抑制性寡脱氧核苷酸对染矽尘小鼠早期炎症的影响及其机制探讨

发布时间：2018-04-21 03:16

本文选题：二氧化硅 + 矽肺　；参考：《重庆医科大学》2013年硕士论文

【摘要】：矽肺是因长期大量吸入游离二氧化硅(SiO2)粉尘所引起的以肺组织弥漫性纤维化为主要特征的全身性疾病。根据2011年4月18日卫生部向社会公布的《2010年职业病防治工作情况和2011年重点工作》，2010年新发职业病27240例，其中尘肺病23812例。目前尘肺病仍是我国最严重的职业病，2010年报告的尘肺病例数占职业病报告总例数的87.42%。有专家调查发现，能够做职业病诊断并在疾控中心登记的，仅为尘肺病患者的10%-20%，因此估计尘肺病实际发生的病例数不少于100万人。因此，进一步探讨矽肺的发病机制和寻找新型的治疗药物对矽肺的预防、治疗乃至最终消除矽肺具有重要的意义。目前的研究表明，T淋巴细胞由树突状细胞和巨噬细胞等抗原递呈细胞处理修饰二氧化硅后激活。虽然Th1/Th2极化在矽肺的发病机制中仍不完全明确，但研究表明，Th1型细胞因子在矽肺的炎症阶段起主要作用，而Th2型细胞因子在矽肺的纤维化阶段起主要作用。IFN-γ（γ-干扰素，Interferon-γ）具有促进初始T淋巴细胞向Th1型细胞分化以及活化巨噬细胞和NK细胞的作用，是典型的Th1型细胞因子。Th1型细胞的分化成熟需要IFN-γ依赖的信号转导子和转录激活子1（signaltransducers and activators of transcription1,STAT1）STAT1和STAT4的磷酸化信号级联反应。实验研究证实，富含重复“TTAGGG”基序的抑制性寡核苷酸（suppressive oligodeoxynucleotides, Sup ODN）具有下调Th1型细胞因子和前炎症介质产生的作用。因此，我们可以将这类ODN用于矽肺的研究中，观察其对矽肺早期炎症的影响，以及炎症细胞因子的产生影响。研究目的观察Sup ODN对染矽尘小鼠早期炎症的影响，探讨Sup ODN作用机制并评价其应用价值。研究方法雌性Balb/c小鼠60只随机分4组，分别为空白对照组、矽尘组、Sup ODN组、对照寡核苷酸（control oligodeoxynucleotides, Con ODN)组。除空白对照组气管注入0.1mL生理盐水外，其余各组采用气管暴露法一次性注入0.1mL SiO2悬液（5g/L），Sup ODN组和Con ODN组在造模前3h分别腹腔注射Sup ODN、Con ODN各0.3mL(1mg/mL)。以建模术时为0d记，模型建立7d后处死动物，，采用瑞氏染色作血清白细胞分类计数；ELISA法检测血清IFN-γ和TNF-α水平；免疫组织化学法检测肺组织中IFN-γ和pSTAT4的表达,并作定量分析；HE染色观察肺组织病理变化；天狼猩红染色观察肺组织Ⅰ、Ⅲ型胶原表达。研究结果 1.HE染色证实气管暴露注入二氧化硅粉尘悬液成功建立了小鼠矽肺模型。 2.Sup ODN组与矽尘组、Con ODN组相比，血清中IFN-γ表达明显降低（P0.01）；矽尘组小鼠肺组织间隔水肿并增厚，Sup ODN组小鼠肺组织炎症较矽尘组减轻；与空白对照组比较，矽尘组小鼠肺组织中IFN-γ表达增加(P0.05)，与矽尘组比较，Sup ODN组小鼠肺组织内IFN-γ表达减少(P0.05);与空白对照组比较，矽尘组小鼠肺组织中pSTAT4表达显著增加(P0.01)，与矽尘组比较，Sup ODN组小鼠肺组织内pSTAT4表达显著减少(P0.01)。研究结论 1.矽尘可诱导染矽尘小鼠血清中IFN-γ和TNF-α表达增加，SupODN可减少染矽尘小鼠IFN-γ的产生，而对TNF-α无影响。 2.Sup ODN可能通过抑制IFN-γ和pSTAT4的表达，抑制小鼠Th1型免疫应答，最终实现其对染矽尘小鼠肺炎症的保护作用。综上所述，本研究结果初步表明Sup ODN对染矽尘小鼠早期炎症有一定的阻碍作用，为矽肺炎症的防治及辅助治疗，开拓了新的途径。
[Abstract]:Silicosis is a systemic disease characterized by diffuse fibrosis of free silica (SiO2) for a long time. According to the status of occupational disease prevention and Prevention published in April 18, 2011 by the Ministry of health and the key work in 2011, 27240 new occupational diseases were found in 2010, including 23812 cases of pneumoconiosis. At present, pneumoconiosis is still the most serious occupational disease in China. The number of pneumoconiosis cases reported in 2010 accounted for 87.42%. of the total number of occupational diseases reported by experts. It was found that the number of cases of pneumoconiosis diagnosed and registered at the CDC was only 10%-20% of pneumoconiosis patients, so the number of cases of pneumoconiosis was estimated to be not less than 1 million. One step to explore the pathogenesis of silicosis and the search for new therapeutic drugs is of great significance for the prevention of silicosis, treatment and even eventually eliminating silicosis. The present study shows that T lymphocytes are activated by dendritic cells and macrophages and other antigen presenting cells to treat the modified silica. Although Th1/Th2 polarization is in the pathogenesis of silicosis The system is still not completely clear, but the study shows that type Th1 cytokines play a major role in the inflammatory stage of silicosis, while Th2 type cytokines play a major role in the fibrosis stage of silicosis..IFN- gamma (interferon gamma, Interferon- gamma) can promote the differentiation of initial T lymphocytes to Th1 cells and the activation of macrophages and NK cells. The differentiation of typical Th1 type cytokine.Th1 type cells requires IFN- gamma dependent signal transducers and transcriptional activator 1 (signaltransducers and activators of transcription1, STAT1) STAT1 and STAT4 phosphorylation signal cascade reaction. Ligodeoxynucleotides, Sup ODN) has the role of down-regulation of Th1 type cytokines and proinflammatory mediators. Therefore, we can use this kind of ODN in silicosis research to observe its effect on early silicosis and the effect of inflammatory cytokines.
research objective
To observe the effect of Sup ODN on the early inflammation of mice exposed to silica dust, to explore the mechanism of Sup ODN and evaluate its application value.
60 female Balb/c mice were randomly divided into 4 groups: blank control group, silica dust group, Sup ODN group and control oligonucleotide (control oligodeoxynucleotides, Con ODN) group. Except the blank control group, the trachea was injected into the 0.1mL physiological saline, the rest of the other groups were injected into the 0.1mL SiO2 suspension (5g/L) by tracheal exposure method. The anterior 3H was intraperitoneally injected with Sup ODN and Con ODN 0.3mL (1mg/mL). The animal was killed after modeling, and the animal was killed after the model was established. The serum white blood cell count was counted by Rayleigh staining. The ELISA method was used to detect the level of IFN- gamma and TNF- alpha in serum; the immunohistochemical method was used to detect the expression of gamma ray in lung tissue and quantitative analysis. The pathological changes of lung tissue were observed. The expression of type I and III collagen in lung tissue was observed by Sirius red staining.
Research results
1.HE staining confirmed that the silicosis model was successfully established by injecting silica dust into the trachea.
In group 2.Sup ODN, the expression of IFN- gamma in serum was significantly lower than that in Con ODN group (P0.01), and the pulmonary tissue was thicker and thicker in the silico dust group, and the lung tissue inflammation in the Sup ODN group was less than that in the silica dust group. Compared with the blank control group, the expression of IFN- gamma in the lung tissue of the silicodust mice increased (P0.05), and the Sup ODN group lung was compared with that of the silica dust group. The expression of IFN- gamma in the tissue was decreased (P0.05), and the expression of pSTAT4 in the lung tissue of the silica dust group was significantly increased (P0.01) compared with the blank control group (P0.01). The expression of pSTAT4 in the lung tissue of the Sup ODN group was significantly reduced (P0.01).
research conclusion
1. silica dust can induce the expression of IFN- gamma and TNF- alpha in serum of mice exposed to silica dust. SupODN can reduce the production of IFN- gamma in mice infected with silica dust, but has no effect on TNF- alpha.
2.Sup ODN may inhibit the Th1 type immune response in mice by inhibiting the expression of IFN- gamma and pSTAT4 and finally realizing its protective effect on pneumonia in mice infected with silica dust. To sum up, the results of this study preliminarily indicate that Sup ODN has a certain hindrance effect on early inflammation in mice infected with silicosis, and has opened a new way for the prevention and treatment of silicosis inflammation and adjuvant therapy. Way.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R135.2

【参考文献】