增龄对小鼠肺纤维化模型上皮间质转化及Snail影响的研究
发布时间：2018-11-20 20:42
【摘要】：目的:研究不同年龄段(青年、老年)小鼠实验性肺纤维化中上皮细胞-间充质细胞转化(epithelial-mesenchymal transition,EMT)相关指标:上皮细胞标志物E-钙粘蛋白(E-Cadherin,E-Cad),间质细胞标志物α平滑肌肌动蛋白(alpha smooth muscle Actin,α-SMA)、波形蛋白(Vimentin)及转录因子Snail随病程进展的表达变化,初步探讨增龄对小鼠肺纤维化病变程度及EMT的影响,为进一步探索增龄促进肺纤维化进展的可能机制提供依据。方法:20周龄雄性C57/BL6小鼠60只,随机分为两组:青年组(20周龄)、老年组(饲养至26周龄)。上述两组每组再随机分为5组,其中1组作为生理盐水对照组(NS),给予气管内注入0.9%氯化钠注射液,于第28d处死;其余4组为肺纤维化模型组,按5mg/kg剂量,予气管内注入博来霉素(bleomycin,BLM),分别于造模后第7d、14d、21d及28d处死,取左肺制备石蜡切片,右肺液氮冻存后行相关蛋白RT-PCR检测。石蜡切片行HE染色及Masson染色,通过肺组织形态改变从而判断肺纤维化病变进展程度;免疫组织化学染色检测EMT相关指标E-Cadherin、Vimentin、α-SMA蛋白的表达水平,平均光灰度值使用美国Universal Imaging Porporation图像分析系统测量,反映病程中EMT的动态变化;RT-PCR法检测液氮冻存肺组织转录因子Snail1、Snail2及其调控蛋白E-cad的m RNA的表达水平。使用SPSS19.0对上述数据进行统计分析,所有数据均采取“均数标准差”表示,P0.05,差异有统计学意义。结果:HE染色及Masson染色结果示BLM诱导的小鼠肺纤维化模型组,随病程进展,肺实质破坏、胶原生成量较对照组增加明显,表明造模成功。将老年与青年小鼠同时间点的纤维化病变程度进行对比,前者更严重。免疫组化法结果显示,E-Cadherin主要表达于肺泡上皮细胞、支气管黏膜上皮细胞。青年、老年两组小鼠对照组表达较多,各时间点模型组的表达较对照组减少,且随病程进展,减少更为明显,差异有统计学意义(P0.05);进一步将两组模型小鼠的各个对应时间点表达量进行比较,老年较青年组表达较少(P0.05),而两组对照组无明显差异。Vimentin主要表达于支气管黏膜上皮及肺间质细胞,α-SMA在对照组只表达于支气管及血管平滑肌细胞,而在模型组肺间质肌成纤维细胞中还可观测到其表达。上述两个指标在各时间点模型组表达较对照组增加,随病程进展,增多更显著(P0.05);同时间点老年模型组较青年模型组表达较多(P0.05),两组对照组无明显差异。RT-PCR法结果示在青年、老年两组小鼠中,随时间推移,Snail1及Snail2的表达均呈现先升高后下降的趋势(相较对照组,模型组14d表达量明显增加,28d较14d表达明显减少,P0.05)。对比两组小鼠同时间点的表达情况,老年模型组较青年模型组于14d增加更为明显,28d减少更为缓慢(P0.05),两组对照组无明显差异。E-Cadherin模型组14d、28d的表达较对照组减少,且随病程进展,减少更为明显(P0.05)。相较青年模型组,同时间点老年模型组表达较少(P0.05),两组对照组无明显差异,与免疫组化结果基本一致。结论:BLM气管内注入诱导小鼠肺纤维化模型成功,同时期老年模型组相较青年模型组肺纤维化及EMT程度更严重,而对照组无明显差异。老年模型组Snail1、Snail2、E-Cadherin较青年模型组变化更为明显。增龄可能通过影响小鼠肺纤维化模型EMT进程中转录因子Snail1、Snail2及其下游调节蛋白E-Cadherin等表达从而加重肺纤维化病变程度。
[Abstract]:Objective: To study the related indexes of epithelial cell-mesenchymal transition (EMT) in experimental pulmonary fibrosis of mice with different age groups (young and old): E-cadherin (E-Cadiin, E-Cad). The effect of increasing age on the degree of pulmonary fibrosis and EMT in mice was studied. To further explore the possible mechanism of increasing age to promote the progression of pulmonary fibrosis. Methods: 60-week-old male C57/ BL6 mice were randomly divided into two groups: the young group (20 weeks old) and the old group (26 weeks old). The two groups were randomly divided into 5 groups, one group was used as the normal saline control group (NS), and 0.9% sodium chloride injection was injected into the trachea, and was sacrificed on day 28d; the remaining 4 groups were pulmonary fibrosis model group, and the bleomycin (BLM) was injected into the trachea according to the dose of 5mg/ kg. The left lung was used to prepare the paraffin section and the right lung liquid nitrogen was frozen and the related protein RT-PCR was detected. The paraffin sections were stained with HE and Masson, and the degree of progression of pulmonary fibrosis was determined by the change of the morphology of the lung. The expression levels of E-Cadherin, Vimentin, and S-SMA were measured by immunohistochemical staining. The average light gray values were measured using the American Universal Imaging Porptron image analysis system. The dynamic changes of EMT were reflected in the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the disease. The above data were statistically analyzed using SPSS19.0, and all the data were expressed in 鈥渕ean standard deviation鈥，
本文编号：2345962
[Abstract]:Objective: To study the related indexes of epithelial cell-mesenchymal transition (EMT) in experimental pulmonary fibrosis of mice with different age groups (young and old): E-cadherin (E-Cadiin, E-Cad). The effect of increasing age on the degree of pulmonary fibrosis and EMT in mice was studied. To further explore the possible mechanism of increasing age to promote the progression of pulmonary fibrosis. Methods: 60-week-old male C57/ BL6 mice were randomly divided into two groups: the young group (20 weeks old) and the old group (26 weeks old). The two groups were randomly divided into 5 groups, one group was used as the normal saline control group (NS), and 0.9% sodium chloride injection was injected into the trachea, and was sacrificed on day 28d; the remaining 4 groups were pulmonary fibrosis model group, and the bleomycin (BLM) was injected into the trachea according to the dose of 5mg/ kg. The left lung was used to prepare the paraffin section and the right lung liquid nitrogen was frozen and the related protein RT-PCR was detected. The paraffin sections were stained with HE and Masson, and the degree of progression of pulmonary fibrosis was determined by the change of the morphology of the lung. The expression levels of E-Cadherin, Vimentin, and S-SMA were measured by immunohistochemical staining. The average light gray values were measured using the American Universal Imaging Porptron image analysis system. The dynamic changes of EMT were reflected in the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the disease. The above data were statistically analyzed using SPSS19.0, and all the data were expressed in 鈥渕ean standard deviation鈥，
本文编号：2345962
本文链接：https://www.wllwen.com/yixuelunwen/huxijib/2345962.html
最近更新
教材专著
