PBA抑制博莱霉素诱导肺纤维化过程中的上皮—间质转化

发布时间：2018-11-25 20:07

【摘要】：目的:为了研究PBA对于博莱霉素诱导的小鼠肺纤维化及肺纤维化发生过程中肺泡上皮细胞上皮-间叶转化的影响;方法:取成年ICR雄性小鼠(28-32g)。随机分为四组:空白对照组(Control),单纯PBA对照组(PBA),肺纤维化模型组(BLM),PBA保护组(BLM+PBA)。在肺纤维化模型组中,一次性给予小鼠气管内注射博莱霉素(3.0mg/kg)。在PBA保护组中,同时给予小鼠每天腹腔注射PBA(150mg/kg),给予小鼠博莱霉素21天后处死小鼠,取出肺组织。留取部分肺组织福尔马林固定,石蜡包埋后切片HE染色了解各组小鼠肺组织中炎症浸润情况,天狼星红染色了解各组肺组织纤维化情况,同时留取切片用于免疫组织化学检测了解α-SMA及NF-κb在各组小鼠肺组织中的表达。提取肺组织总蛋白用于测定内质网应激通路相关蛋白GRP78、CHOP、p-e IF2α、e IF2α、IRE1α、p-IRE1α及EMT特异性分子α-SMA等在肺组织中的表达。提取肺组织RNA,利用RT-PCR测定TNF-α、IL-1β、IFN-γ和TGF-β1等炎性因子及胶原基因Col1α1和Col1α2在各组小鼠肺中的表达。结果:在肺纤维化模型组中,给予小鼠气管内注入博莱霉素21天后,蛋白印迹试验证实纤维化模型组小鼠肺组织中内质网应激相关蛋白GRP78、CHOP、p-e IF2α、p-IRE1α表达较正常对照小鼠明显升高,而给予PBA后可以明显抑制小鼠肺组织中内质网应激相关蛋白的水平的升高;HE染色证明肺纤维化模型组小鼠肺组织中炎症浸润明显,给予PBA后可以抑制小鼠肺组织中炎症浸润程度;免疫组化提示纤维化模型组小鼠中NF-κb阳性细胞明显增多,而给予PBA后可以明显减少NF-κb阳性细胞的数量;RT-PCR结果提示肺纤维化模型组小鼠肺组织中TNF-α、IL-1β、IFN-γ和TGF-β1基因表达水平明显升高,给予PBA后可以明显抑制这些炎性因子基因的表达;免疫组化及蛋白印迹实验证实肺纤维化模型组小鼠肺组织中α-SMA表达明显升高,PBA可以明显抑制α-SMA在小鼠肺组织中的表达;天狼星红染色提示给予小鼠气管内注射博莱霉素21天后可见肺组织中胶原蛋白明显增多,PBA可以减轻博莱霉素所致的肺纤维化程度;同时RT-PCR提示肺纤维化模型组中胶原基因Col1α1和Col1α2表达水平明显升高,而PBA可以抑制Col1α1和Col1α2表达的升高。结论:PBA可能抑制博莱霉素诱导的小鼠肺纤维化过程中的内质网应激介导的上皮-间质转化。
[Abstract]:Aim: to study the effect of PBA on alveolar epithelial epithelial-mesenchymal transformation in bleomycin induced pulmonary fibrosis and pulmonary fibrosis in mice. Methods: adult ICR male mice (28-32 g) were selected. Four groups were randomly divided into four groups: blank control group, (Control), control group, simple PBA control group, (PBA), pulmonary fibrosis model group, (BLM), PBA protection group, (BLM PBA). In the pulmonary fibrosis model group, mice were given intratracheal injection of bleomycin (3.0mg/kg). In the PBA protection group, mice were given intraperitoneal injection of PBA (150mg/kg) every day, and the mice were killed 21 days after Bleomycin was given, and lung tissue was taken out. Partial lung tissue was fixed by formalin, HE staining of paraffin embedded sections was used to investigate the inflammatory infiltration in lung tissue of mice in each group, and Sirius red staining was used to investigate the fibrosis of lung tissue in each group. The expression of 伪-SMA and NF- 魏 b in lung tissues of each group was detected by immunohistochemistry. Lung tissue total protein was extracted to determine the expression of endoplasmic reticulum stress pathway related proteins GRP78,CHOP,p-e IF2 伪, IRE1 伪, p-IRE1 伪 and EMT specific molecule 伪 -SMA in lung tissue. The expression of TNF- 伪, IL-1 尾, IFN- 纬 and TGF- 尾 1 and collagen genes Col1 伪 1 and Col1 伪 2 in the lungs of mice were determined by RT-PCR. Results: in the pulmonary fibrosis model group, the endoplasmic reticulum stress-related protein (GRP78,CHOP,p-e IF2 伪) was confirmed by Western blotting after intratracheal administration of bleomycin 21 days later. The expression of p-IRE1 伪 was significantly higher than that of normal control mice, but the level of endoplasmic reticulum stress-related protein in lung tissue was significantly inhibited by PBA. HE staining showed that the inflammatory infiltration in lung tissue was obvious in the model group of pulmonary fibrosis, and the degree of inflammatory infiltration in the lung tissue could be inhibited by PBA. Immunohistochemical staining showed that the number of NF- 魏 b positive cells in the fibrotic model group was significantly increased, but the number of NF- 魏 b positive cells was significantly decreased after the administration of PBA. The results of RT-PCR indicated that the expression of TNF- 伪, IL-1 尾, IFN- 纬 and TGF- 尾 1 in lung tissue of pulmonary fibrosis model group was significantly increased, and the expression of these inflammatory factor genes could be significantly inhibited by PBA. Immunohistochemistry and Western blot analysis showed that the expression of 伪-SMA in lung tissue of the model group was significantly increased, and that PBA could inhibit the expression of 伪-SMA in the lung tissue of mice. Sirius red staining showed that the collagen in lung tissue was significantly increased 21 days after intratracheal injection of bleomycin in mice, and PBA could alleviate the degree of pulmonary fibrosis induced by bleomycin. At the same time, RT-PCR indicated that the expression of collagen gene Col1 伪 1 and Col1 伪 2 in pulmonary fibrosis model group was significantly increased, while PBA could inhibit the increase of Col1 伪 1 and Col1 伪 2 expression. Conclusion: PBA may inhibit the epithelial-interstitial transformation mediated by endoplasmic reticulum stress during bleomycin induced pulmonary fibrosis in mice.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R563

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