钙结合蛋白S100A8、S100A9在慢性阻塞性肺疾病大鼠肺泡巨噬细胞中的表达及作用研究

发布时间：2018-03-28 06:16

本文选题：COPD　切入点：S100A8　出处：《川北医学院》2017年硕士论文

【摘要】：目的:探讨钙结合蛋白S100A8、S100A9在慢性阻塞性肺疾病(COPD)大鼠肺泡巨噬细胞中的表达及其作用。方法:1.将12只健康成年雄性大鼠随机分为两组:COPD组和正常组。采用烟熏加气管内注射内毒素方法1个月建立COPD大鼠模型。显微镜下观察两组大鼠肺组织病理形态,图像分析软件测定肺平均内衬间隔(MLI)、平均肺泡数(MAN)、肺泡腔面积与总面积比(PAA),瑞氏染色法检测两组大鼠支气管肺泡灌洗液(BLAF)中细胞总数、肺泡巨噬细胞、淋巴细胞、中性粒细胞的绝对值和比例,分离、培养COPD组和正常组大鼠肺泡巨噬细胞,分别予以不同剂量S100A8、S100A9处理6h、12h。采用酶联免疫吸附法(ELSA)检测大鼠肺泡巨噬细胞上清液中IL-6、IL-8和TNF-a的浓度,原位杂交方法和免疫组化方法观察离体培养的大鼠肺泡巨噬细胞中S100A8、S100A9mRNA和S100A8/A9蛋白的表达情况。结果:(1)与正常对照组比较,COPD组大鼠肺组织出现了典型的COPD病理改变,且MLI、PAA比正常对照组明显增高(P0.05),而MAN则显著低于正常对照组(P0.05)。(2)COPD组BALF中细胞总数与各炎症细胞的绝对值较正常对照组显著增高(P均0.05),其中,肺泡巨噬细胞绝对值升高最为明显。(3)原位杂交显示S100A8、S100A9mRNA主要表达在大鼠肺泡巨噬细胞细胞质与细胞膜,统计分析示COPD组大鼠肺泡巨噬细胞中S100A8、S100A9mRNA表达较正常对照组显著增加(P0.05)。(4)免疫组化显示S100A8/A9蛋白也主要表达在大鼠肺泡巨噬细胞细胞质和胞膜,统计分析示COPD组大鼠肺泡巨噬细胞中S100A8/A9蛋白表达量亦较正常对照组明显增加(P0.05))。(5)ELISA结果显示:S100A8、S100A9刺激两组大鼠肺泡巨噬细胞释放炎症因子IL-6、IL-8、TNF-α呈剂量和时间依赖性增加。在相同浓度的S100A8、S100A9作用相同时间时,COPD组大鼠肺泡巨噬细胞上清液中IL-6、IL-8、TNF-α的浓度均高于正常对照组(P均0.05)。相同剂量S100A8与S100A9作用大鼠肺泡巨噬细胞比较,S100A8处理两组大鼠肺泡巨噬细胞后上清液中IL-8和TNF-α的浓度均高于S100A9处理两组大鼠肺泡巨噬细胞后上清液中IL-8和TNF-α的浓度(P均0.05);而较高剂量S100A8刺激COPD及正常大鼠肺泡巨噬细胞分泌IL-6的量也均高于S100A9刺激两组大鼠肺泡巨噬细胞分泌IL-6的量(P均0.05)。结论:(1)COPD大鼠模型可以通过烟熏加气管内注射内毒素方法建成。(2)COPD大鼠肺泡巨噬细胞内S100A8、S100A9mRNA以及S100A8/A9蛋白的表达量较正常对照组明显增加。(3)S100A8、S100A9呈时间、浓度依赖性刺激大鼠肺泡巨噬细胞分泌炎症因子IL-6、IL-8和TNF-α增多。(4)S100A8、S100A9刺激COPD大鼠肺泡巨噬细胞分泌IL-6、IL-8和TNF-α增多作用较正常大鼠肺泡巨噬细胞更明显。(5)S100A8刺激大鼠肺泡巨噬细胞分泌IL-6、IL-8和TNF-α增多作用明显强于S100A9。
[Abstract]:Objective: to investigate the expression and role of calcium binding protein S100A8 (S100A8) S100A9 in alveolar macrophages of rats with chronic obstructive pulmonary disease (COPD). Methods: 1. Twelve healthy adult male rats were randomly divided into two groups: the control group and the control group. COPD rat model was established by endotoxin injection into trachea for 1 month. The pathological morphology of lung tissue was observed under microscope in two groups. The image analysis software was used to measure the mean lining interval (MLI), the average number of alveoli (MAN), the ratio of alveolar cavity area to total area (PAA). The total number of cells, alveolar macrophages and lymphocytes in bronchoalveolar lavage fluid (BLAF) of the two groups were detected by Rayleigh staining. The absolute value and proportion of neutrophils, isolation and culture of alveolar macrophages in COPD group and normal group were treated with different doses of S100A8 / S100A9 for 6 h and 12 h respectively. The concentrations of IL-6IL-8 and TNF-a in the supernatant of rat alveolar macrophages were detected by enzyme-linked immunosorbent assay (Elisa). The expression of S100A8, S100A9 mRNA and S100A9 protein in cultured rat alveolar macrophages was observed by in situ hybridization and immunohistochemistry. Compared with the normal control group, the total number of BALF cells and the absolute values of the inflammatory cells in the MAN group were significantly higher than those in the normal control group (P 0.05), especially in the normal control group (P < 0.05), and the total number of BALF cells and the absolute values of the inflammatory cells in the BALF group were significantly higher than those in the normal control group (P < 0.05). In situ hybridization showed that S100A8 and S100A9 mRNA were mainly expressed in the cytoplasm and cell membrane of alveolar macrophages in rats. Statistical analysis showed that the expression of S100A8, S100A9 mRNA in alveolar macrophages in COPD group was significantly higher than that in normal control group (P 0.05, P 0.05, P < 0.05). Immunohistochemical staining showed that S100A8/A9 protein was also mainly expressed in the cytoplasm and membrane of alveolar macrophages in rats. Statistical analysis showed that the expression of S100A8/A9 protein in alveolar macrophages in COPD group was also significantly higher than that in normal control group. The results of Elisa showed that the inflammatory factor IL-6 and IL-8 TNF- 伪 were increased in a dose-and time-dependent manner after stimulated by S100A8 and S100A9 in two groups. At the same time, the concentration of IL-6 and IL-8 TNF- 伪 in alveolar macrophage supernatant of COPD group was higher than that of normal control group (P < 0.05). The comparison of alveolar macrophages treated with S100A8 and S100A9 in the same dose of S100A8 and S100A9 was made. The concentrations of IL-8 and TNF- 伪 in supernatants of alveolar macrophages were higher than those of IL-8 and TNF- 伪 in the supernatants of alveolar macrophages treated with S100A9 in rats (P < 0.05), while the concentrations of IL-6 secreted by COPD and normal alveolar macrophages were stimulated by high dose S100A8. The amount of IL-6 secreted by alveolar macrophages stimulated by S100A9 was also higher than that of both groups (P < 0.05). Conclusion the S100A8 S100A9 mRNA and the surface of S100A8/A9 protein in alveolar macrophages can be established by smoking and endotoxin injection into the alveolar macrophages. Compared with the normal control group, the volume of S100A8 and S100A9 increased significantly. In a dose-dependent manner, the cytokines IL-6, IL-8 and TNF- 伪 from alveolar macrophages were stimulated in a dose-dependent manner. S100A8 and S100A9 stimulated the production of IL-6, IL-8 and TNF- 伪 by alveolar macrophages in COPD rats, which were more obvious than those in normal rat alveolar macrophages. 5S100A8 stimulated alveolar macrophages in rats. The increase of IL-6, IL-8 and TNF- 伪 was stronger than that of S100A9.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.9

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