经鼻给予热休克蛋白60诱导免疫耐受对动脉粥样硬化的影响及其机制研究

发布时间：2018-08-16 10:33

【摘要】：第一部分鼻饲热休克蛋白60对动脉粥样硬化形成及进展的影响目的：研究HSP60经鼻给药后对动脉粥样硬化的形成及发展的影响,为临床应用抗原诱导免疫耐受治疗慢性炎性免疫性疾病提供依据。方法：将6周龄的ApoE-/-雄性小鼠随机分为HSP60组和PBS组,HSP60组与PBS组再分别随机分为7周龄给药组(早期组)和16周龄给药组(晚期组)。HSP60组ApoE-/-小鼠经鼻给予HSP60,对照组经鼻给予PBS,在16周龄或24周龄时,冰冻切片后,用HE和油红O染色法观察处理组和对照组小鼠主动脉根部粥样斑块的面积大小,比较两者之间是否有统计学差异。结果：对于7周龄时给药的ApoE-/-小鼠,在16周龄时,HSP60组小鼠较PBS对照组小鼠其主动脉根部斑块面积明显减少,达53.7%之多(226432±29676μm2vs489250±31339μm2;P0.01)。而对于10周龄时给药ApoE-/-小鼠,在24周龄时,HSP60组小鼠与PBS对照组小鼠主动脉根部粥样斑块面积无显著统计学差异(845917士61180μm2vs800685±39654μm2；P0.05)。结论：经鼻给予HSP60可降低ApoE-/-小鼠动脉粥样硬化的形成,而对动脉粥样硬化的进展似乎没有明显抑制作用,且短暂给予HSP60后能保持长期的动脉粥样硬化抑制效果(至少10周),为临床上提供了一种潜在的动脉粥样硬化治疗方法。第二部分鼻饲热休克蛋白60抑制动脉粥样硬化的潜在机制目的：研究经鼻给予HSP60诱导免疫耐受减少动脉粥样硬化形成的机制,探讨Treg细胞及细胞因子TGF-β在免疫耐受诱导中的作用。方法：6周龄ApoE-/-雄性小鼠高质饮食一周后经鼻给予HSP60或PBS作为对照,鼻饲后第4天和第14天,应用流式细胞学技术检测ApoE-/-小鼠颈部淋巴结(CLNs)和脾脏中CD4+LAP+和CD4+CD25+Foxp3+Treg细胞的数量及组成比例；ELISA被用于鼻饲后第4天、第14天和第8周细胞因子TGF-β及NF-γ等水平的测定；鼻饲后8周,用Real-time PCR实验方法测定ApoE-/-小鼠主动脉斑块内TGF-β、INF-γ、CD25、 Foxp3、MCP-1等细胞因子的水平。结果：流式细胞学发现鼻饲后4天,小鼠脾脏和CLNs中CD4+CD25+LAP+T细胞占所有CD4+T细胞的百分比在HSP60与PBS两组间没有显著差异,但是,鼻饲后14天,我们观察到HSP60处理组小鼠脾脏和CLNs中CD4+CD25-LAP+T细胞占所有CD4+T细胞的百分比较PBS处理组小鼠有明显升高(P＜0.01)；ELISA发现鼻饲后4天,14天和8周三个时间点,HSP60组小鼠CLNs的TGF-β水平较PBS组都有明显升高(P0.01),鼻饲后8周INF-γ水平HSP60处理组小鼠较PBS处理组小鼠有显著性降低(P0.05):Real-time PCR发现HSP60处理组两种炎性标志物MCP-1和INF-γ的表达较对照组有明显降低(P0.05),对于Treg细胞特异性标志物TGF-β,其相对mRNA表达水平在鼻饲HSP60组比PBS组有显著性升高(P0.05)。结论：鼻饲HSP60致动脉粥样硬化形成减轻可能与诱导Treg细胞生成有关,尤其是CD4+CD25-LAP+T细胞,且这一有益作用的潜在机制也许和Treg细胞分泌的细胞因子TGF-β关系密切。
[Abstract]:Part I effects of nasal feeding of heat shock protein 60 on the formation and progression of atherosclerosis objective: to study the effect of HSP60 on the formation and development of atherosclerosis after nasal administration. To provide evidence for clinical application of antigen-induced immune tolerance in the treatment of chronic inflammatory immune diseases. Methods: six week-old ApoE-r-male mice were randomly divided into HSP60 group, PBS group, HSP60 group and PBS group. They were randomly divided into 7-week-old group (early group) and 16-week-old group (late group). HSP60 group was given intranasal administration of HSP60, and the control group was treated with HSP60. Nasal administration of PBSs at 16 or 24 weeks of age, After frozen sections, HE and oil red O staining were used to observe the size of atherosclerotic plaques in the root of aorta of the treated and control mice, and to compare whether there were statistical differences between the two groups. Results: the aortic root plaque area in HSP60 group was significantly decreased to 53.7% (226432 卤29676 渭 m2vs489250 卤31339 渭 m 2P0.01) compared with that in PBS control group at the age of 16 weeks (226432 卤29676 渭 m2vs489250 卤31339 渭 m ~ (2) P 0.01). However, there was no significant difference in the area of aortic root atherosclerotic plaque between HSP60 group and PBS control group at 24 weeks of age (845917 卤61180 渭 m2vs800685 卤39654 渭 m ~ (-2) P 0.05). Conclusion: nasal administration of HSP60 can reduce the formation of atherosclerosis in ApoE-r-mice, but it does not seem to inhibit the progression of atherosclerosis. Short administration of HSP60 can maintain long-term atherosclerotic inhibition (at least 10 weeks), which provides a potential therapy for atherosclerosis. Part two the potential mechanism of heat shock protein 60 (HSP60) in inhibiting atherosclerosis by nasal feeding objective: to study the mechanism of immune tolerance induced by nasal administration of HSP60 to reduce the formation of atherosclerosis. To investigate the role of Treg cells and cytokine TGF- 尾 in the induction of immune tolerance. Methods after a week of high quality diet, HSP60 or PBS were given to the male mice at the age of 6 weeks as control, and on the 4th and 14th days after nasal feeding, the mice were treated with HSP60 or PBS. The number and proportion of CD4 LAP and CD4 CD25 Foxp3 Treg cells in cervical lymph nodes and spleen of ApoE-r-mice were detected by flow cytometry. The levels of cytokines TGF- 尾 and NF- 纬 were determined by Elisa on the 4th, 14th and 8th week after nasal feeding. At 8 weeks after nasal feeding, the levels of cytokines such as TGF- 尾 -INF- 纬 CD25, Foxp3 and MCP-1 in aortic plaques of ApoE-r-mice were measured by Real-time PCR assay. Results: on day 4 after nasal feeding, the percentage of CD4 CD25 LAP T cells in spleen and CLNs in all CD4 T cells was not significantly different between HSP60 and PBS groups. However, 14 days after nasal feeding, there was no significant difference in the percentage of CD4 CD25 LAP T cells between HSP60 and PBS groups. We observed that the percentage of CD4 CD25-LAP T cells in spleen and CLNs of HSP60 treated mice was significantly higher than that of PBS treated mice (P < 0. 01). ELISA found that the level of TGF- 尾 in HSP60 group was significantly higher than that in PBS group at 14 days and 8 weeks after nasal feeding (P0.01), and the level of INF- 纬 in HSP60 group was significantly lower than that in PBS group at 8 weeks after nasal feeding (P0.05). Compared with the control group, the expression of MCP-1 and INF- 纬 in the two inflammatory markers were significantly decreased (P0.05). For TGF- 尾, the relative mRNA expression level in the HSP60 group was significantly higher than that in the PBS group (P0.05). Conclusion: the reduction of atherosclerosis induced by nasal feeding of HSP60 may be related to the induction of Treg cells, especially CD4 CD25-LAP T cells, and the potential mechanism of this beneficial effect may be closely related to the cytokine TGF- 尾 secreted by Treg cells.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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