EMT在MARCO介導(dǎo)大鼠矽肺纖維化中的作用及機(jī)制研究
發(fā)布時間：2018-09-13 05:49
【摘要】：目的通過對吸入式氣管滴注SiO_2法誘導(dǎo)的SD大鼠肺組織纖維化的矽肺動物模型進(jìn)行研究,探討:(1)SiO_2能否通過誘導(dǎo)上皮間質(zhì)轉(zhuǎn)化參與肺組織發(fā)生纖維化;(2)EMT在多聚鳥嘌呤核苷酸(Poly G)減輕矽肺纖維化的分子生物學(xué)機(jī)制。(3)Poly G預(yù)防性和治療性干預(yù)措施對染矽塵大鼠EMT的影響以及兩種不同干預(yù)措施的效果。方法無特定病原體級健康雄性SD大鼠96只,體重180~220g,按體重隨機(jī)分為生理鹽水組(32只)、矽肺模型組(32只)、Poly G預(yù)防組(16只)和Poly G(28天)治療組(16只)。用乙醚將大鼠麻醉后,生理鹽水組經(jīng)支氣管滴注1ml生理鹽水,其余各組均采用吸入式氣管滴注法一次性滴注濃度為50g/L的SiO_2懸濁液1ml。Poly G預(yù)防組大鼠于造模當(dāng)天以尾靜脈注射方式一次性給予Poly G2.5mg/Kg體重(劑量由造模當(dāng)天大鼠的體重來確定),Poly G治療組大鼠于造模28天后以尾靜脈注射方式一次性給予Poly G2.5mg/Kg體重(劑量由造模28天后大鼠的體重來確定)。Poly G預(yù)防組和治療組分別于相應(yīng)給藥后第28天、第56天處死大鼠各8只;矽肺模型組及生理鹽水組也均于相同時間點處死各組大鼠8只。取適量左側(cè)肺組織勻漿,細(xì)胞裂解后,采用Western blot法檢測MARCO、Ecadherin、α-SMA、vimentin以及Ⅰ型和Ⅲ型膠原等蛋白的含量;采用實時定量熒光聚合酶鏈?zhǔn)椒磻?yīng)(Real-time PCR)法檢測E-cadherin、α-SMA、vimentin以及Ⅰ型和Ⅲ型前膠原mRNA的相對表達(dá)水平;右側(cè)肺中葉組織經(jīng)4%多聚甲醛固定,常規(guī)制片,HE染色和Masson染色觀察肺組織病理變化;免疫組織化學(xué)法檢測Ecadherin、α-SMA和vimentin在肺組織中的定位表達(dá)。結(jié)果1不同組別大鼠肺組織病理變化:生理鹽水組大鼠肺組織結(jié)構(gòu)正常,周圍有少量炎性細(xì)胞浸潤,間質(zhì)無明顯藍(lán)色膠原纖維分布,隨著時間的延長,大鼠肺組織結(jié)構(gòu)及膠原沉積情況無明顯變化;矽肺模型組大鼠肺組織內(nèi)有大量炎細(xì)胞浸潤,并出現(xiàn)了典型的纖維性結(jié)節(jié),結(jié)節(jié)由巨噬細(xì)胞和成纖維細(xì)胞組成,有些區(qū)域的肺泡結(jié)構(gòu)尚存在,但肺泡壁和小血管壁出現(xiàn)不同程度的增厚,并在肺間質(zhì)出現(xiàn)大量的藍(lán)色膠原沉積;隨著粉塵暴露時間的延長,纖維性結(jié)節(jié)增多,部分結(jié)節(jié)融合變大,嚴(yán)重者甚至發(fā)生玻璃樣變,間質(zhì)內(nèi)藍(lán)色膠原纖維也不斷增多;Poly G預(yù)防組及治療組大鼠肺組織內(nèi)均可見普遍地炎細(xì)胞浸潤現(xiàn)象,細(xì)胞性結(jié)節(jié)或矽結(jié)節(jié)數(shù)目較模型組減少,藍(lán)色膠原纖維沉積較模型組明顯減少;預(yù)防給藥組大鼠的病變程度均較治療給藥組輕。免疫組化結(jié)果顯示:模型組α-SMA和vimentin陽性細(xì)胞明顯增多,而Ecadherin陽性細(xì)胞明顯減少。給予Poly G干預(yù)后,可以顯著增加E-cadherin陽性細(xì)胞的數(shù)量以及減少α-SMA和vimentin陽性細(xì)胞的數(shù)量。2不同組別MARCO與EMT相關(guān)蛋白在大鼠肺組織中的表達(dá):各時間點生理鹽水組間大鼠肺組織MARCO、E-cadherin、α-SMA和vimentin的表達(dá)水平差異均無統(tǒng)計學(xué)意義(P0.05),模型組大鼠肺組織E-cadherin隨觀察時間延長而降低,其他蛋白水平均隨觀察時間的延長而增加(P0.05)。MARCO、α-SMA和vimentin蛋白表達(dá)水平在Poly G干預(yù)組(預(yù)防性和治療性)大鼠肺組織中均低于同期矽肺模型組,高于生理鹽水組,但E-cadherin均高于同期矽肺模型組(P0.05)。3不同組別Ecadherin、α-SMA和vimentin的mRNA在大鼠肺組織中表達(dá)水平的比較:各組大鼠肺組織E-cadherin、α-SMA和vimentin的mRNA表達(dá)水平變化趨勢均與相應(yīng)蛋白表達(dá)相同。4不同組別Ⅰ、Ⅲ型膠原蛋白以及Ⅰ、Ⅲ型前膠原mRNA在大鼠肺組織中的表達(dá):各個時間點生理鹽水組間大鼠肺組織Ⅰ、Ⅲ膠原蛋白表達(dá)水平無顯著差異(P0.05);矽肺模型組大鼠肺組織Ⅰ、Ⅲ膠原蛋白表達(dá)水平均隨染塵時間延長而增加(P0.05)。Poly G干預(yù)組(預(yù)防性和治療性)Ⅰ、Ⅲ膠原蛋白表達(dá)水平均高于同期生理鹽水組,低于矽肺模型組(P0.05)。Ⅰ、Ⅲ型前膠原mRNA的變化趨勢與Ⅰ、Ⅲ膠原蛋白表達(dá)相同。結(jié)論1矽肺模型組E-cadherin蛋白含量及mRNA相對表達(dá)水平降低,α-SMA、vimentin的蛋白含量及mRNA相對表達(dá)水平升高,表明矽肺發(fā)生發(fā)展過程中存在上皮間質(zhì)轉(zhuǎn)化現(xiàn)象。2抑制MARCO與SiO_2結(jié)合后,α-SMA、vimentin的蛋白含量及mRNA相對表達(dá)水平顯著降低,E-cadherin的蛋白含量及mRNA相對表達(dá)水平明顯上調(diào);Ⅰ、Ⅲ膠原蛋白含量以及Ⅰ、Ⅲ型前膠原mRNA的表達(dá)水平均下降且肺組織病理改變均得到不同程度地減輕。3給予Poly G干預(yù)(預(yù)防性和治療性)能夠有效抑制EMT的進(jìn)程,進(jìn)而延緩肺組織纖維化的形成,且以早期給予預(yù)防性干預(yù)效果較好。
[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.
【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R135.2
本文編號：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.
【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R135.2
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