EMT在MARCO介导大鼠矽肺纤维化中的作用及机制研究
发布时间：2018-09-13 05:49
【摘要】：目的通过对吸入式气管滴注SiO_2法诱导的SD大鼠肺组织纤维化的矽肺动物模型进行研究,探讨:(1)SiO_2能否通过诱导上皮间质转化参与肺组织发生纤维化;(2)EMT在多聚鸟嘌呤核苷酸(Poly G)减轻矽肺纤维化的分子生物学机制。(3)Poly G预防性和治疗性干预措施对染矽尘大鼠EMT的影响以及两种不同干预措施的效果。方法无特定病原体级健康雄性SD大鼠96只,体重180~220g,按体重随机分为生理盐水组(32只)、矽肺模型组(32只)、Poly G预防组(16只)和Poly G(28天)治疗组(16只)。用乙醚将大鼠麻醉后,生理盐水组经支气管滴注1ml生理盐水,其余各组均采用吸入式气管滴注法一次性滴注浓度为50g/L的SiO_2悬浊液1ml。Poly G预防组大鼠于造模当天以尾静脉注射方式一次性给予Poly G2.5mg/Kg体重(剂量由造模当天大鼠的体重来确定),Poly G治疗组大鼠于造模28天后以尾静脉注射方式一次性给予Poly G2.5mg/Kg体重(剂量由造模28天后大鼠的体重来确定)。Poly G预防组和治疗组分别于相应给药后第28天、第56天处死大鼠各8只;矽肺模型组及生理盐水组也均于相同时间点处死各组大鼠8只。取适量左侧肺组织匀浆,细胞裂解后,采用Western blot法检测MARCO、Ecadherin、α-SMA、vimentin以及Ⅰ型和Ⅲ型胶原等蛋白的含量;采用实时定量荧光聚合酶链式反应(Real-time PCR)法检测E-cadherin、α-SMA、vimentin以及Ⅰ型和Ⅲ型前胶原mRNA的相对表达水平;右侧肺中叶组织经4%多聚甲醛固定,常规制片,HE染色和Masson染色观察肺组织病理变化;免疫组织化学法检测Ecadherin、α-SMA和vimentin在肺组织中的定位表达。结果1不同组别大鼠肺组织病理变化:生理盐水组大鼠肺组织结构正常,周围有少量炎性细胞浸润,间质无明显蓝色胶原纤维分布,随着时间的延长,大鼠肺组织结构及胶原沉积情况无明显变化;矽肺模型组大鼠肺组织内有大量炎细胞浸润,并出现了典型的纤维性结节,结节由巨噬细胞和成纤维细胞组成,有些区域的肺泡结构尚存在,但肺泡壁和小血管壁出现不同程度的增厚,并在肺间质出现大量的蓝色胶原沉积;随着粉尘暴露时间的延长,纤维性结节增多,部分结节融合变大,严重者甚至发生玻璃样变,间质内蓝色胶原纤维也不断增多;Poly G预防组及治疗组大鼠肺组织内均可见普遍地炎细胞浸润现象,细胞性结节或矽结节数目较模型组减少,蓝色胶原纤维沉积较模型组明显减少;预防给药组大鼠的病变程度均较治疗给药组轻。免疫组化结果显示:模型组α-SMA和vimentin阳性细胞明显增多,而Ecadherin阳性细胞明显减少。给予Poly G干预后,可以显著增加E-cadherin阳性细胞的数量以及减少α-SMA和vimentin阳性细胞的数量。2不同组别MARCO与EMT相关蛋白在大鼠肺组织中的表达:各时间点生理盐水组间大鼠肺组织MARCO、E-cadherin、α-SMA和vimentin的表达水平差异均无统计学意义(P0.05),模型组大鼠肺组织E-cadherin随观察时间延长而降低,其他蛋白水平均随观察时间的延长而增加(P0.05)。MARCO、α-SMA和vimentin蛋白表达水平在Poly G干预组(预防性和治疗性)大鼠肺组织中均低于同期矽肺模型组,高于生理盐水组,但E-cadherin均高于同期矽肺模型组(P0.05)。3不同组别Ecadherin、α-SMA和vimentin的mRNA在大鼠肺组织中表达水平的比较:各组大鼠肺组织E-cadherin、α-SMA和vimentin的mRNA表达水平变化趋势均与相应蛋白表达相同。4不同组别Ⅰ、Ⅲ型胶原蛋白以及Ⅰ、Ⅲ型前胶原mRNA在大鼠肺组织中的表达:各个时间点生理盐水组间大鼠肺组织Ⅰ、Ⅲ胶原蛋白表达水平无显著差异(P0.05);矽肺模型组大鼠肺组织Ⅰ、Ⅲ胶原蛋白表达水平均随染尘时间延长而增加(P0.05)。Poly G干预组(预防性和治疗性)Ⅰ、Ⅲ胶原蛋白表达水平均高于同期生理盐水组,低于矽肺模型组(P0.05)。Ⅰ、Ⅲ型前胶原mRNA的变化趋势与Ⅰ、Ⅲ胶原蛋白表达相同。结论1矽肺模型组E-cadherin蛋白含量及mRNA相对表达水平降低,α-SMA、vimentin的蛋白含量及mRNA相对表达水平升高,表明矽肺发生发展过程中存在上皮间质转化现象。2抑制MARCO与SiO_2结合后,α-SMA、vimentin的蛋白含量及mRNA相对表达水平显著降低,E-cadherin的蛋白含量及mRNA相对表达水平明显上调;Ⅰ、Ⅲ胶原蛋白含量以及Ⅰ、Ⅲ型前胶原mRNA的表达水平均下降且肺组织病理改变均得到不同程度地减轻。3给予Poly G干预(预防性和治疗性)能够有效抑制EMT的进程,进而延缓肺组织纤维化的形成,且以早期给予预防性干预效果较好。
[Abstract]:Objective To investigate the effects of inhalation of SiO_2 on lung fibrosis in SD rats, and to explore the molecular biological mechanism of EMT in polyguanine nucleotide (Poly G) alleviating silicosis fibrosis. Methods 96 healthy male SD rats of no specific pathogen grade, weighing 180-220 g, were randomly divided into saline group (32 rats), silicosis model group (32 rats), Poly G prevention group (16 rats) and Poly G (28 days) treatment group (16 rats). After anesthesia, rats in the normal saline group were given 1 ml of normal saline by bronchial infusion, while rats in the other groups were given 1 ml of SiO_2 suspension of 50 g/L by inhalation tracheal infusion. Poly G2.5 mg/kg body weight by tail vein injection on the day of modeling (the dose was determined by the body weight of rats on the day of modeling). Poly G group and treatment group were given Poly G2.5mg/kg body weight by tail vein injection 28 days after modeling (the dose was determined by the weight of rats 28 days after modeling). Poly G prevention group and treatment group were given corresponding drug 28 days, 56 days after the death of rats each 8; silicosis model group and physiological saline group were also at the same time point. Eight rats in each group were sacrificed. After cell lysis, the contents of MARCO, E cadherin, alpha-SMA, vimentin, collagen type I and III were detected by Western blot, and E-cadherin, alpha-SMA, vimentin and procollagen type I and III were detected by real-time quantitative polymerase chain reaction (Real-time PCR). The relative expression level of mRNA; the right middle lobe of the lung was fixed by 4% paraformaldehyde, routinely made slices, HE staining and Mason staining were used to observe the pathological changes of the lung; immunohistochemical method was used to detect the localized expression of Ecadherin, alpha-SMA and vimentin in the lung tissue. The structure was normal, a few inflammatory cells infiltrated around the interstitium without obvious blue collagen fibers distribution, with the extension of time, there was no significant change in the lung tissue structure and collagen deposition in rats; in the silicosis model group, there were a large number of inflammatory cells infiltrated in the lung tissue, and there were typical fibrous nodules, which were composed of macrophages and fibroblasts. Cell composition, alveolar structure still exists in some areas, but alveolar wall and small vessel wall thicken in varying degrees, and a large number of blue collagen deposits appear in the interstitium of the lung; with the prolongation of dust exposure, fibrous nodules increase, some nodules fuse and become larger, serious cases even hyaline change, interstitial blue collagen fibers also do not occur. The number of cellular nodules or silica nodules was less than that of the model group, and the deposition of blue collagen fibers was less than that of the model group. The degree of pathological changes in the preventive group was lighter than that in the treatment group. Poly G treatment significantly increased the number of E-cadherin positive cells and decreased the number of alpha-SMA and vimentin positive cells. There was no significant difference in the expression of MARCO, E-cadherin, alpha-SMA and vimentin (P 0.05). E-cadherin decreased with the prolongation of observation time, and other protein levels increased with the prolongation of observation time (P 0.05). Sex) The expression of E-cadherin, alpha-SMA and vimentin mRNA in lung tissues of rats was lower than that of silicosis model group and higher than that of normal saline group, but E-cadherin was higher than that of silicosis model group (P 0.05). 3 Comparison of the expression levels of E-cadherin, alpha-SMA and vimentin mRNA in lung tissues of rats in different groups The expression of collagen type I, III and procollagen type I, III mRNA in lung tissue of rats was the same as that in corresponding protein expression. The expression of collagen I and III in Poly G intervention group (preventive and therapeutic) was higher than that in normal saline group, and lower than that in silicosis model group (P The protein content and mRNA relative expression of alpha-SMA and vimentin were significantly decreased, while the protein content and mRNA relative expression of E-cadherin were significantly increased after inhibiting the binding of MARCO to SiO_2. Poly G intervention (preventive and therapeutic) can effectively inhibit the process of EMT, and then delay the formation of pulmonary fibrosis, and the effect of early preventive intervention is better.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R135.2
本文编号：2240266
[Abstract]:Objective To investigate the effects of inhalation of SiO_2 on lung fibrosis in SD rats, and to explore the molecular biological mechanism of EMT in polyguanine nucleotide (Poly G) alleviating silicosis fibrosis. Methods 96 healthy male SD rats of no specific pathogen grade, weighing 180-220 g, were randomly divided into saline group (32 rats), silicosis model group (32 rats), Poly G prevention group (16 rats) and Poly G (28 days) treatment group (16 rats). After anesthesia, rats in the normal saline group were given 1 ml of normal saline by bronchial infusion, while rats in the other groups were given 1 ml of SiO_2 suspension of 50 g/L by inhalation tracheal infusion. Poly G2.5 mg/kg body weight by tail vein injection on the day of modeling (the dose was determined by the body weight of rats on the day of modeling). Poly G group and treatment group were given Poly G2.5mg/kg body weight by tail vein injection 28 days after modeling (the dose was determined by the weight of rats 28 days after modeling). Poly G prevention group and treatment group were given corresponding drug 28 days, 56 days after the death of rats each 8; silicosis model group and physiological saline group were also at the same time point. Eight rats in each group were sacrificed. After cell lysis, the contents of MARCO, E cadherin, alpha-SMA, vimentin, collagen type I and III were detected by Western blot, and E-cadherin, alpha-SMA, vimentin and procollagen type I and III were detected by real-time quantitative polymerase chain reaction (Real-time PCR). The relative expression level of mRNA; the right middle lobe of the lung was fixed by 4% paraformaldehyde, routinely made slices, HE staining and Mason staining were used to observe the pathological changes of the lung; immunohistochemical method was used to detect the localized expression of Ecadherin, alpha-SMA and vimentin in the lung tissue. The structure was normal, a few inflammatory cells infiltrated around the interstitium without obvious blue collagen fibers distribution, with the extension of time, there was no significant change in the lung tissue structure and collagen deposition in rats; in the silicosis model group, there were a large number of inflammatory cells infiltrated in the lung tissue, and there were typical fibrous nodules, which were composed of macrophages and fibroblasts. Cell composition, alveolar structure still exists in some areas, but alveolar wall and small vessel wall thicken in varying degrees, and a large number of blue collagen deposits appear in the interstitium of the lung; with the prolongation of dust exposure, fibrous nodules increase, some nodules fuse and become larger, serious cases even hyaline change, interstitial blue collagen fibers also do not occur. The number of cellular nodules or silica nodules was less than that of the model group, and the deposition of blue collagen fibers was less than that of the model group. The degree of pathological changes in the preventive group was lighter than that in the treatment group. Poly G treatment significantly increased the number of E-cadherin positive cells and decreased the number of alpha-SMA and vimentin positive cells. There was no significant difference in the expression of MARCO, E-cadherin, alpha-SMA and vimentin (P 0.05). E-cadherin decreased with the prolongation of observation time, and other protein levels increased with the prolongation of observation time (P 0.05). Sex) The expression of E-cadherin, alpha-SMA and vimentin mRNA in lung tissues of rats was lower than that of silicosis model group and higher than that of normal saline group, but E-cadherin was higher than that of silicosis model group (P 0.05). 3 Comparison of the expression levels of E-cadherin, alpha-SMA and vimentin mRNA in lung tissues of rats in different groups The expression of collagen type I, III and procollagen type I, III mRNA in lung tissue of rats was the same as that in corresponding protein expression. The expression of collagen I and III in Poly G intervention group (preventive and therapeutic) was higher than that in normal saline group, and lower than that in silicosis model group (P The protein content and mRNA relative expression of alpha-SMA and vimentin were significantly decreased, while the protein content and mRNA relative expression of E-cadherin were significantly increased after inhibiting the binding of MARCO to SiO_2. Poly G intervention (preventive and therapeutic) can effectively inhibit the process of EMT, and then delay the formation of pulmonary fibrosis, and the effect of early preventive intervention is better.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R135.2
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