游离二氧化硅诱导大鼠肺泡Ⅱ型上皮细胞转分化及其时间—效应关系

发布时间：2018-03-17 14:06

  本文选题：肺泡Ⅱ型上皮细胞　切入点：巨噬细胞　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景和目的有关肝、肾等纤维化疾病的研究表明,肌成纤维细胞是分泌胶原纤维的主效应细胞,上皮间质转分化是其主要来源之一。矽肺的发病机制并不完全清楚,但已知矽肺的纤维化过程中,肌成纤维细胞同样是合成、分泌胶原纤维的主效应细胞。肌成纤维细胞的来源除了肺内固有的成纤维细胞外,其他来源也同样受到重视。肺泡Ⅱ型上皮细胞具有一定的分化能力,有可能是肌成纤维细胞的潜在来源之一。迄今为止,在矽肺过程中,二氧化硅(Si O2)粉尘如何诱导肺泡Ⅱ型上皮细胞转分化为肌成纤维细胞的机制、效率以及转分化标志物的时间-效应关系尚不确定。因此,本研究构建了体外共培养模型和大鼠矽肺模型,应用电子透射电镜、实时荧光定量PCR、激光共聚焦成像、Western blot等实验技术,探索体内外肺泡Ⅱ型上皮细胞发生EMT的过程及其时间-效应关系,为矽肺的机制阐释提供了新的思路,为矽肺的监测和干预提供了参考。方法1.提取原代肺泡Ⅱ型上皮细胞(AECⅡ)和AM,购买肺泡Ⅱ型上皮细胞株(RLE-6TN),并对AECⅡ和RLE-6TN进行电子透射鉴定。通过CCK-8实验,用1~140μg/mL的SiO2浓度梯度染尘24、48和72h,确定染尘浓度。2.建立体外共培养模型,AECⅡ细胞分为对照组和实验组,RLE-6TN分为直接对照组、直接染尘组、间接对照组和间接染尘组。采用western blot和qRT-PCR技术测定不同分组24、48和72h时AECⅡ和RLE-6TN中GAPDH、E-cad和α-SMA的蛋白和mRNA的相对表达水平;采用酶联免疫吸附法(ELISA)技术测定24、48和72h时AECⅡ、RLE-6TN的细胞培养上清液中TGF-β1的含量。3.建立大鼠矽肺模型,选择SPF级4~6周龄的雄性健康SD大鼠84只,体重在160~180g之间,购于河南省实验动物中心,通过非气管暴露法进行SiO2染尘,实验分为对照组和染尘组,对照给予等量的生理盐水,每组6只,并在实验的1、2、3、4、6、9和12周时处死相应的大鼠。采用HE染色法判断大鼠矽肺模型是否成功;采用激光共聚焦荧光双染技术,检测肺组织冰冻切片中E-cad和α-SMA的定位及表达水平,采用ELISA测定大鼠矽肺组织研磨液中TGF-β1的含量。4.统计分析:采用SPSS21.0软件分析实验数据。计量资料经正态性检验服从正态分布,数据以均值加减标准差(χ±s)表示,双因素数据采用重复测量资料的方差分析,单因素数据采用单因素方差分析(one-way ANOVA),均数的两两比较采用最小显著差法(LSD检验),检验水准α=0.05。结果1.原代AECⅡ和RLE-6TN的电子透射电镜显示,细胞结构内均可见特异性的板层小体。CCK-8测定SiO2对细胞抑制率显示,同一染毒条件下随着时间的延长,抑制作用越强,同一染尘时间时,随着染尘浓度的增大抑制作用增强,约在最长染尘时间72h时抑制率达到50%,此时的SiO2浓度约为100μg/mL。2.原代AECⅡ的实验组和对照组相比,蛋白E-cad和α-SMA的相对表达水平的差异显著(P0.05),实验组E-cad和α-SMA的相对表达水平随着时间的延长呈现明显的时间-效应关系。实验组和对照组培养细胞的上清液中TGF-β1的含量的差异具有统计学意义(P0.05),并且实验组TGF-β1的变化具有明显的时间-效应关系,其最大值为1.42ng/mL。3.RLE-6TN细胞的直接染尘组与直接对照组、间接染尘组与间接对照组相比,E-cad和α-SMA的蛋白和mRNA水平的差异均具有统计学意义(P0.05),且具有明显的时间-效应关系;直接染尘组和间接染尘组均可使E-cad的蛋白和mRNA水平表达下降而α-SMA的表达上升,间接染尘组E-cad下降和α-SMA上升的时间早于直接染尘组。细胞培养上清液中,TGF-β1的含量在直接染尘组与直接对照组、间接染尘组与间接对照组差异显著(P0.05),间接染尘组和直接染尘组中均具有明显的时间-效应关系,其最大值为1.81ng/mL。4.HE染色显示,在2周时可见染尘组肺组织有明显的炎性细胞聚集,3周时肌纤维逐渐减少,胶原纤维增多,特别是在3周以后,可见胶原纤维聚集成团,形成细胞性结节,6周有纤维性结节出现,之后结节逐渐增大融合,甚至出现肺间隔断裂。染尘组肺组织研磨液中TGF-β1的含量在2周时达到最大值,呈现先升高后下降的趋势;免疫荧光双染显示对照组和实验组在2周时E-cad的表达达到最大值,之后逐渐下降,而α-SMA呈现随时间逐渐上升的趋势。结论游离SiO2粉尘可诱导肺泡Ⅱ型上皮细胞发生EMT,转分化为肌成纤维细胞。在巨噬细胞同时存在的条件下更易于发生EMT,这可能与巨噬细胞分泌的细胞因子TGF-β有关。
[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.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R135.2
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本文编号：1625070