去甲肾上腺素对NK92-MI细胞迁移及趋化因子受体表达的影响

发布时间：2018-02-06 06:08

本文关键词： 去甲肾上腺素 NK92-MI 迁移 CCR5 CX3CR1　出处：《中国医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 前言应激这一概念最初由内分泌学家H. Selye在20世纪30年代提出,是指机体在受到各种内外环境因素刺激时所出现的非特异性全身反应,可以由不同的生理、心理及综合因素引起。应激可诱导机体心血管系统、中枢神经系统、免疫系统等的变化,严重时可发生疾病甚至引起死亡。近年来,随着社会发展、环境变化、生活节奏加快,应激已成为威胁人类健康的不可忽视的因素。以往一直认为,应激损害机体的免疫功能,但现有研究表明,慢性应激具有免疫抑制作用,而短期急性应激可以促进免疫功能和健康,并且这种保护作用部分归因于应激激素对免疫细胞迁移的影响。急性应激时人外周血中NK细胞的数量迅速增加,源自于机体各种NK细胞池中NK细胞的释放,但有关应激是通过何种途径影响NK细胞此种再分布的报道较少。为此,我们检测了去甲肾上腺素(norepinephrine,NE)对NK92-MI细胞迁移及趋化因子受体表达的影响,以研究应激引起NK细胞再分布的可能机制。材料和方法 1、Transwell法检测不同浓度NE (5×10-12M、5×10-10M、5×10-9M、5×10-8M、5×10-6M)对NK92-MI细胞迁移的影响。 2、不同浓度NE (5×10-12M、5×10-10M、5×10-9M、5×10-8M)处理NK92-MI细胞20分钟、40分钟、1小时、2小时、4小时。Real-Time PCR检测各实验组趋化因子受体CCR5、CCR7、CXCR3、CXCR4、CX3CR1 mRNA的表达水平,并检测加入β-肾上腺素能受体阻滞剂普萘洛尔后趋化因子受体mRNA的表达水平。 3、不同浓度NE (5×10-10M、5×10-9M、5×10-8M、)处理NK92-MI细胞1小时。流式细胞术检测各实验组NK92-MI细胞表面趋化因子受体CCR5的表达情况。 4、MTT法测定不同浓度NE处理1小时对NK92-MI细胞杀伤活性的影响。结果 1、高浓度的NE (5×10-6M、5×10-8M、5×10-9M)对NK92-MI细胞的迁移有明显的促进作用(P0.05),随着NE浓度的降低,这种促进作用逐渐减弱甚至消失。 2、Real-Time PCR法检测到五种趋化因子受体中CCR5和CX3CR1 mRNA的变化明显且变化趋势较一致。各处理组中以5×10-9M浓度作用最为明显,当该浓度NE处理NK92-MI细胞1小时后,CCR5和CX3CR1 mRNA表达最高。5×10-7M普萘洛尔能阻断5×10-9M NE的促表达作用。 3、流式检测结果表明,NK92-MI细胞表面无CCR5的表达。 4、效靶比为4：1时,高浓度的NE (5×10-5M、5×10-6M)对NK92-MI细胞的杀伤活性具有明显的抑制作用(P0.05),其余浓度的NE (5×10-7M、5×10-8M、5×10-9M、5×10-10M、5×10-11M、5×10-12M)虽然能降低NK92-MI细胞的杀伤活性,但与对照组相比无统计学意义(P0.05)。结论应激浓度的NE可以直接促进NK92-MI细胞迁移,并且对其杀伤活性无明显的抑制作用。应激浓度的NE作用1小时可以显著增加趋化因子受体CCR5和CX3CR1 mRNA表达,而这种促表达作用可被非选择性β-肾上腺素能受体阻滞剂普萘洛尔阻断。在NK92-MI细胞表面没有检测到趋化因子受体CCR5的表达。
[Abstract]:Foreword The concept of stress, originally proposed by endocrinologist H. Selye in 1930s, refers to the nonspecific systemic response of the body when stimulated by various internal and external environmental factors. Stress can induce changes in the cardiovascular system, central nervous system, immune system, etc. In recent years, severe diseases and even death may occur. With the development of society, the change of environment and the quickening of life rhythm, stress has become a factor that can not be ignored as a threat to human health. It has always been thought that stress impairs the immune function of the body, but the existing research shows that chronic stress has immunosuppressive effect, while short-term acute stress can promote immune function and health. This protective effect is partly due to the effect of stress hormones on the migration of immune cells. The number of NK cells in peripheral blood increases rapidly during acute stress. Although NK cells are released from various NK cell pools, there are few reports about how stress affects the redistribution of NK cells. We examined the effects of norepinephrineine (NE) on migration and chemokine receptor expression in NK92-MI cells. To study the possible mechanism of NK cell redistribution induced by stress. Materials and methods 1Transwell method was used to detect 5 脳 10 ~ (-12) M ~ (5 脳 10 ~ (-10)) M ~ (-1) and 5 脳 10 ~ (-9) M ~ (5 脳 10 ~ (-9)) M ~ (-1). The effect of 5 脳 10 ~ (-6) M) on the migration of NK92-MI cells. (2) NK92-MI cells were treated with 5 脳 10 -12M (5 脳 10 -10M) and 5 脳 10 -9M (5 脳 10 -8M) for 20 minutes, 40 minutes and 1 hour. The chemokine receptor CCR5, CCR7, CXCR3 and CXCR4 were detected by Real-Time PCR. The expression level of CX3CR1 mRNA and the expression of chemokine receptor mRNA were detected after 尾 -adrenoceptor blocker was added to propranolol. 3, 5 脳 10 ~ (-10) M ~ (5 脳 10 ~ (-9)) M ~ (5 脳 10 ~ (-9) M) ~ 5 脳 10 ~ (-8) M. NK92-MI cells were treated for 1 hour. The expression of chemokine receptor (CCR5) on the surface of NK92-MI cells was detected by flow cytometry. 4 MTT assay was used to determine the effect of different concentrations of NE on the cytotoxicity of NK92-MI cells for 1 hour. Results 1. The high concentration of NE 5 脳 10 -6 M (5 脳 10 -8 MN 5 脳 10 -9 M) significantly promoted the migration of NK92-MI cells (P 0.05). With the decrease of NE concentration, the promoting effect gradually weakened or even disappeared. 2. Detection of CCR5 and CX3CR1 in five chemokine receptors by Real-Time PCR. The change of mRNA was obvious and the change trend was the same. The effect of 5 脳 10 ~ (-9) M concentration was the most obvious in each treatment group. NK92-MI cells were treated with NE for 1 hour. The expression of CCR5 and CX3CR1 mRNA was up to 5 脳 10 ~ (-7) M propranolol which blocked the expression of 5 脳 10 ~ (-9) M NE. 3. Flow cytometry showed that there was no expression of CCR5 on the surface of NK92-MI cells. (4) when the effective / target ratio was 4: 1, the high concentration of NE 5 脳 10 -5 M (5 脳 10 -6 M) inhibited the cytotoxicity of NK92-MI cells significantly (P 0.05). Other concentrations of NE 5 脳 10 -7 M ~ (5 脳 10 ~ (-8)) M ~ (5 脳 10 ~ (-9)) M ~ (5) 脳 10 ~ (-9) MN ~ (5) 脳 10 ~ (-10) M ~ (-1). Although 5 脳 10 ~ (-12) M) could reduce the killing activity of NK92-MI cells, there was no significant difference between the control group and the control group (P 0.05). Conclusion The stress concentration of NE can directly promote the migration of NK92-MI cells. No significant inhibitory effect was found on the cytotoxic activity. The expression of chemokine receptor CCR5 and CX3CR1 mRNA was significantly increased by the stress concentration of NE for 1 hour. The expression was blocked by propranolol, a non-selective 尾 -adrenergic receptor blocker. No chemokine receptor CCR5 expression was detected on the surface of NK92-MI cells.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.6

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