【作者】 李娟；

【导师】 姚武；

【作者基本信息】 郑州大学 ， 劳动卫生与环境卫生学， 2017， 博士

【摘要】 背景和目的矽肺是由于吸入游离二氧化硅（SiO₂）粉尘引起的以肺组织慢性炎症和进展性纤维化为特征的全身性疾病,发病机制尚不明确。国内外研究发现,矽肺的发生发展是一个涉及多种细胞参与、多因子调控的复杂过程。大量研究结果证实活化的成纤维细胞/肌成纤维细胞是分泌胶原蛋白、最终导致肺组织广泛纤维化的主要终端效应细胞。既往矽肺研究中主要关注肺成纤维细胞,近年来研究发现循环纤维细胞（circulating fibrocyte,CF）具有炎症趋化、类干细胞分化的特性,可能是成纤维细胞/肌成纤维细胞的又一主要来源。研究证实CF参与特发性肺纤维化等疾病发病过程,但其是否参与矽肺纤维化的发生发展,尚未见报道。因此,通过建立大鼠矽肺模型,辅以体外实验,采用流式细胞术、免疫荧光、电子显微镜观察等实验技术,系统研究循环纤维细胞在矽肺炎症与纤维化形成期动态变化及可能作用与机制,进一步深化对矽肺发病机制的认识,寻找矽肺潜在的生物学标志及可能的关键靶点,为矽肺的预防、早发现及有效干预提供新的思路和理论依据。材料和方法1.二氧化硅诱导循环纤维细胞转分化的体外研究取SPF级雄性SD大鼠分离大鼠外周血单个核细胞,以含20%胎牛血清的DMEM调整细胞浓度2.5×106/ml,接种于6孔板后持续培养12 d,获得CF。大鼠肺泡巨噬细胞（alveolar macrophage,AM）的获取采用支气管肺泡灌洗法。免疫荧光双染法观察不同培养天数CF,流式细胞术检测不同培养天数CF的比例。RT-PCR半定量法检测Collagen I、Collagen III和α-SMA mRNA表达,酶联免疫吸附测定法检测TGF-β1细胞因子分泌。免疫组织化学法检测纯化后继续培养至第20 d的CFα-SMA蛋白表达。CCK8法测定不同浓度SiO₂粉尘在不同暴露时间下对AM和CF存活率的影响,确定后续染毒浓度。建立AM和培养6 d的外周血单个核细胞共培养模型探讨SiO₂粉尘暴露对其向CF转分化的影响,染毒24 h、48 h后流式细胞术检测CF比例;qRT-PCR和免疫荧光法检测SiO₂粉尘暴露后CF的Collagen I、Collagen III和α-SMA m RNA和蛋白表达;酶联免疫吸附法检测SiO₂暴露后CF培养上清中Collagen I、Collagen III和TGF-β1蛋白分泌水平。建立AM和纯化后培养至20 d的CF共培养模型,染毒48 h,免疫组织化学法检测CFα-SMA蛋白表达。2.实验性矽肺大鼠循环纤维细胞分布规律和趋化行为的动态观察随机将72只SD雄性大鼠分为矽肺组和对照组。非暴露气管滴注染尘法建立大鼠矽肺模型。建模后1、2、3、6、9、12周处死大鼠（矽肺组和对照组各6只）,收集外周血、肺泡灌洗液和肺组织。HE和Masson染色观察不同时段大鼠肺组织形态学变化和胶原沉积情况,鉴定大鼠矽肺模型。流式细胞术检测大鼠外周血、肺组织中CF比例;免疫荧光双染法检测肺组织中CF分布及CF来源Collagen I分泌细胞所占比例及CF来源肌成纤维细胞比例和肺泡II型上皮细胞来源的肌成纤维细胞比例;免疫组织化学法检测肺组织中CXCR4表达,ELISA法测定肺组织中CXCR4的配体CXCL12表达;Western blot检测肺组织p-PI3K、p-Ser473-Akt、p-mTOR的蛋白表达;流式细胞术检测肺泡灌洗液中Th1、Th2细胞数量变化,ELISA测定肺组织中Th1、Th2型细胞因子IFN-γ和IL-4及TGF-β1和IL-1β的表达。另选10只SD雄性大鼠观察CF从外周血向肺内迁移情况,CFSE活细胞荧光染料标记CF,尾静脉注射入大鼠体内,立即对大鼠进行SiO₂染毒处理。1周后处死大鼠,免疫荧光法观察肺组织中是否存在荧光标记的CF及其分布,流式细胞术检测肺组织中CF所占比例。3.统计学分析运用SPSS17.0对实验数据进行统计学分析,分析结果以c±s表示,两组独立样本间均数比较采用Student’s t检验,多组独立样本间比较采用ANOVA,LSD检验用于进一步进行两两比较,相关分析采用Pearson相关分析,检验水准α=0.05。结果1.CF原代培养、鉴定及转分化特性的观察体外培养外周血单个核细胞,显微镜下可见淋巴样圆形细胞减少,6 d后梭形及不规则样细胞增多并开始增殖,12 d体积明显增大呈典型的成纤维样;随着培养天数增加,CF细胞比例明显升高,12 d后可以达到74.25%。随着体外培养时间的延长,Collagen I、Collagen III的m RNA表达升高,9d和12 d最高,差异均有统计学意义（P<0.05）,α-SMA mRNA表达则呈初期高后期低趋势;纯化后的CF培养至第20 d开始表达α-SMA蛋白。2.SiO₂粉尘暴露对CF转分化的观察SiO₂粉尘染毒可以抑制AM和CF的活性。在相同染毒时间下,AM和CF的存活率均随着SiO₂染毒浓度的升高而下降,差异具有统计学意义（P<0.05）。对照组和染尘组CF比例均随培养时间的延长而升高（与0 h组相比,P<0.05）;而相同时间段内,染尘组CF比例明显高于对照组,差异具有统计学意义（与同时段对照组相比,P<0.05）。与对照组和0 h组相比,48 h染尘组Collagen I和Collagen III mRNA表达和细胞培养上清中Collagen I和Collagen III分泌均明显升高,差异均有统计学意义（P<0.05）。同时观察到Collagen I和Collagen III定位于CF细胞质中,未观察到α-SMA蛋白表达。染尘组和对照组细胞培养上清中TGF-β1的表达均随培养时间延长而升高,48 h组均达到最高,且染尘组比对照组明显升高,差异具有统计学意义（P<0.05）。纯化后的CF培养至20 d与AM建立共培养体系,染尘组与对照组相比,α-SMA蛋白表达明显升高,差异具有统计学意义（P<0.05）。3.大鼠肺组织形态学及纤维化程度观察染尘1、2周后肺泡壁明显增宽,小气道周围及肺泡腔出现大量炎性细胞浸润;染尘3周后可见肺泡腔出现明显的细胞性结节,以炎性细胞聚集为主;6周后可见细胞性结节和纤维性结节形成,并出现结节融合现象;9、12周后可见大量纤维性结节出现,肺泡结构破坏,间隔断裂及融合。矽肺大鼠肺组织胶原沉积面积百分比均高于对照组,且随染尘时间的增加呈升高趋势,差异具有统计学意义（P<0.05）。4.大鼠外周血和肺组织中CF比例及其来源的Collagen I分泌细胞比例和分布与对照组相比,染尘2周至12周矽肺大鼠外周血中CF的比例均明显升高,差异具有统计学意义（P<0.05）。与对照组相比,不同染尘时段矽肺大鼠肺组织单细胞悬液中CF的比例均明显升高,差异具有统计学意义（P<0.05）。与对照组相比,不同染尘时段矽肺大鼠肺组织中CF占Collagen I分泌细胞比例均显著升高（10%¹6%）,差异具有统计学意义（P<0.05）。并且染尘初期CF主要位于血管和炎细胞聚集处,纤维化期可见CF存在于纤维结节内。5.三种不同来源的肌成纤维细胞权重及变化趋势与对照组相比,不同染尘时段矽肺大鼠肺组织中CF和II型肺泡上皮细胞来源的肌成纤维细胞均显著升高,差异均具有统计学意义（P<0.05）。染尘不同时段,CF来源的肌成纤维细胞比例约15%³5%,而II型肺泡上皮细胞来源的肌成纤维细胞约9%²1%,计算肺成纤维细胞来源肌成纤维细胞的比例约为40%⁶5%。循环纤维细胞和肺泡II型上皮细胞来源肌成纤维细胞在染尘初期（1周、2周、3周）呈升高趋势,染尘后期（6周、9周、12周）呈下降趋势,肺成纤维细胞来源的肌成纤维细胞与上两者趋势相反。6.SiO₂粉尘暴露对CF体内迁移及肺组织中CXCR4、CXCL12和PI3K/AKT/mTOR信号通路蛋白表达的影响染尘1周,CF可以从外周血迁移至肺内,与对照组相比,矽肺组大鼠肺组织中CFSE+CF占肺总细胞比例明显升高,差异具有统计意义（P<0.05）,并且CF主要分布于血管周围。与对照组相比,不同染尘时段矽肺大鼠肺组织中CXCR4的表达均明显升高,1周至9周差异具有统计学意义（P<0.05）,并且1周最高。与对照组相比,1周、2周和3周矽肺组大鼠肺组织中CXCL12表达均升高,差异具有统计学意义（P<0.05）。与对照组相比,矽肺组大鼠肺组织中p-PI3K、p-Ser473-Akt、p-m TOR的蛋白表达在均在1周和2周明显升高,差异均具有统计学意义（P<0.05）。7.大鼠肺泡灌洗液Th1、Th2细胞数量及肺组织中相关细胞因子的变化随着染尘时间的延长,不同时段矽肺组大鼠肺泡灌洗液中Th1细胞比例呈下降趋势,Th2细胞呈升高趋势,Th1/Th2比值逐渐下降,差异具有统计学意义（P<0.05）。与对照组相比,Th1细胞比例1、2、3周升高,Th2细胞比例1、2周下降、9周和12周升高,Th1/Th2比值1、2、3周升高,12周下降,差异均具有统计学意义（P<0.05）。与对照组相比,矽肺组大鼠肺组织中INF-γ表达在1周明显升高,差异具有统计学意义（P<0.05）;IL-4表达水平1周降低,12周升高,差异均具有统计学意义（P<0.05）。与对照组相比,矽肺组大鼠肺组织中TGF-β1表达在2周明显升高,而IL-1β表达在染尘1到9周均显著升高,差异具有统计学意义（P<0.05）。8.CF与Th1/Th2及相关细胞因子的关联性分析染尘1周,CF来源Collagen I分泌细胞比例与Th1/Th2比值和肺内INF-γ的水平均呈显著正相关（r=0.879,P=0.021;r=0.942,P=0.005）。结论1.CF具有类干细胞分化特性,SiO₂体外和体内暴露均促进外周血单个核细胞向CF转分化,并促进CF向肌成纤维细胞转分化。2.在实验性矽肺的发生发展过程中,CF可以由外周血迁移至肺部,炎症反应期肺内CF来源的肌成纤维细胞增多最明显,比例最高可达35%,是肌成纤维细胞的重要来源。更多还原

【Abstract】 Exposure to free silica（SiO₂）induces silicosis characterized by pulmonary chronic inflammation and progressive fibrosis and its mechanisms is still unclear.It is well known that the pathological process of silicosis is involved in interaction of multi-type cells and cytokines.However,activated fibroblasts / myofibroblasts are regarded as a critical effector cell which can secrete collagen and lead to extensive fibrosis in lung.The previous studies on silicosis focused on lung resident fibroblasts.Recently,circulating fibrocyte（CF）was found to have the ability of inflammatory chemotaxis and differentiation like stem cell,which may be another source of fibroblast/myofibroblast.CF is proved to play a pivotal role in several lung fibrosis diseases including idiopathic pulmonary fibrosis,but whether CF participates in the process of silicosis is rarely reported.This study aimed to explore the dynamic change and mechanism of CF during the progress of rat silicosis couple with experimental model in vitro,flow cytometry,immunofluorescence,electron microscope and so on.It is helpful to find the potential biological mechanism and explore the potential biomarker in silicosis if we have a better understanding on how CF activated,differentiated and tracked to lung.It may provide a new insight and theory to prevent,discovery and intervene silicosis.Materials and Methods1.The study of circulating fibrocyte differentiation when exposed to SiO₂ in vitroPeripheral blood mononuclear cells（PBMC）were obtained from SPF male SD rats.PBMC were incubated in DMEM consists of 20% fetal calf serum and seeded in6-well plate at cell density of 2.5×106/ml.CF was obtain after cultured for 12 days.Alveolar macrophages（AM）were obtained by bronchoalveolar lavage fluids（BALF）.Immunofluorescence double staining was used to observe CF and Flow cytometry（FCM）was used to detect the proportion of CF.The expression of Collagen I,Collagen III and α-SMA m RNA and TGF-β1 protein were detected by semi-quantitative RT-PCR and ELISA.Immunohistochemistry staining was used to detect α-SMA expression of CF（20 d）.CCK8 method was used to determine the viability of CFs and AMs exposed to SiO₂.The co-culture system was established with CF and AM.The AMs were exposed to SiO₂ for 0 hours,24 hours and 48 hours.After SiO₂ exposure,the proportion of CF was detected by FCM.Collagen I,Collagen III and α-SMA mRNA expression and protein location were examined by qRT-PCR and immunofluorescence.The levels of Collagen I,Collagen III and TGF-β1 in cell supernatant were measured by ELISA.AM and CF（20 d）were used to establish a same co-culture system and AMs exposed to SiO₂ for 48 hours,immunohistochemistry staining was used to detect α-SMA expression of CF.2.Dynamic observation of distribution and chemotactic behavior of circulating fibrocytes in process of experimental rat silicosis.72 SPF male SD rats were randomly divided into model group and control group.The rat silicosis model was established by intratracheal instillation of crystalline SiO₂ suspension and equal amount of saline was instilled to control group rats.After SiO₂ exposure,rats were sacrificed at week 1,2,3,6,9,12 and collected peripheral blood,BALF and lung tissue.HE and Masson staining were used to observe the pathological changes and collagen deposition in silicosis rats to identify model.The proportion of CF in peripheral blood and lung were detected by FCM.The percentage of CF derived Collagen I secreting cells or CF derived and type II alveolar epithelial cellsderived myofibroblasts in lung was detected by immunofluorescence.CXCR4 and CXCL12 were detected by immunohistochemistry and ELISA.The p-PI3 K,pSer473-Akt and p-mTOR protein expression in lung was detected by Western blot.The number of Th1 and Th2 cells in BALF was measured by FCM.The levels of IFN-γ,IL-4,TGF-β1 and IL-1β were measured by ELISA.CFSE labeled CF were injected into another 10 SD male rats by tail vein.The rats were exposed to SiO₂ or saline immediately.After 1 week,the rats were sacrificed,then proportion of CFSE+CF in lung and CF location were detected by immunofluorescence.FCM was used to examined the proportion of CF in lung of rats.3.Statistical analysisSPSS 17.0 was used to analysze the data.The data was expressed as mean ± SD.Student’s t-test was used to compare two comparisons.ANOVA was used to compare the multiple samples.The LSD post-hoc tests was used to further compare the two groups.Correlation analysis used Pearson correlation analysis.Inspection level of alpha = 0.05.Results1.The observation and identification of primary cultured CFThe PBMC were cultured for 12 days,lymphocyte-like cell decreased and numerous CF can be observed with a spindle shape.The cells began to proliferate at day 6 and own a fibroblast-like morphology at day 12.The proportion of CF was increased and reached 74.25% at day 12.Collagen I and Collagen III m RNA expression was increased and reached a higher level at day 9 and 12.The differences were statistically significant（P<0.05）.α-SMA mRNA expression was decreased during cultured period.The CF began to expression α-SMA protein at day 20.2.The observation of differentiation of CF exposed to SiO₂ in vitroSiO₂ exposure can inhibit the survival of AM and CF and it has a dosedependent manner,the differences were statistically significant（P <0.05）.Proportion of CF in both control and SiO₂ exposure group were increased by a time-dependent manner（Compared with 0 h group,P <0.05）.Proportion of CF of SiO₂ exposure group was higher than that of control group at a same period（P<0.05）.Compared with control and 0 h group,Collagen I and Collagen III mRNA expression and protein levels in culture supernatant of 48 h SiO₂ exposure group were significantly higher（P <0.05）.Meanwhile,Collagen I and Collagen III protein were localized in CF cytoplasm and no α-SMA protein was observed.The level of TGF-β1 in culture supernatant increased by a time-dependent manner in both two groups and highest at 48 h group.Meanwhile,SiO₂ exposure group was higher than that of control group at 48 hours,the difference was statistically significant（P <0.05）In co-culture system of AM and CF（20 d）,the α-SMA protein expression was significantly increased than that of control group after exposed to SiO₂ for 48 hours,the difference was statistically significant（P <0.05）.3.The observation of morphology and fibrosis level in lungThe alveolar wall became widened and plenty of inflammatory cells infiltrated around the small airway and alveolar cavity after exposed to SiO₂ for 1 and 2 weeks.Cell nodules which is mainly consist of inflammatory cells were formed at week 3.After 6 weeks,small nodules merged into larger one,and then alveolar structure damage and fusion,with a number of fibrous nodules formed.The percentage of collagen deposition area in lung tissue of silicosis rats was higher than that of control group,the difference was statistically significant（P <0.05）.4.The proportion of CF and CF derived Collagen I secreting cellsCompared with control group,the proportion of CF in peripheral blood was significantly higher from week 2 to week 12,the differences were statistically significant（P <0.05）.The proportion of CF in lung of silicosis rat was significantly raised than that of control group,the difference was statistically significant（P <0.05）.The proportion of CF derived Collagen I secreting cells in lung of silicosis rat was significantly increased than that of control group,and difference was statistically significant（P <0.05）.5.The weight and trend of myofibroblasts from three different sourcesCompared with control group,CF derived and alveolar epithelial type II cells derived myofibrobasts in lung of silicosis rat were increased,and differences were statistically significant（P <0.05）.Proportion of CF derived myofibroblasts is about 15% to 35%,and that of type II alveolar epithelial cells is 9% to 21%,lung fibroblast derived myofibroblast is about 40% to 65%.The former two sources of myofibroblast showed a upward trend in the early stage of silicosis（week 1,2,3）,and downward trend in the late stage（week 6,9,12）.The lung fibroblast derived myofibroblast has a reverse trend.6.Tracking migration of CF and changes of CXCR4,CXCL12 and PI3 K / AKT /mTOR signaling protein.CF migrated from peripheral blood to pulmonary after SiO₂ exposure for 1 week.Compared with control group,the proportion of CFSE+CF in lung of silicosis rat was significantly raised and the difference was statistically significant（P < 0.05）.The expression of CXCR4 in lung of silicosis rat was higher than control group from week 1 to week 9,and highest at week 1,the differences were statistically significant（P <0.05）.The expression of CXCL12 in lung of silicosis rat was increased at week 1,2,3than that of control group,and the difference was significant（P <0.05）.Compared with control group,the levels of p-PI3 K,p-Ser473-Akt and p-mTOR in lung of silicosis rat were significantly raised at week 1 and 2,the differences were statistically significant（P < 0.05）.7.The changes of Th1,Th2 and cytokines in bronchoalveolar lavage fluid and lungWith prolonged period of SiO₂ exposure,proportion of Th1 cells was decreased and TH2 cells was raised,with the ratio of Th1/Th2 was decreased in BALF,the differences were statistically significant（P <0.05）.Compared with control group,Th1 cells increased at week 1,2,3,Th2 cells decreased at week 1,2 and increased at week9,12,the ratio of Th1 / Th2 was increased at week 1,2,3 and decreased at week 12,the differences were statistically significant（P <0.05）.Compared with control group,the expression of INF-γ in lung tissue of silicosis rat increased at week 1,and the level of IL-4 decreased at week 1 and increased at week 12,the differences were statistically significant（P <0.05）.Compared with the control group,the expression of TGF-β1 in lung of silicosis rat was significantly increased at week 2,and that of IL-1β was increased from week1 to week 9,the differences were statistically significant（P < 0.05）.8.The correlation analysis between CF and Th1 / Th2 and related cytokinesAfter exposure to SiO₂ for 1 week,there is a obvious positive correlation between CF derived Collagen I secreting cells and Th1 / Th2 ratio or level of INF-γ.（r=0.879,P=0.021;r=0.942,P=0.005）Conclusions1.CF has ability of differentiation like stem cell,SiO₂ can promote PBMC differentiate to CF and further differentiate to myofibroblast.2.CF migrated from peripheral blood to lung in the process of silicosis;the accumulation of CF derived myofibroblast in lung is obvious in inflammatory reaction period.CF derived myofibroblast contributes to 35% and it is a pivotal original of myofibroblast.更多还原