【作者】 张欢；

【导师】 姚武；

【作者基本信息】 郑州大学 ， 劳动卫生与环境卫生学， 2009， 硕士

【摘要】 矽肺是在生产过程中长期吸入大量含游离二氧化硅生产性粉尘所引起的以肺部广泛的结节性纤维化为主的全身性疾病。肺纤维化前期主要表现为肺泡巨噬细胞,中性粒细胞和淋巴细胞等炎性细胞增多,后期则表现为大量的间质细胞增生及基质成分增多、基质胶原进行性积聚。在增生的细胞中,除肺泡上皮细胞外主要为成纤维细胞。目前认为,矽肺形成过程中巨噬细胞是游离SiO₂粉尘的直接效应细胞。巨噬细胞受刺激后可释放多种细胞因子如白细胞介素、转化生长因子β、肿瘤坏死因子、纤维粘连蛋白等,刺激成纤维细胞增生、网织纤维及胶原纤维的合成。虽然肺组织已有的纤维化病变难以消除,但肺纤维化是一个慢性、进行性的病理反应过程,探索肺纤维化发病的分子机制以延缓或阻断纤维化发展仍具有重要的实际意义。在纤维化的发展过程中,肺成纤维细胞转化为肌成纤维细胞起关键作用。肌成纤维细胞是种在超微结构和功能上兼有成纤维细胞和平滑肌细胞两者特征的非典型的成纤维细胞,其特征性表达为仅α-平滑肌肌动蛋白。大量研究证明,肺纤维化形成过程中,转化生长因子β发挥重要作用。胰岛素样生长因子Ⅱ受体在哺乳动物多个组织均有表达,IGF-ⅡR可以结合前转化生长因子β并刺激前转化生长因子β裂解和活化形成转化生长因子β。也有研究提示IGF-Ⅱ可能通过直接作用于自身或邻近癌细胞的IGF-ⅡR,放大细胞持续生长信号,加速癌细胞的自主性生长。本研究拟通过体外分离培养SD大鼠腹腔巨噬细胞、肺成纤维细胞,观察肺成纤维细胞暴露于不同浓度的游离SiO₂粉尘刺激巨噬细胞后的培养上清,鉴定肺成纤维细胞是否可被诱转为肌成纤维细胞;而后从mRNA、蛋白水平水平上分析诱导后的肺成纤维细胞IGF-ⅡR表达,探讨IGF-ⅡR在诱导后的肺成纤维细胞中的表达水平及意义,为进一步研究矽肺的发生、发展机制及治疗措施提供参考依据。方法1.建立大鼠腹腔巨噬细胞和成纤维细胞体外原代培养模型。成熟SD大鼠腹腔灌洗获取巨噬细胞;用胰蛋白酶消化肺组织,分离和纯化肺成纤维细胞,在37℃、5%CO₂条件下进行培养。利用MTT法描述肺成纤维细胞生长曲线。2.肺成纤维细胞转化鉴定。以无血清DMEM培养基为溶剂配制游离SiO₂粉尘工作液,终浓度分别为0μg/ml、25μg/ml、50μg/ml、75μg/ml、100μg/ml,对离体原代培养的大鼠巨噬细胞染毒24h,离心收集M_φ培养上清,并作用于肺成纤维细胞24h,免疫组织化学法检测细胞α-SMA蛋白的表达。3.游离SiO₂粉尘刺激巨噬细胞的上清对IGF-ⅡR基因转录和蛋白表达的影响。游离SiO₂粉尘工作液对离体原代培养的大鼠巨噬细胞染毒24h,离心收集M_φ培养上清,并作用于肺成纤维细胞24h:（1）提取成纤维细胞内总RNA,进行逆转录聚合酶链反应（RT-PCR）,以GAPDH为内参照,检测成纤维细胞内IGF-ⅡRmRNA水平。（2）免疫组织化学法检测细胞IGF-ⅡR蛋白的表达。4.统计学分析。运用SPSS 12.0统计软件的单因素方差分析（analysis ofvariance,ANOVA）、Bonfferoni两两比较、Levene方差齐性检验对数据进行统计学分析（检验水准α=0.05）。结果1.本研究分离培养的SD大鼠腹腔巨噬细胞、肺成纤维细胞体外生长良好,可以用于体外实验研究。2.肺成纤维细胞转化鉴定:以0μg/ml、25μg/ml、50μg/ml、75μg/ml、100μg/ml游离SiO₂粉尘刺激M_φ的培养上清作用于肺成纤维细胞后24h后,免疫组织化学法检测发现,高浓度刺激组α-SMA表达明显高于对照组,且随游离SiO₂粉尘浓度增高,肺成纤维细胞表达α-SMA的量也逐渐增多,3.肌成纤维细胞IGF-ⅡR mRNA表达结果:不同浓度游离SiO₂粉尘刺激M_φ的培养上清作用于肺成纤维细胞24h后,实验组肺成纤维细胞IGF-ⅡR mRNA相对表达量均高于对照组,低剂量组（0μg/ml、25μg/ml、50μg/ml）与对照组比较差异无统计学意义（P＞0.05）;75μg/ml、100μg/ml游离SiO₂粉尘组与对照组比较差异有统计学意义（P＜0.05）。4.肌成纤维细胞IGF-ⅡR蛋白表达结果:不同浓度游离SiO₂粉尘刺激M_φ的培养上清作用于肺成纤维细胞后24h,结果显示随游离SiO₂粉尘浓度增高肺成纤维细胞表达IGF-ⅡR的量也逐渐增高。结论1.游离SiO₂粉尘刺激M_φ的培养上清可诱导肺成纤维细胞转化为肌成纤维细胞。2.游离SiO₂粉尘诱转的肌成纤维细胞存在IGF-R的表达增多。更多还原

【Abstract】 Silicosis is a system disease with a wide range of nodular fibrosis in lung,which is induced by inhaling much free silica dust for a long time.The increasing of inflammatory cells,such as pulmonary alveolar macrophages,neutrophils and lymphocytes,is the main behaviour in the prophase of pulmonary fibrosis.And proliferation of mesenchymal cell,increasing of matrix and accumulation of matrix collagen are in the later stage.Most of the proliferation cells are fibroblasts besides the alveolar epithelial cells.At present,it is believed that macrophage is the direct effect cells of free silica dust at Silicosis,Macrophages could secrete much cytokines after stimulated,such as interleukin,transtbrming growth factorβ,tumor necrosis factor,fibronectin,etc. These cytokines could stimulate the proliferate of fibroblasts and increase the synthesize of reticulum fiber and collagen fiber.The pathological changes of the lung tissue was difficult to eliminate.The pulmonary fibrosis is a chronic,progressive pathological process,so exploring the molecular mechanisms of pulmonary fibrosis to delay or block the development of fibrosis is still important.The process that fibroblasts transform into myofibroblasts plays a key role in the development of fibrosis.Myofibroblast is an atypical fibroblast with the characteristics of both fibroblasts and smooth muscle cells in ultrastructure and function,which express a-smooth muscle actin distinctively.Large number of studies have proved that transforming growth factor-βplays an important role in the process of pulmonary fbrosis.Most of organizations in mammalian can express Insulin-like growth factorⅡreceptor,which can be combined to the pro-transforming growth factorβand stimulate it cleavage and activate.Some researches suggested that IGF-Ⅱmay amplify signal to promote cells proliferate continuously and accelerate cancer cells to grow spontaneously,by combining with IGF-ⅡR on itself or surrounding cells.In this study,the rat enterocoelia macrophages and lung fibroblasts in vitro were cultrured.Then the lung fibroblasts were exposed to the macrophages culture supematant which was stimulated by different concentration of flee silica dust. Analyze the lung fibroblasts whether transformate to myofibroblasts or not,Study the changes of mRNA and protein levels of IGF-ⅡR in myofibroblasts,to explore the significance of IGF-ⅡR,to provide reference for the mechanisms of occurance and development and the treatment measure of Silicosis,further.Methods1.Establish the primary culture model of rat enterocoelia macrophage and fibroblast in vitro.The enterocoelia macrophages were obtained by lavaging the Mature SD rat and the lung tissue was digested by trypsin,then the cells were separated and puriflcated and followed by cultured at the 37℃,5%CO₂ conditions. MTT method was used to describe the growth curve of lung fibroblast.2.Identify the transformation of lung fibroblast.The serum-free DMEM medium was used for preparation of the free silica dust fluid.The final concentrations of free silica dust were 0μg/ml,25μg/ml,50μg/ml,75μg/ml,100μg/ml,respectively.The free silica dust fluid was used to stimulate M_φfor 24h,then the culture supernatant was collected.After that,the fibroblast was stimulated by culture supernatant for 24h,then the expression of theα-SMA protein was detected by using immunohistochemistry.3.The changes of the IGF-ⅡR in gene transcription and protein expression after stimulation.（1）The total RNA extracted from flbroblasts was undergone the reverse transcription polymerase chain reaction（RT-PCR）,with GAPDH as the internal reference to detect the expression level of IGF-ⅡR of fibroblasts.（2） The expression of theα-SMA protein was camed out by using immunohistochemistry.4.Statistical analysis.SPSS12.0 was used to statistically analyze the datas.A difference at P＜0.05 was considered statistically significant.Result1.Peritoneal macrophages and lung fibroblasts isolated from rat for primary culture grew well,and can be used in vitro experiment.2.Identifing the transformation of lung fibroblast:The fibroblasts was stimulated by 0μg/ml,25μg/ml,50μg/ml,75μg/ml,100μg/ml culture supematant after free silica dust stimulating the macrophages.The difference ofα-SMA expression was significant between high concentration group and control group（P＜0.05）.With the concentration of free silica dust increasing,the expression ofα-SMA was also increased.3.Expression of IGF-ⅡR mRNA in myofibroblasts:After the supernatant of M_φwhich stimulated by different concentrations of free silica dust working on lung fibroblasts for 24h,the expression of IGF-ⅡR mRNA in all of experimental groups were higher than that of control group.The differences of IGF-ⅡR mRNA expression were significant between 75μg/ml,100μg/ml concentration groups and control group （P＜0.05）.4.Expression of IGF-ⅡR protein in myofibroblasts:The supernatant of M_φwhich stimulated by different concentrations of free silica dust worked on lung fibroblasts for 24h.The results showed that with the concentration of free silica dust increasing,the expression of IGF-ⅡR was also increased.Conclusion1.The culture supernatant of M_φstimulated by free silica dust can induce the lung fibroblasts transformate into myofibroblasts.2.Expression of IGF-ⅡR was increased in myofibroblasts induced by free silica dust.更多还原