过敏毒素C5a刺激肥大细胞脱颗粒促进肝细胞损伤

发布时间：2018-06-25 13:55

本文选题：肥大细胞 + CD88　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的探讨建立CD88表型肥大细胞的方法,研究融合蛋白C5a刺激肥大细胞脱颗粒情况,利用氯化钴刺激肝实质细胞,建立肝实质细胞缺氧损伤模型,将脱颗粒的肥大细胞与肝实质细胞AML12共培养,观察肥大细胞脱颗粒对受损肝实质细胞的影响。方法 (1)提取骨髓源肥大细胞和皮肤源肥大细胞,流式鉴定肥大细胞表型以及甲苯胺蓝法鉴定肥大细胞;(2)应用PMA或者离子霉素处理骨髓源肥大细胞和P815细胞后,流式鉴定其CD88表达情况,并寻找PMA和离子霉素刺激肥大细胞表达CD88的合适处理时间以及剂量;(3)蛋白免疫印迹法(Western blot)分析检测肥大细胞CD88、β-actin蛋白的表达水平的影响;(4)应用C5a融合蛋白刺激CD88表型的肥大细胞,β-内酰胺酶法检测肥大细胞脱颗粒情况;(5)应用氯化钴处理肝实质细胞AML12建立肝缺氧模型;(6)将脱颗粒的肥大细胞与受损的肝实质细胞AML12共培养,观察肥大细胞脱颗粒对受损肝实质细胞的影响。结果(1)通过细胞因子诱导分化,一段时间后可以获得大量高纯度的肥大细胞,皮肤提取肥大细胞未能获得肥大细胞;(2)骨髓源肥大细胞经过PMA或者离子霉素刺激后,流式检测其CD88表达明显上升,在时间和剂量的对比试验中发现,PMA和离子霉素刺激肥大细胞表达CD88的最适时间为24h,最适宜浓度分别为50ng/ml和1000ng/ml,而P815肥大细胞株未出现明显的CD88表达;(3)蛋白免疫印迹法结果提示:与未经PMA或离子霉素刺激的肥大细胞相比,经过刺激的肥大细胞,其CD88蛋白的表达明显增加;(4)融合蛋白C5a刺激CD88表型肥大细胞后,应用β-内酰胺酶法测肥大细胞脱颗粒发现,与未加C5a的对照组相比,其脱颗粒明显增加,且随融合蛋白C5a加入浓度的增加而增加,呈现出浓度依赖性;(5)根据文献报道,经过预实验,寻找合适的氯化钴浓度来建立肝实质细胞的缺氧损伤;(6)应用氯化钴建立肝实质细胞受损模型后发现,在轻微的肝实质细胞受损情况下,肥大细胞脱颗粒能够刺激肝实质细胞反应性的增生,而在重度肝实质细胞受损的情况下,肥大细胞脱颗粒能够进一步促进损伤的发生,发挥抑制肝实质细胞增殖的作用。结论通过细胞因子诱导可以获得大量高纯度的肥大细胞,经过PMA或离子霉素刺激后,其表面CD88表达明显上升,经C5a刺激后,肥大细胞脱颗粒并呈现浓度依赖性,脱颗粒的肥大细胞可以抑制重度受损的肝实质细胞增殖。
[Abstract]:Objective to investigate the method of establishing CD88 phenotypic mast cells, to study the degranulation of mast cells stimulated by fusion protein C5a, and to establish a model of hypoxia-induced liver parenchymal cells injury by using cobalt chloride to stimulate liver parenchymal cells. The effects of degranulation of mast cells on injured hepatic parenchymal cells were observed by co-culture of degranulated mast cells and hepatic parenchymal cells (AML12). Methods (1) Bone marrow-derived mast cells and skin derived mast cells were extracted, mast cell phenotypes were identified by flow cytometry and mast cells were identified by toluidine blue, (2) bone marrow-derived mast cells and P815 cells were treated with PMA or ionomycin. The expression of CD88 was detected by flow cytometry. The appropriate treatment time and dose of PMA and ionomycin to stimulate the expression of CD88 in mast cells were found; (3) Western blot analysis was used to detect the expression level of CD88 and 尾 -actin in mast cells; (4) C5a fusion protein was used to stimulate the expression of CD88. Type A mast cells, 尾 -lactamases were used to detect degranulation of mast cells; (5) liver hypoxia model was established by cobalt chloride treatment of hepatic parenchymal cells; (6) degranulated mast cells were co-cultured with damaged hepatic parenchymal cells (AML12). To observe the effect of mast cell degranulation on injured hepatic parenchymal cells. Results (1) after inducing differentiation by cytokines, a large number of high purity mast cells could be obtained after a period of time, but the mast cells extracted from the skin could not obtain mast cells, (2) the bone marrow-derived mast cells were stimulated by PMA or ionomycin. The expression of CD88 was significantly increased by flow cytometry. The optimal time for CD88 expression of mast cells stimulated by PMA and ionomycin was 24 h, and the optimal concentration was 50ng/ml and 1000ng / ml, respectively, but no CD88 expression was found in P815 mast cell line; (3) Western blot. The results suggest that compared with mast cells without PMA or ionomycin stimulation, (4) after stimulation of CD88 phenotypic mast cells by fusion protein C5a, the degranulation of mast cells was detected by 尾 -lactamase method, and it was found that the degranulation of mast cells was significantly increased compared with the control group without C5a. The concentration of C5a protein increased in a concentration-dependent manner. (5) according to the literature, the concentration of the fusion protein C5a increased. (6) the liver parenchymal cell damage model was established by using cobalt chloride to establish the liver parenchymal cell damage model, and it was found that the liver parenchymal cells were damaged in the case of mild hepatic parenchymal cell damage. Mast cell degranulation can stimulate the reactive proliferation of hepatic parenchyma cells, but in the case of severe hepatic parenchymal cell injury, mast cell degranulation can further promote the occurrence of injury and play a role in inhibiting the proliferation of hepatic parenchymal cells. Conclusion A large number of high purity mast cells can be obtained by cytokine induction. After stimulation with PMA or ionomycin, the expression of CD88 on the surface of mast cells increased significantly. After C5a stimulation, mast cells were degranulated in a concentration-dependent manner. Degranulated mast cells inhibit the proliferation of severely damaged hepatic parenchymal cells.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R657.3

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