。 結(jié)果抗Tim4抗體能抑制混合淋巴細胞培養(yǎng)體系中同種反應(yīng)性T細胞增殖,延長移植物存活時間。供體Tim4基因缺陷明顯減輕排斥反應(yīng),減少移植物內(nèi)效應(yīng)性T細胞浸潤,增加調(diào)節(jié)性T細胞數(shù)量,從而顯著延長移植物存活時間(P＜0.01)。Tim4基因缺陷能減少活化的效應(yīng)性T細胞而增加Treg的數(shù)量,但是對效應(yīng)性T細胞和Treg的功能無明顯影響。聯(lián)合小劑量雷帕霉素能誘導(dǎo)Tim4基因缺陷移植物長期存活。Tim4基因缺陷或/和小劑量雷帕霉素延長移植物存活時間依賴Treg的存在。 結(jié)論Tim4在同種異體免疫反應(yīng)中發(fā)揮重要的免疫調(diào)節(jié)作用,阻斷Tim4-Timl通路能明顯延長心臟移植物存活時間。
[Abstract]:Objective to establish a simple and effective method to separate infiltrating lymphocytes from cardiac allografts of mice, and to detect the subtypes of infiltrating lymphocytes in transplanted heart by flow cytometry.
Methods model of cervical heterotopic heart transplantation in mice, divided into experimental group (allograft group: donor Balb/c mice C57BL/6 mice as receptor) and control group (isograft group as donor and recipient C57BL/6 mice). 3 days after transplantation, 5 days and 7 days for heart transplantation, which will be part of the graft the pathological observation of rejection, graft and residual shear by type II collagenase digestion improved (250 U/ml, 30-40 min) digestion heart, then using Ficoll density gradient centrifugation (800g * 20 min) mononuclear cells were separated by calcium from the analysis of cell activity with fluorescence dye Indo-1, finally CD8, CD44 labeled antibodies of CD4, CD62L, Foxp3, flow cytometry staining and flow cytometry in the graft infiltrating lymphocyte subsets.
The number of mononuclear cells in the graft stable separation in 1 x 106, seventh days after operation the number reached a peak, separated from the number of mononuclear cells and the severity of rejection. Lymphocytes isolated from mononuclear cells of the ratio of (31.9 + 2.3)% and (95.1 + 2.1 to live%.) graft infiltrating T cells for the majority of effector T cells, the ratio of CD4+/CD8+ decreased gradually with the rejection.
Conclusion the method of single heart transplantation by collagenase digestion, using Ficoll density gradient centrifugation, reduce the damage of heart allografts infiltrating lymphocytes in plants, the cells can be further used for flow cytometric analysis of the phenotype, for direct detection of infiltration into the graft within the lymphocyte subtypes provides an effective and reliable method, is a worthy of study on the immunology method.
Objective to detect the expression changes of Tim3 mRNA in grafts and the expression of Tim3 in different parts of T lymphocytes in recipients, and to explore the relationship between them and acute rejection.
Methods mouse heart transplantation model, divided into experimental group (allograft group: donor Balb/c mice C57BL/6 mice as receptor) and control group (isograft group as donor and recipient C57BL/6 mice). After transplantation for 3 days, 5 days, 7 days and 9 days for the graft, using quantitative real-time RT-PCR detection of Tim3 mRNA expression in shift in plants. After 3 days and 6 days isolated from peripheral blood, spleen, lymph nodes, graft lymphocytes was detected by flow cytometry and Tim3 positive cells in CD4+ and CD8+T cells.
Results in isograft group the expression of Tim3 was very low. The expression of allogeneic transplantation after third days in the graft Tim3mRNA significantly increased the expression of Tim3 and mRNA with the rejection significantly increased (P0.01), to completely exclude the graft before reaching the peak, then the expression decreased gradually after transplantation by Tim3. The proportion of positive cells in peripheral blood and spleen had no significant change (P0.05), lymph node in Tim3+/CD4+ ratio increased slightly (P < 0.05), graft of Tim3+/CD4+ and Tim3+/CD8+ were significantly increased (P0.01). After transplantation for third days and sixth days of drainage Tim3+/CD4+ ratio difference in lymph nodes was not statistically significant (P0.05), and sixth days after surgery in the graft of Tim3+/CD4+ and Tim3+/CD8+ ratio were significantly higher than that of third days (P0.01).
Conclusion the expression of Tim3 mRNA is closely related to the progress of allograft heart transplantation rejection in allografts. The proportion of Tim3+ cells in the draining lymph nodes and the grafts in the allogeneic recipients is increased, and the increase in the graft is more significant.
Objective To observe the effect of Galectin-9 on the survival time of allograft cardiac allograft, and to explore the immunomodulatory effect of Galectin-9 activated Tim3-Tim3L pathway in mouse heart transplantation model.
Methods mouse cardiac allograft model, divided into experimental group and control group. The stability of Galectin-9 protein was administered for 7 consecutive days experimental group after operation, the control group was given PBS. to observe the cardiac allograft survival time. Seventh days after operation for the experimental group and the control group of cardiac allograft rejection, pathology observation, immunity histochemical observation of infiltrating CD4+ and CD8+T cells shift in plants. The number of real-time RT-PCR detection in the graft Tim3, IFN- expression and IL-17mRNA. Flow cytometry and lymph nodes in the graft ratio of Tim3+ cells and Th1 in peripheral blood and Th17 cell ratio.
Results Galectin-9 protein treatment group the mean survival time of cardiac allograft for 22.7 days, the control group was only 7.2 days, the Galectin-9 treatment group significantly prolong graft survival time (P0.05). The pathological sections showed that Galectin-9 treatment group significantly reduce graft rejection, infiltration of CD4+ and CD8+T were significantly decreased after treatment of.Galectin-9 graft in Tim3. Reduced expression of IFN- gamma and IL-17mRNA. The draining lymph node and Tim3+ cell ratio in plants to reduce the shift, Th1 in peripheral blood and Th17 cell ratio also decreased.
Conclusion short term Galectin-9 therapy can significantly prolong the survival time of allograft cardiac allograft, and its mechanism is to reduce lymphocyte infiltration in graft and reduce the Th1 and Th17 cell responses in vivo by triggering programmed cell death of Tim3+ cells.
Objective To observe the effect of blocking Tim4 on the survival time of cardiac allograft in mice and to explore the role of Tim4 in the induction of immune tolerance in the Tim4-Timl pathway.
Methods mouse cardiac allograft model, given by anti Tim4 antibody, to observe its effect on graft survival time, effect of mixed lymphocyte culture in vitro detection of anti Tim4 antibody on allogeneic T cell proliferation. Tim4 gene deficient and wild-type graft in recipients survival time and pathological judgment rejection, observed by immunofluorescence staining in the graft infiltrating lymphocytes. The expression of real time quantitative RT-PCR detection of shift of immune related genes in plants, the change of flow cytometry graft infiltrating lymphocyte subsets. Adoptive transfer recipients of effector T cells and regulatory T cells to Babl/c SCID mice to observe its effect the skin graft survival time. Give the effect of a small dose of rapamycin observed in Tim4 deficient graft survival time, and through the preoperative administration of anti CD25 The role of Treg in this experiment was scavenged in the body of the recipient.
The anti Tim4 antibody can inhibit the mixed lymphocyte culture system of alloreactive T cell proliferation, prolong graft survival. Donor Tim4 gene defects significantly reduce rejection, reduce the displacement effect of T cell infiltration in plants, increase the number of regulatory T cells, thereby significantly prolong graft survival time (P < 0.01).Tim4 gene the defect can reduce the activation of effector T cells and increase the number of Treg, but the effect on T cells and the function of Treg has no obvious effect. Combined with small dose of rapamycin could induce Tim4 gene defects in long-term graft survival of.Tim4 gene defect or / and small dose of rapamycin to prolong graft survival depends on the presence of Treg.
Conclusion Tim4 plays an important role in immunomodulating in allogeneic immune response, and blocking the Tim4-Timl pathway can significantly prolong the survival time of heart graft.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392

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