茴香霉素对小鼠淋巴细胞行为及其同种异基因皮肤移植的影响

发布时间：2017-12-31 19:07

本文关键词：茴香霉素对小鼠淋巴细胞行为及其同种异基因皮肤移植的影响　出处：《暨南大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的：研究茴香霉素对小鼠淋巴细胞行为及其同种异基因皮肤移植的影响。方法：以活体染料羧基荧光素乙酰乙酸琥珀酰亚胺酯染色，建立在多克隆刺激剂刀豆蛋白A(ConA)刺激下评价小鼠淋巴细胞增殖的模型，通过流式细胞术和MTT法分析茴香霉素在不同剂量下对淋巴细胞增殖的作用；采用碘化丙锭染色分析茴香霉素对ConA或佛波醇酯(PDB)加离子霉素(Ion)刺激的小鼠淋巴细胞周期变化的作用；利用荧光标记的单克隆抗体双染技术和流式细胞术观察茴香霉素对小鼠CD3~+T细胞早期及中期活化标志分子CD69和CD25表达的影响。用MTT法检测茴香霉素作用下淋巴细胞在单向或双向混合淋巴细胞反应(MLR)中的反应性、对大鼠肝癌细胞(7919)的杀伤效应以及茴香霉素对原代淋巴细胞和7919细胞系的直接细胞毒作用。建立小鼠同种异基因皮肤移植模型，观察不同剂量茴香霉素对皮肤移植存活时间、迟发性超敏反应(DTH)及移植后小鼠淋巴细胞反应性的影响。结果：茴香霉素浓度在0.01～0.5ng／ml时，能够增强ConA对淋巴细胞的促增殖作用，以0.5ng／ml茴香霉素促进作用最明显；1.0～25.0ng／ml茴香霉素则能抑制ConA对淋巴细胞的促增殖作用。进一步发现，0.01～0.5ng／ml茴香霉素能够促进ConA诱导的淋巴细胞周期进入G2／M期，促进PDB加Ion刺激的淋巴细胞周期进入S期：1.0～25.0ng／ml茴香霉素能使ConA或PDB加Ion刺激的淋巴细胞周期停滞于G0／G1期，阻止其进入S期，以10.0ng／ml和25.0ng／ml茴香霉素的抑制作用最为明显。据此选用最佳剂量10.0ng／ml，茴香霉素能够明显抑制CD3~+T细胞表面分子CD69和CD25的表达(P均＜0.01)，，且能抑制单向或双向MLR中淋巴细胞的反应性(P均＜0.01)，其抑制作用与地塞米松相比无明显差异(P＞0.05)；茴香霉素剂量从0.1ng／ml逐渐增至100.0ng／ml，淋巴细胞对大鼠肝癌细胞的杀伤效应逐渐减弱，以10.0ng／ml和100.0ng／ml剂量组抑制作用最为明显(P＜0.01)；在上述剂量下，茴香霉素对小鼠原代淋巴细胞和大鼠肝癌7919细胞系无直接细胞毒作用。体内研究进一步发现，15.0mg／kg茴香霉素能够显著延长小鼠皮肤移植存活时间、抑制DTH发展及体内小鼠淋巴细胞的反应性，且上述作用明显强于环孢素A(CsA) (P＜0.01或P＜0.05)。结论：茴香霉素能明显抑制小鼠淋巴细胞的增殖、周期、活化、抗原反应性和杀伤效应等，抑制小鼠同种异基因皮肤移植的排斥反应，且作用优于CsA，这些发现可能为其作为新的免疫抑制剂的研发提供理论和实验依据。
[Abstract]:Objective: to study the effect of anisomycin on lymphocyte behavior and allogeneic skin transplantation in mice. Methods: in vivo dye carboxyl fluorescein acetoacetate succinimide ester staining was used. A model was established to evaluate lymphocyte proliferation in mice stimulated by concanavalin Cona, a polyclonal stimulator. The effect of anisomycin on lymphocyte proliferation was analyzed by flow cytometry and MTT. Using iodized propyl ingot staining, the effect of anisomycin on lymphocyte cycle changes induced by ConA or phorbol ester PDBs plus ionomycin Ion (Ion) in mice was analyzed. Observation of the effect of anisomycin on mouse CD _ 3 ~ ~ by fluorescence labeled monoclonal antibody double staining technique and flow cytometry. The expression of CD69 and CD25 in the early and middle stage of T cell activation. The lymphocyte reaction in unidirectional or bidirectional mixed lymphocyte was detected by MTT method. The reactivity in MLR. The cytotoxicity of anisomycin on primary lymphocytes and 7919 cell line was studied. The allogeneic skin transplantation model of mice was established. The survival time of different doses of anisomycin on skin transplantation was observed. Delayed hypersensitivity reaction (DTH) and lymphocyte reactivity after transplantation. Results: the concentration of anisome was 0. 01 ~ 0. 5 ng / ml. The effect of ConA on lymphocyte proliferation was enhanced, especially by 0.5 ng / ml anisamycin. Anisomycin at a dose of 25.0ng / ml inhibited the proliferation of lymphocytes induced by ConA. 0. 01 ~ 0. 5 ng / ml anisomycin could promote lymphocyte cycle induced by ConA into G 2 / M phase. To promote the lymphocyte cycle stimulated by PDB and Ion into S phase:. 1.0 ng / ml anisomycin could arrest the lymphocyte cycle induced by ConA or PDB plus Ion in G _ 0 / G _ 1 phase. The inhibitory effect of anisomycin was the most obvious in 10.0ng / ml and 25.0ng / ml of anisomycin, and the best dose was 10.0ng / ml. Anisamycin could significantly inhibit the expression of CD69 and CD25 on CD3T cells (P < 0.01). The reactivity of lymphocytes in unidirectional or bidirectional MLR was inhibited (P < 0.01), and there was no significant difference compared with dexamethasone (P > 0.05). The dose of anisomycin increased from 0.1 ng / ml to 100.0 ng / ml, and the killing effect of lymphocytes on hepatoma cells decreased gradually. The inhibitory effect of 10.0ng / ml and 100.0ng / ml groups was the most obvious (P < 0.01). Under the above dosage, anisomycin had no direct cytotoxic effect on primary lymphocytes of mice and rat hepatoma 7919 cell line. Anisomycin 15.0mg / kg could significantly prolong the survival time of mouse skin graft and inhibit the development of DTH and the reactivity of lymphocytes in vivo. The above effects were significantly stronger than that of cyclosporine (P < 0.01 or P < 0.05). Conclusion: anisomycin can significantly inhibit the proliferation, cycle and activation of lymphocytes in mice. The antigenic reactivity and killing effect could inhibit the rejection of allogeneic skin transplantation in mice and its effect was superior to that of CSA. These findings may provide a theoretical and experimental basis for the development of new immunosuppressants.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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