【作者】 李怡芳；

【导师】 栗原；

【作者基本信息】 暨南大学 ， 免疫肿瘤药理学， 2010， 硕士

【摘要】 目的：在本实验中,将小鼠放置在狭窄的拘束环境中,限制自由运动,使其烦躁和焦虑来模拟心理应激状态。在此应激模型上研究营养型鸡精(Brands Essence of Chicken, BEC)对小鼠免疫功能的保护作用及其机制,旨在为营养型鸡精作为保健品的广泛应用提供有益的实验依据,同时可为“治未病”提供现代医学的评价方法和治疗手段。方法：(1)本研究建立小鼠拘束应激模型,观察拘束负荷后0、3、5、7、10天小鼠免疫器官指数、脾脏和胸腺淋巴细胞总数,以及测定小鼠脾脏NK细胞的活性,以观察拘束应激对免疫系统的影响以及确定拘束负荷的最佳恢复时间。(2)其次,研究了营养型鸡精(BEC)对拘束小鼠免疫功能的保护作用。实验将KM小鼠随机分为正常对照组,拘束应激组,BEC-L(12 ml/kg)组及BEC-H(24 ml/kg)组,每组7只。除正常对照组外,其余各组连续灌胃给药5天后拘束负荷18 h,拘束恢复3 d后取胸腺和脾脏分别测定免疫器官指数、脾脏和胸腺淋巴细胞总数、脾脏和胸腺淋巴细胞亚群Th(CD3+CD4+)与Ts(CD3+CD8+)的百分比、脾淋巴细胞NK细胞的活性。(3)最后,深入研究了BEC对拘束应激诱导的小鼠脾淋巴细胞凋亡的改善作用,以此来探讨BEC改善拘束小鼠免疫功能的机制。实验将KM小鼠随机分为正常对照组,拘束应激组,RU486(糖皮质激素受体拮抗剂,25mg/kg)组,BEC-L(12 ml/kg)组及BEC-H(24 ml/kg)组,每组10只。除正常对照组外,其余各组连续灌胃给药5天后拘束负荷18 h,拘束恢复3 d后取血取脾以测定免疫器官指数、脾淋巴细胞总数、血浆皮质酮的含量、线粒体膜电位、线粒体和细胞质CytC的含量以及GR mRNA、Bax mRNA和Bcl-2 mRNA的表达。结果：(1)小鼠拘束应激后,脾脏和胸腺明显萎缩,脾脏和胸腺细胞数目明显较少,NK细胞活性也明显下降,且拘束恢复3天后这种免疫抑制作用最为明显(2) BEC-L和BEC-H能够显著提高免疫器官指数和增加脾脏和胸腺淋巴细胞总数；BEC-L和BEC-H明显升高拘束小鼠脾脏Th(CD3+CD4+)亚群百分率和降低Ts(CD3+CD8+)亚群百分率,使小鼠脾脏T淋巴细胞中的Th/Ts比值升高,使应激小鼠的脾脏免疫细胞亚群比值趋向正常状态；同时,BEC-H可明显降低拘束小鼠胸腺Th(CD3+ CD4+)或Ts(CD3+CD8+)百分率,使其恢复正常；BEC-H能显著增强脾NK细胞的活性。(3)BEC和RU486可以明显改善拘束应激对小鼠脾脏的损伤和提高脾脏指数和脾淋巴细胞总数；BEC能显著降低应激小鼠血浆中皮质酮的含量和升高GR mRNA的基因表达；同时,BEC和RU486能显著下调拘束小鼠脾淋巴细胞中促凋亡因子Bax mRNA到水平和上调凋亡抑制因子Bcl-2 mRNA的水平,明显提高线粒体膜电位,增加线粒体CytC的含量和减少细胞质CytC的含量。结论：BEC有明显的抗应激作用,能够抑制应激小鼠淋巴细胞的凋亡以维护淋巴细胞的数目和功能,从而发挥免疫保护作用。创新点：首次研究和探讨了营养型鸡精能缓解拘束应激对小鼠的免疫抑制作用,且发现营养型鸡精的免赋活作用与其对神经内分泌系统的调节作用有关。更多还原

【Abstract】 Objective:To investigate the protective effects of BEC(Brands Essence of Chicken) on immune function in mice loaded with restraint stress.Methods:(1)The changes of spleen and thymus index, the total number of spleen and thymus lymphocytes as well as the activity of NK cells in the spleen of mice sacrificed 0,3,5, 7 and 10 days after restraint stress were observed and compared.(2) In order to study the effects of BEC on immune system in restraint stressed mice, KM mice were randomly divided into 4 groups with 7 mice each group:normal control group, restraint control group, BEC-L (12 ml/kg) group and BEC-H (24 ml/kg)group. All mice were restraint for 18 h except normal group. All mice were sacrificed 3 days after restraint. The index of immune organs, the total number of spleen and thymus lymphocytes, the subsets of lymphocytes Th(CD3+CD4+) and Ts(CD3+CD8+) in spleen and thymus, the activity of NK cells in the spleen were investigated.(3) KM mice were randomly divided into 5 groups with 10 mice each group:normal control group, restraint control group and RU486 (GR antagonist) group, BEC-L group and BEC-H group. All mice were restraint for 18 h except normal group. All mice were sacrificed 3 days after restraint. Blood was distilled and spleens were received to prepare lymphocytes for testing spleen index, total number of spleen lymphocytes, the content of plasma corticosterone, mitochondrion membrane potential (△Ψmt), the contents of CytC in mitochondrion and cytoplasma, the expression of GR, Bax and Bcl-2 mRNAs in spleen lymphocytes.Results:(1) The results showed that, after restraint stress, the spleen and thymus index, the total number of spleen and thymus lymphocytes obviously decreased, and the activity of NK cells in the spleen significantly decreased, indicating the suppressive effect of restraint stress on immune system of mice especially 3 days after restraint. (2) BEC-L and BEC-H could significantly elevate the index of immune organs and the total number of spleen and thymus lymphocytes in restraint stressed mice. BEC-L and BEC-H could obviously elevate the percentage of Th(CD3+CD4+) and reduce the percentage of Ts(CD3+CD8+) in spleen lymphocytes of stressed mice, resulting in the increase of Th/Ts ratios which indicated the recovery of immune function in spleen of stressed mice. BEC-H could obviously reduce the percentages of both Th and Ts in thymus of stressed mice, resulting in the recovery of immune function in thymus of stressed mice. BEC-H could also increasethe activity of NK cells in the spleen. All these results indicated the recovery effects of BEC on immune function.(3) It is showed BEC and RU486 and significantly elevated spleen index and the total number of spleen lymphocytes. BEC also obviously reduced the content of plasma corticosterone in restraint mice. RT-PCR analysis showed BEC and RU486 could markedly up-regulate the expression of GR and Bcl-2 mRNAs, down-regulate the expression of Bax mRNA in spleen lymphocytes of stressed mice. Significantly, reduced mitochondrion membrane potential (△Ψmt) in spleen lymphocytes was elevated when treated with BEC and RU486. Moreover, BEC and RU486 recovered the contents of CytC mitochondrion and cytoplasm in stressed mice. all the above demonstrated that BEC could inhibit the stress-induced apoptosis of lymphocytes in spleen.Conclusion:BEC had obvious anti-stress effects on the function of immune system. The mechanism may be related with its regulation on neuroendocrine system.Novel points:This study firstly investigated and analyzed the effects of BEC on immune function of restraint stressed mice. Moreover, the effects of BEC on immune function were found to relate with the activity of BEC on neuroendocrine system.更多还原