JNK信号通路对肺纤维化大鼠成纤维细胞表型的影响

发布时间：2018-05-08 07:46

本文选题：JNK丝裂原活化蛋白激酶类 + 肺纤维化　；参考：《山西医科大学》2012年硕士论文

【摘要】：研究背景：肺纤维化是一种病因不明的渐进性加重的致命性弥漫性肺疾病。传统治疗主要是抑制炎症反应，但临床疗效甚微。为了显著提高肺纤维化患者的生存率，进一步探讨其发病机制成为必然。成纤维细胞表型分化为特征性表达α-平滑肌肌动蛋白（α-SMA）的肌成纤维细胞为肺纤维化的关键步骤，但其发生机制不甚明了。近年来，信号通路成为其研究的热点。c-jun氨基末端激酶（JNK）是丝裂原活化蛋白激酶家族的重要一员，JNK信号通路参与调控许多细胞增殖、分化与凋亡。已有研究表明JNK信号通路参与人肺成纤维细胞向肌成纤维细胞分化的过程,而关于体内实验JNK信号通路是否在肺纤维化转分化过程中发挥作用尚未见相关研究报道。JNK激酶抑制剂SP600125可特异性阻断JNK信号通路。本实验使用SP600125进行干预，通过测定大鼠肺组织中α-SMA和p-JNK蛋白的表达水平，从分子信号角度探讨肺纤维化中JNK信号通路与肺纤维化成纤维细胞表型改变的关系，探讨该途径在肺纤维化中的地位。目的：利用博莱霉素（BLM）诱导的大鼠肺纤维化模型，通过观察健康对照组、模型组和SP600125组肺组织病理形态学的变化，测定肺组织羟脯氨酸（HYP）的含量，观察肺组织中α-SMA和p-JNK蛋白的表达，进一步分析JNK信号通路在肺纤维化成纤维细胞表型转分化病理过程中的作用。方法：将54只健康雄性Wistar大鼠（体重为200g±20g）随机分为对照组、模型组和SP600125组，每组各18只。模型组于气管内滴注BLM生理盐水(5mg/kg），，造模当日腹腔内注射二甲基亚砜（DMSO）液；SP600125组用同样方法造模，造模当日腹腔内注射SP600125溶液（15mg/kg，溶于DMSO液中），一次性给药；对照组气管内滴注生理盐水，造模当日腹腔内注射DMSO液。分别于造模后第7、14、28天处死大鼠，取肺组织病理切片行苏木素-伊红（HE）染色和Masson染色，光镜下观察大鼠肺组织病理学变化；碱水解法检测肺组织HYP含量；应用免疫组织化学法检测肺组织中α-SMA和p-JNK蛋白表达水平。结果：1、模型组第7天肺泡炎程度严重，于第28天形成显著肺纤维化，SP600125组各期病理变化较模型组减轻。2、模型组和SP600125组的HYP含量随时间延长逐渐升高。第7天模型组较对照组升高（P＜0.01)，SP600125组与模型组和对照组比较差异无统计学意义，第14、28天模型组较对照组明显升高（P＜0.01),SP600125组较模型组有所减低（P＜0.01)，但仍高于对照组。3、模型组各时间点α-SMA与p-JNK蛋白均明显表达，以第14天最明显。对照组及SP600125组第14、28天α-SMA与p-JNK蛋白表达均较模型组降低(P＜0.05)。4、模型组α-SMA与p-JNK蛋白含量呈显著正相关(r=0.927，P＜0.05)。结论：肺纤维化病理过程可能与p-JNK蛋白表达增加并活化α-SMA的表达有关，应用SP600125对肺纤维化有抑制作用。
[Abstract]:Background: pulmonary fibrosis is a fatal diffuse pulmonary disease with unknown etiology and progressive exacerbation. The traditional treatment is mainly to suppress inflammatory reaction, but the clinical effect is very little. In order to improve the survival rate of patients with pulmonary fibrosis, it is necessary to further explore its pathogenesis. Phenotypic differentiation of fibroblasts into myofibroblasts expressing 伪 -smooth muscle actin (伪 -SMA) is a key step in pulmonary fibrosis, but its mechanism is not clear. In recent years, the signal pathway has become the focus of its research. C-jun amino-terminal kinase (JNKK) is an important member of the mitogen-activated protein kinase family. JNK signaling pathway plays an important role in regulating the proliferation, differentiation and apoptosis of many cells. It has been shown that JNK signaling pathway is involved in the differentiation of pulmonary fibroblasts into myofibroblasts. However, whether the JNK signaling pathway plays a role in the process of pulmonary fibrosis transdifferentiation has not been reported in vivo. JNK kinase inhibitor SP600125 can specifically block the JNK signaling pathway. The expression of 伪 -SMA and p-JNK protein in lung tissue of rats was measured by SP600125. The relationship between JNK signaling pathway and phenotypic changes of fibroblasts in pulmonary fibrosis was studied from the molecular signal perspective. To explore the role of this pathway in pulmonary fibrosis. Objective: to observe the pathological changes of lung tissue in control group, model group and SP600125 group, and to determine the content of hydroxyproline (HYP) in lung tissue of rats induced by bleomycin (BLM). To observe the expression of 伪 -SMA and p-JNK protein in lung tissue and to further analyze the role of JNK signaling pathway in the pathological process of phenotypic transdifferentiation of pulmonary fibrosis fibroblasts. Methods: 54 healthy male Wistar rats (body weight was 200g 卤20g) were randomly divided into control group, model group and SP600125 group with 18 rats in each group. The model group was treated with intratracheal instillation of BLM saline (5 mg / kg) and intraperitoneal injection of dimethyl sulfoxide (DMSO) solution in SP600125 group on the same day. On the same day, SP600125 solution was injected intraperitoneally with 15 mg / kg SP600125 solution and dissolved in DMSO solution. In the control group, normal saline was injected into trachea and DMSO solution was injected intraperitoneally on the day of modeling. The rats were killed on day 7, 1428, respectively. The pathological sections of lung tissue were stained with hematoxylin-eosin) and Masson staining. The pathological changes of lung tissue were observed under light microscope, and the content of HYP in lung tissue was detected by alkaline hydrolysis. The expression of 伪 -SMA and p-JNK protein in lung tissue was detected by immunohistochemical method. Results the severity of alveolitis in the model group was severe on the 7th day. The pathological changes of each stage in SP600125 group were lighter than those in the model group on the 28th day. The content of HYP in the model group and SP600125 group gradually increased with the prolongation of time. On the 7th day, there was no significant difference between the model group and the model group and the control group (P < 0.01). On the 14th day, the expression of 伪 -SMA and p-JNK protein in model group was significantly higher than that in control group (P < 0.01), but still higher than that in control group (P < 0.01). The expression of 伪 -SMA and p-JNK protein in model group was the most obvious on the 14th day. The expression of 伪 -SMA and p-JNK protein in the control group and SP600125 group was significantly lower than that in the model group on the 28th day (P < 0.05). There was a significant positive correlation between 伪 -SMA and p-JNK protein content in the model group (P < 0.05). Conclusion: the pathological process of pulmonary fibrosis may be related to the increased expression of p-JNK protein and activation of 伪 -SMA expression. The application of SP600125 can inhibit pulmonary fibrosis.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.9

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