本文選題：肺泡Ⅱ型上皮細(xì)胞　切入點(diǎn)：巨噬細(xì)胞　出處：《鄭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景和目的有關(guān)肝、腎等纖維化疾病的研究表明,肌成纖維細(xì)胞是分泌膠原纖維的主效應(yīng)細(xì)胞,上皮間質(zhì)轉(zhuǎn)分化是其主要來源之一。矽肺的發(fā)病機(jī)制并不完全清楚,但已知矽肺的纖維化過程中,肌成纖維細(xì)胞同樣是合成、分泌膠原纖維的主效應(yīng)細(xì)胞。肌成纖維細(xì)胞的來源除了肺內(nèi)固有的成纖維細(xì)胞外,其他來源也同樣受到重視。肺泡Ⅱ型上皮細(xì)胞具有一定的分化能力,有可能是肌成纖維細(xì)胞的潛在來源之一。迄今為止,在矽肺過程中,二氧化硅(Si O2)粉塵如何誘導(dǎo)肺泡Ⅱ型上皮細(xì)胞轉(zhuǎn)分化為肌成纖維細(xì)胞的機(jī)制、效率以及轉(zhuǎn)分化標(biāo)志物的時(shí)間-效應(yīng)關(guān)系尚不確定。因此,本研究構(gòu)建了體外共培養(yǎng)模型和大鼠矽肺模型,應(yīng)用電子透射電鏡、實(shí)時(shí)熒光定量PCR、激光共聚焦成像、Western blot等實(shí)驗(yàn)技術(shù),探索體內(nèi)外肺泡Ⅱ型上皮細(xì)胞發(fā)生EMT的過程及其時(shí)間-效應(yīng)關(guān)系,為矽肺的機(jī)制闡釋提供了新的思路,為矽肺的監(jiān)測(cè)和干預(yù)提供了參考。方法1.提取原代肺泡Ⅱ型上皮細(xì)胞(AECⅡ)和AM,購買肺泡Ⅱ型上皮細(xì)胞株(RLE-6TN),并對(duì)AECⅡ和RLE-6TN進(jìn)行電子透射鑒定。通過CCK-8實(shí)驗(yàn),用1~140μg/mL的SiO2濃度梯度染塵24、48和72h,確定染塵濃度。2.建立體外共培養(yǎng)模型,AECⅡ細(xì)胞分為對(duì)照組和實(shí)驗(yàn)組,RLE-6TN分為直接對(duì)照組、直接染塵組、間接對(duì)照組和間接染塵組。采用western blot和qRT-PCR技術(shù)測(cè)定不同分組24、48和72h時(shí)AECⅡ和RLE-6TN中GAPDH、E-cad和α-SMA的蛋白和mRNA的相對(duì)表達(dá)水平;采用酶聯(lián)免疫吸附法(ELISA)技術(shù)測(cè)定24、48和72h時(shí)AECⅡ、RLE-6TN的細(xì)胞培養(yǎng)上清液中TGF-β1的含量。3.建立大鼠矽肺模型,選擇SPF級(jí)4~6周齡的雄性健康SD大鼠84只,體重在160~180g之間,購于河南省實(shí)驗(yàn)動(dòng)物中心,通過非氣管暴露法進(jìn)行SiO2染塵,實(shí)驗(yàn)分為對(duì)照組和染塵組,對(duì)照給予等量的生理鹽水,每組6只,并在實(shí)驗(yàn)的1、2、3、4、6、9和12周時(shí)處死相應(yīng)的大鼠。采用HE染色法判斷大鼠矽肺模型是否成功;采用激光共聚焦熒光雙染技術(shù),檢測(cè)肺組織冰凍切片中E-cad和α-SMA的定位及表達(dá)水平,采用ELISA測(cè)定大鼠矽肺組織研磨液中TGF-β1的含量。4.統(tǒng)計(jì)分析:采用SPSS21.0軟件分析實(shí)驗(yàn)數(shù)據(jù)。計(jì)量資料經(jīng)正態(tài)性檢驗(yàn)服從正態(tài)分布,數(shù)據(jù)以均值加減標(biāo)準(zhǔn)差(χ±s)表示,雙因素?cái)?shù)據(jù)采用重復(fù)測(cè)量資料的方差分析,單因素?cái)?shù)據(jù)采用單因素方差分析(one-way ANOVA),均數(shù)的兩兩比較采用最小顯著差法(LSD檢驗(yàn)),檢驗(yàn)水準(zhǔn)α=0.05。結(jié)果1.原代AECⅡ和RLE-6TN的電子透射電鏡顯示,細(xì)胞結(jié)構(gòu)內(nèi)均可見特異性的板層小體。CCK-8測(cè)定SiO2對(duì)細(xì)胞抑制率顯示,同一染毒條件下隨著時(shí)間的延長(zhǎng),抑制作用越強(qiáng),同一染塵時(shí)間時(shí),隨著染塵濃度的增大抑制作用增強(qiáng),約在最長(zhǎng)染塵時(shí)間72h時(shí)抑制率達(dá)到50%,此時(shí)的SiO2濃度約為100μg/mL。2.原代AECⅡ的實(shí)驗(yàn)組和對(duì)照組相比,蛋白E-cad和α-SMA的相對(duì)表達(dá)水平的差異顯著(P0.05),實(shí)驗(yàn)組E-cad和α-SMA的相對(duì)表達(dá)水平隨著時(shí)間的延長(zhǎng)呈現(xiàn)明顯的時(shí)間-效應(yīng)關(guān)系。實(shí)驗(yàn)組和對(duì)照組培養(yǎng)細(xì)胞的上清液中TGF-β1的含量的差異具有統(tǒng)計(jì)學(xué)意義(P0.05),并且實(shí)驗(yàn)組TGF-β1的變化具有明顯的時(shí)間-效應(yīng)關(guān)系,其最大值為1.42ng/mL。3.RLE-6TN細(xì)胞的直接染塵組與直接對(duì)照組、間接染塵組與間接對(duì)照組相比,E-cad和α-SMA的蛋白和mRNA水平的差異均具有統(tǒng)計(jì)學(xué)意義(P0.05),且具有明顯的時(shí)間-效應(yīng)關(guān)系;直接染塵組和間接染塵組均可使E-cad的蛋白和mRNA水平表達(dá)下降而α-SMA的表達(dá)上升,間接染塵組E-cad下降和α-SMA上升的時(shí)間早于直接染塵組。細(xì)胞培養(yǎng)上清液中,TGF-β1的含量在直接染塵組與直接對(duì)照組、間接染塵組與間接對(duì)照組差異顯著(P0.05),間接染塵組和直接染塵組中均具有明顯的時(shí)間-效應(yīng)關(guān)系,其最大值為1.81ng/mL。4.HE染色顯示,在2周時(shí)可見染塵組肺組織有明顯的炎性細(xì)胞聚集,3周時(shí)肌纖維逐漸減少,膠原纖維增多,特別是在3周以后,可見膠原纖維聚集成團(tuán),形成細(xì)胞性結(jié)節(jié),6周有纖維性結(jié)節(jié)出現(xiàn),之后結(jié)節(jié)逐漸增大融合,甚至出現(xiàn)肺間隔斷裂。染塵組肺組織研磨液中TGF-β1的含量在2周時(shí)達(dá)到最大值,呈現(xiàn)先升高后下降的趨勢(shì);免疫熒光雙染顯示對(duì)照組和實(shí)驗(yàn)組在2周時(shí)E-cad的表達(dá)達(dá)到最大值,之后逐漸下降,而α-SMA呈現(xiàn)隨時(shí)間逐漸上升的趨勢(shì)。結(jié)論游離SiO2粉塵可誘導(dǎo)肺泡Ⅱ型上皮細(xì)胞發(fā)生EMT,轉(zhuǎn)分化為肌成纖維細(xì)胞。在巨噬細(xì)胞同時(shí)存在的條件下更易于發(fā)生EMT,這可能與巨噬細(xì)胞分泌的細(xì)胞因子TGF-β有關(guān)。
[Abstract]:Background and purpose of the liver, kidney and other fibrotic diseases research showed that myofibroblasts are the main effect cells secrete the collagen fibers, epithelial mesenchymal transition is one of the main sources. The pathogenesis of silicosis is not entirely clear, but in the process known as silicosis fibrosis, myofibroblast is also the main cell synthesis the effect of the secretion of collagen fibers. The source of myofibroblast in pulmonary fibroblasts intrinsic, other sources are also valued. Type II alveolar epithelial cells have ability to differentiate, there may be one potential source of muscle fiber cells. So far, in the process of silicosis and silica (Si O2) dust how to induce type II alveolar epithelial cells to differentiate into cells of muscle fiber mechanism, efficiency and transdifferentiation of markers of time effect relationship is still uncertain. Therefore, this study constructed in vitro The model and the silicotic rats were cultured using transmission electron microscopy, fluorescence quantitative real-time PCR, laser confocal imaging, Western blot experimental technology, exploring the process and time - in type II alveolar epithelial cells EMT effect, provides a new way to explain the mechanism of silicosis, provides a reference for monitoring and the intervention of silicosis. The primary method of extraction of 1. type II alveolar epithelial cells (AEC II) and AM, the purchase of type II alveolar epithelial cell line (RLE-6TN), and transmission electron identification of AEC II and RLE-6TN. Through CCK-8 test, with SiO2 concentration gradient 1~140 g/mL 24,48 stain and 72h, determine the dye the dust concentration of.2. in vitro co culture model, AEC II cells were divided into control group and experimental group, RLE-6TN control group divided into direct, indirect direct dye dust group, control group and experimental groups. The indirect determination of different groups of 24,48 by Western blot and qRT-PCR Technology When 72h and AEC II and RLE-6TN GAPDH, the relative expression of E-cad and alpha -SMA protein and mRNA levels; using enzyme-linked immunosorbent assay (ELISA) determination of 24,48 and 72h technology of AEC II,.3. content of TGF- beta 1 to establish the rat model of silicosis in the supernatant of RLE-6TN cells, male healthy SD SPF 4~6 rats 84 weeks old, weighing between 160~180g, purchased from the experimental animal center of Henan Province, SiO2 exposed to dust by tracheal method, were divided into control group and silica group, normal saline injected, 6 rats in each group were killed, and the corresponding experimental 1,2,3,4,6,9 in rats and at 12 weeks. Using HE staining method to determine the rat model of silicosis was successful; using laser confocal fluorescence double staining technique to detect frozen lung tissue localization of E-cad and alpha -SMA sections and expression level, determine the content of.4. TGF- beta 1 polishing silicosis tissue in rats with ELISA Statistical analysis: using SPSS21.0 software to analyze the experimental data. The measurement data by normality test data obey the normal distribution, with mean and standard deviation (x + s) said, two-way analysis of variance of data using repeated measurement data, single factor data using single factor analysis of variance (one-way ANOVA), mean 22 by using the least significant difference method (LSD test), a =0.05. level test results of electron transmission electron microscope 1. AEC II and RLE-6TN showed that the cell structure were observed in the specificity of lamellar bodies was determined by.CCK-8 SiO2 on the cell inhibition rate showed the same exposure conditions with the extension of time, the stronger the inhibition, the same a time when exposed to dust, with the increase of the inhibition concentration of dust in the dust, about long time 72h inhibition rate reached 50%, the concentration of SiO2 at about 100 g/mL.2. AEC II in the experimental group compared with control group, E- protein CAD and alpha -SMA relative expression levels had significant difference (P0.05), experimental group E-cad and alpha -SMA relative expression level with the extension of time showed obvious time effect relationship. The experimental group and the control group were cultured in TGF- 1 beta cells in the supernatant with statistical significance (P0.05), and the change group TGF- beta 1 has obvious time effect relationship, the maximum value of 1.42ng/mL.3.RLE-6TN cells directly exposed group and control group, the experimental groups compared with the indirect indirect control group, the difference between E-cad and alpha -SMA protein and mRNA levels were statistically significant (P0.05), and has the obvious time effect relationship; direct and indirect dust dust group groups can make E-cad protein and mRNA expression decreased and the expression of alpha -SMA increased, indirect dust exposure group E-cad decreased and alpha -SMA rise time earlier than the direct dye dust group. Cell culture In the supernatant, the content of TGF- beta 1 in direct silicotic group and control group, indirect dust group and indirect control group had significant difference (P0.05), indirect and direct exposure to dust dust group group had obvious time effect relationship, the maximum value of 1.81ng/mL.4.HE staining showed that in the 2 week visible staining of lung tissue dust group had obvious accumulation of inflammatory cells, decreased muscle fiber 3 weeks, collagen fibers increased, especially after 3 weeks, the collagen fibers together, forming cell nodules, 6 week fibrous nodules, nodules increased gradually after fusion, even the pulmonary septal rupture. Dye content TGF- beta 1 in lung tissue of group dust slurry to the maximum in the 2 week, increased and then decreased; double immunofluorescent staining showed that the control group and the experimental group at 2 weeks, the expression of E-cad reached the maximum value, then decreased gradually, while the alpha -SMA appears gradually with time Conclusion the free SiO2 dust can induce EMT in the alveolar type II epithelial cells and differentiate into myofibroblasts. EMT is more likely to occur in the presence of macrophages, which may be related to the cytokines TGF- beta secreted by macrophages.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R135.2
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本文編號(hào)：1625070