供者来源凋亡胸腺细胞输注延长皮肤移植物存活及其机制

发布时间：2018-01-21 20:04

本文关键词： 细胞凋亡同种皮肤移植移植排斥反应树突状细胞调节性T细胞　出处：《华中科技大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:以异基因小鼠皮肤移植为模型,探讨供者来源凋亡胸腺细胞输注诱导同种异体移植物免疫耐受的效应、细胞学基础与分子机制,为设计防治移植排斥反应的新策略提供线索。方法:用地塞米松诱导供者小鼠(Balb/c,H-2d)胸腺细胞凋亡:采用DNA Ladde凝胶电泳法以及Annexin V / PI细胞染色后流式细胞术检测细胞凋亡;BALB/c小鼠胸腺细胞以PKH67染色,诱导凋亡后,经尾静脉过继输注MHC型别完全不匹配的受者小鼠(C57BL/6, H-2b),12个小时后,取其脾细胞以抗CD11c-PE抗体标记树突状细胞,激光共聚焦显微镜观察树突状细胞吞噬凋亡的胸腺细胞;细胞过继7天后,将BALB/c小鼠皮肤移植给C57BL/6小鼠,观察移植物存活时间;皮肤移植后7天,取移植皮片,组织学检测移植物组织中炎性细胞浸润;混合淋巴细胞反应评价供者脾细胞刺激受者小鼠T细胞增殖的作用;RT-PCR检测受者小鼠脾脏细胞TGF-β、IL-10、Foxp3的mRNA表达水平;流式细胞术检测受者小鼠脾脏树突状细胞亚类。结果: 一、诱导供者胸腺细胞凋亡并过继输注,及受者DC吞噬凋亡胸腺细胞的作用 1.DNA Ladder试验和Annexin V / PI染色检测证实,地塞米松可显著增强小鼠胸腺细胞凋亡:经地塞米松诱导细胞凋亡率达58.7%,未经诱导的胸腺细胞作为对照,在体外培养相同时间凋亡率为32.4%。 2.BALB/c小鼠胸腺细胞以PKH67预先染色,诱导凋亡后经尾静脉输注至C57BL/6小鼠体内,12个小时后,取其脾细胞以抗CD11c-PE抗体标记DC,激光共聚焦显微镜下可见PKH67+CD11c+,提示凋亡胸腺细胞被DC吞噬。二、供者凋亡胸腺细胞过继输注延长异基因小鼠皮肤移植物存活时间及其机制以BALB/c小鼠为供者,C57BL/6小鼠为受者,进行背部皮肤移植。移植术前实验组通过尾静脉输注2.5×10~7供者凋亡胸腺细胞,并以PBS、地塞米松和第三方小鼠(C3H小鼠)作为对照。 1.移植物存活时间供者凋亡细胞过继输注,能明显延长皮肤移植物存活时间:凋亡细胞处理组平均存活时间为17.750±3.956天,PBS组为10.250±1.389天,地塞米松组为15.625±1.685天,C3H组为10.500±1.927天。(n=8,p㩳0.01) 2.移植皮片组织学检查移植术后7天,取移植皮片制石蜡切片,常规HE染色,观察移植物组织中炎性细胞浸润。与PBS对照组和C3H组相比,凋亡胸腺细胞组移植组织中单个核细胞浸润明显减少,未出现毛细血管淤血、间质出血、动脉炎和静脉炎等现象;地塞米松组移植组织中单个核细胞浸润也比PBS对照组和C3H组略为减少。 3.受者淋巴细胞增殖功能移植术后7天,取供者脾细胞再次刺激受者脾脏T细胞,检测后者对供者同种抗原的再次应答能力。结果显示:凋亡胸腺细胞组受者脾脏T细胞对供者脾细胞的增殖反应显著低于PBS对照组和C3H组。 4.受者脾脏细胞因子mRNA表达移植术后7天,取供者脾细胞和淋巴结,提取总RNA,借助RT-PCR检测TGF-β、IL-10、Foxp3的mRNA表达水平。结果显示:凋亡胸腺细胞组脾脏TGF-β、IL-10、Foxp3的mRNA表达水平均较对照组升高,差异有显著性;地塞米松组脾脏IL-10、Foxp3的mRNA表达水平增高。(p㩳0.05) 5.受者脾脏中DC亚类检测移植术后7天,取供者脾细胞流式细胞术检测DC亚类。结果显示:凋亡胸腺细胞组CD11c+B220+DC比例显著增高;凋亡胸腺细胞组和地塞米松组CD11c+CD45RB+DC比例均增高。结论: 供者来源凋亡胸腺细胞过继输注,可促进受者体内产生具有免疫抑制功能的DC和调节性T细胞,促进IL-10、TGF-β表达;导致受者同种反应性T细胞增殖活性下降,移植物组织中单个核细胞浸润减少,并显著延长异基因小鼠皮肤移植物存活时间。该作用具有供者抗原特异性。本研究成果提示,供者凋亡胸腺细胞过继输注可抑制同种移植物排斥反应,具有潜在的临床应用价值。
[Abstract]:Objective: To investigate the effects of donor derived thymocyte infusion on allograft immune tolerance, based on allogeneic skin transplantation in mice, and to provide clues for designing new strategies to prevent graft rejection.
Methods: donor mice induced by dexamethasone (Balb/c, H-2d) on thymocyte apoptosis: using DNA Ladde gel electrophoresis and Annexin V / PI cells after staining by flow cytometry to detect cell apoptosis; BALB/c mouse thymus cells were stained with PKH67 staining, apoptosis induction, intravenous infusion of MHC type adoptive do not match the mice (C57BL/6, H-2b), after 12 hours, with anti CD11c-PE antibody labeled dendritic cells from the spleen cells, confocal microscopy observation of dendritic cells phagocytosis of apoptotic thymocytes; cell adoptive 7 days later, the BALB /c mouse skin transplanted to C57BL/6 mice, observe the survival time of the grafts; 7 days after skin transplantation take, skin graft, infiltration of inflammatory cells in histological examination of tissue graft; mixed lymphocyte reaction evaluation of donor spleen cells stimulated the proliferation of T cells in mice; RT-PCR recipient mice spleen fine detection The mRNA expression level of TGF- beta, IL-10, Foxp3, and splenic dendritic cells in the recipient mice were detected by flow cytometry.
Result:
1. Inducing apoptosis and adoptive infusion of donor thymus cells, and the effect of DC phagocytosis of apoptotic thymocytes
1.DNA Ladder test and Annexin V / PI staining test confirmed that dexamethasone could significantly enhance thymocyte apoptosis in mice: the apoptosis rate induced by dexamethasone was 58.7%, and the apoptosis rate of 32.4%. cells was 32.4%. at the same time when cultured in vitro.
2.BALB/c mice thymocytes were pre stained by PKH67 and induced apoptosis. After tail vein infusion into C57BL/6 mice, 12 hours later, spleen cells were labeled with anti CD11c-PE antibody for DC, and PKH67+CD11c+ was observed under confocal laser scanning microscope, indicating that apoptotic thymocytes were phagocytosis by DC.
Two, adoptive infusion of donor apoptotic thymocytes to prolong the survival time and mechanism of skin graft in allogeneic mice
BALB/c mice served as donors and C57BL/6 mice as recipients. Back skin transplantation was performed. Before transplantation, the experimental group was injected with 2.5 * 10~7 apoptotic thymocytes via tail vein, and PBS, dexamethasone and third party mice (C3H mice) were used as controls.
1. graft survival time
Donor cell apoptotic cells infusion can significantly prolong the survival time of skin graft: the average survival time of the apoptotic cell treatment group is 17.750 + 3.956 days, the PBS group is 10.250 + 1.389 days, the dexamethasone group is 15.625 + 1.685 days, and the C3H group is 10.500 + 1.927 days. (n=8, P 0.01).
Histological examination of 2. skin graft
7 days after transplantation, the graft for paraffin section, stained by HE, the infiltration of inflammatory cells to evaluate the graft tissue. Compared with PBS control group and C3H group, infiltration of mononuclear cells significantly reduced thymocyte apoptosis in the transplanted tissue group, without capillary congestion, interstitial hemorrhage, arteritis and phlebitis the phenomenon; infiltration of mononuclear cells than the PBS control group and C3H group decreased slightly in the dexamethasone group transplanted tissue.
Lymphocyte proliferation of 3. recipients
On the 7 day after transplantation, donor spleen cells stimulated the recipient spleen T cells again, and the ability of the latter to respond to donor allo antigen was detected. The results showed that the proliferation of splenic T cells in donor cells was significantly lower than that in PBS control group and C3H group.
Expression of spleen cytokine mRNA in 4. recipients
7 days after transplantation, donor spleen cells and lymph nodes, the extraction of total RNA by RT-PCR detection of TGF- beta, IL-10, the expression level of Foxp3 mRNA. The results showed: the thymocyte apoptosis group of spleen TGF- beta, IL-10, the expression level of Foxp3 mRNA were higher than those in control group, there was significant difference between dexamethasone group; the spleen of IL-10, increased the expression level of Foxp3 mRNA. (P? 0.05)
Detection of DC subclass in the spleen of 5. recipients
On the 7 day after transplantation, donor subgroup splenocytes and flow cytometry were used to detect DC subclasses. Results showed that the proportion of CD11c+B220+DC in apoptotic thymocytes group increased significantly, and the proportion of CD11c+CD45RB+DC in apoptotic thymocyte group and dexamethasone group increased.
Conclusion:
The source of donor apoptotic thymocytes infusion of adoptive recipients, can promote the production and regulatory T cells, promote the immunosuppressive function of DC IL-10, TGF- beta expression; T cell proliferation caused by decreased activity of allogeneic mononuclear cell infiltration, reduce graft tissues and prolong allogeneic skin of mice the survival time of the grafts. The effect with donor antigen specificity. The results of this study suggest that adoptive donor apoptotic thymocytes infusion can inhibit allograft rejection and potential clinical value.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【引证文献】