大鼠骨髓间充质干细胞分泌因子对Ⅳ型变态反应的影响

发布时间：2018-01-01 17:02

本文关键词：大鼠骨髓间充质干细胞分泌因子对Ⅳ型变态反应的影响　出处：《青岛大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的：观察骨髓间充质干细胞分泌因子对迟发型超敏反应的影响,并初步探讨其作用机制,为骨髓间充质干细胞分泌因子在临床上用于过敏性疾病的治疗提供一定的理论依据。方法：体内试验：①分离培养大鼠骨髓间充质干细胞(Bone marrow mesenchymal stem cells, BMSCs),贴壁法纯化,收集第3代细胞融合达85%以上的上清液,获取干细胞分泌因子。②制备小鼠接触性皮炎模型,观察干细胞分泌因子对小鼠耳廓肿胀的抑制作用及胸腺指数和脾脏指数的影响。③MTT比色法反映分泌因子对小鼠脾淋巴细胞增殖的影响。④采用酶联免疫吸附测定法(ELISA法)检测分泌因子对血清IL-2、IFN-γ细胞因子水平的影响。体外实验：①分离培养大鼠BMSCs,贴壁法纯化,收集第三代细胞融合达85%以上的BMSCs上清液,获取干细胞分泌因子。②无菌获取小鼠胸腺、脾脏,制备单细胞悬液,MTT比色法观察高、中、低剂量组干细胞分泌因子对ConA诱导的淋巴细胞增殖的影响；③分离培养大鼠腹腔巨噬细胞,中性红法检测干细胞分泌因子对脂多糖(LPS)刺激后巨噬细胞吞噬活性的影响。结果体内试验：①与对照组相比,干细胞分泌因子和地塞米松(DEX)均能够明显减轻模型小鼠耳廓肿胀程度,降低胸腺、脾脏指数,抑制脾淋巴细胞的增殖(P＜0.05),但分泌因子的抑制作用弱于DEX (P0.05)。②BMSCs分泌因子与DEX均能够显著降低血清中IL-2、IFN-γ水平(P＜0.05)。体外实验：①与对照组相比,细胞分泌因子高、中剂量和DEX对ConA诱导的小鼠胸腺淋巴细胞增殖均有抑制作用(P＜0.05),高剂量组的抑制作用强于中剂量组(P＜0.05),弱于DEX组(P＜0.05)；分泌因子高剂量组和DEX组对ConA诱导的小鼠脾脏淋巴细胞增殖亦有抑制作用(P＜0.05)。②分泌因子能够减弱经LPS刺激后巨噬细胞吞噬中性红的能力(P＜0.05)。结论骨髓间充质干细胞分泌因子具有抗迟发型超敏反应的作用,其抑制特异性T淋巴细胞增殖,降低淋巴细胞分泌IL-2、IFN-γ细胞因子水平,降低活化的巨噬细胞吞噬功能是其发挥作用的机制之一。
[Abstract]:Objective: To observe the effect of bone marrow mesenchymal stem cells secretory factor on delayed type hypersensitivity, and to explore its mechanism of action, providing a theoretical basis for the clinical application of bone marrow mesenchymal stem cells secretory factor in allergic diseases.
Methods: in vivo experiment: the isolation and culture of rat bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMSCs), purified by attachment method, collect the third generation of more than 85% of the supernatant of cell fusion, stem cell factor secretion. Contact dermatitis mouse model by the system, observe the effect of stem cell factor secretion inhibition effect on mouse ear swelling and thymus index and spleen index. The MTT colorimetric method to reflect the influence of cytokines on the proliferation of mouse spleen lymphocytes. 4 by enzyme-linked immunosorbent assay (ELISA) detection of secreted IL-2 clear of blood, the impact factor level of IFN- gamma cells. In vitro: 1. Isolation and culture rat BMSCs, purified by attachment method, collect the third generation of BMSCs was more than 85% cell fusion, stem cell factor secretion. The Sterility from mouse thymus, spleen, preparation of single cell suspension, MTT colorimetric method view The effects of stem cell secretory factors on ConA induced lymphocyte proliferation in rats were observed. (3) the peritoneal macrophages were isolated and cultured, and the effects of stem cell secretory factors on phagocytosis of macrophages stimulated by lipopolysaccharide (LPS) were detected by neutral red method.
The results of experiment in vivo: compared with control group, stem cell factor secretion and dexamethasone (DEX) can significantly reduce the mice ear swelling, reduce thymus, spleen index, inhibit the proliferation of spleen lymphocytes (P < 0.05), but the inhibitory effect of secretory factors is weaker than that of DEX (P0.05). The secretion of BMSCs and factor DEX can significantly reduce the serum IL-2, IFN- levels (P < 0.05). In vitro experiment: compared with control group, cell factor secretion inhibition in high dose and DEX on mouse thymus lymphocyte proliferation were induced by ConA (P < 0.05), a strong inhibiting effect of high dose group in middle dose group (P < 0.05), weaker than the DEX group (P < 0.05); secreted in high dose group and DEX group also have inhibitory effect on mice spleen lymphocyte proliferation induced by ConA (P < 0.05). The secreted factor can decrease after LPS stimulation of macrophage phagocytosis of neutral red can Force (P < 0.05).
Conclusion the secretion factor of bone marrow mesenchymal stem cells has the effect of anti delayed hypersensitivity. It inhibits the proliferation of specific T lymphocytes, lowers the secretion of IL-2 and IFN- gamma cytokines by lymphocytes, and reduces the phagocytosis function of activated macrophages, which is one of the mechanisms of its action.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R758.2

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