脂多糖诱导肺成纤维细胞表型转变及异常增殖的机制研究
发布时间：2018-09-06 15:45
【摘要】：肺纤维化是急性肺损伤（acute lung injury, ALI）和急性呼吸窘迫综合征（acuteespiratory distress syndrome, ARDS）发展的重要病理阶段，以肺成纤维细胞异常增、活化并分泌胶原蛋白，形成肺间质和肺泡内弥散性纤维化为特点。革兰氏阴性菌细胞壁的脂多糖（lipopolysaccharide, LPS）可以作用于肺成纤维细胞的特异性体—Toll样受体4（Toll-like receptor4, TLR4），并直接引起肺成纤维细胞的活化增殖，与肺纤维化发生密切相关。然而LPS对成纤维细胞增殖作用的研究结果很一致，提示不同来源的成纤维细胞亚型可能因存在着不同的表型而产生异质性heterogeneity），其可能是细胞对LPS刺激产生不同生物学效应的重要原因。 Thy-1是成纤维细胞表面由糖磷脂酰肌醇锚定的糖蛋白，，根据细胞表面胸腺细分化抗原-1（thymocyte differentiation antigen1, Thy-1）表达的情况，可以将肺成维细胞分为Thy-1(＋)及Thy-1(－)两种不同的表型。Thy-1在正常肺成纤维细胞面大量表达，但是在特发性肺纤维化（idiopathic pulmonary fibrosis, IPF）患者肺织存在着大量Thy-1呈阴性表达的Thy-1(－)肺成纤维细胞。Thy-1(－)的肺成纤细胞在各种致纤维化因子刺激下具有更活跃的反应能力，与肺纤维化的发生和发密切相关。有研究表明Thy-1基因表达受组蛋白去乙酰化等表观遗传学机制的调，也有研究表明LPS可以通过组蛋白去乙酰化等表观遗传学机制调控多种基因的达。但是，目前尚未明确LPS是否通过表观遗传调控作用抑制肺成纤维细胞Thy-基因的表达而改变细胞的表型，引起肺成纤维细胞的增殖。 本课题拟利用LPS诱导肺成纤维细胞增殖的模型，首先明确LPS直接诱导原代养的Thy-1(＋)肺成纤维细胞发生表型转变的和异常增殖的作用；随后通过RNAi术抑制肺成纤维细胞TLR4的表达，研究LPS通过TLR4对肺成纤维细胞Thy-1因表达以及细胞表型转变和异常增殖的表观遗传学调控作用。 目的：明确在急性肺损伤肺纤维化早期，脂多糖（LPS）诱导肺成纤维细胞增殖及表型转变的作用。 方法：原代培养小鼠肺成纤维细胞，以终浓度为0.01μg/ml、0.1μg/ml和1μg/ml的LPS刺激细胞，并设阴性对照；LPS作用0h、6h、24h、48h和72h后以CCK-8（cell counting kit-8）细胞计数试剂盒检测细胞增殖情况，以real-time PCR检测各组细胞Thy-1mRNA的动态表达情况。 结果：原代培养的小鼠肺成纤维细胞Thy-1基因呈阳性表达；以不同浓度LPS刺激细胞最初24小时内未见细胞明显增殖，48-72小时后细胞增殖速率显著增加，同时伴有Thy-1基因表达量显著下降，经统计学检验差异存在显著性（P0.05）。相对于0.01μg/ml和0.1μg/ml两个浓度，以1μg/ml的LPS刺激细胞产生的细胞增殖及Thy-1基因表达下调的反应更为显著。 结论：LPS可通过抑制肺成纤维细胞Thy-1基因的表达而使细胞发生表型转变并引起异常增殖。 目的：设计、构建和鉴定TLR4-RNAi慢病毒载体（Lentivirus-TLR4-siRNA）用于后续实验。 方法：设计、制备3个针对TLR4基因的siRNA质粒并进行慢病毒包装。转染原代培养的小鼠肺成纤维细胞后通过Real-time PCR筛选RNA干扰最佳靶点。 结果：经PCR鉴定和测序验证，成功构建了3个针对的TLR4基因的siRNA质粒并进行慢病毒包装，形成TLR4-RNAi慢病毒载体（Lentivirus-TLR4-siRNA）；经Real-time PCR筛选出最佳TLR4-RNAi慢病毒载体（Target2＃），其病毒滴度测定结果为8×107TU/ml，抑制效率约80%。 结论：针对TLR4基因的Lentivirus-TLR4-siRNA能有效抑制原代培养的小鼠肺成纤维细胞TLR4基因的表达，用于后续实验。 目的：明确脂多糖调控Thy-1(＋)肺成纤维细胞表型转变及异常增殖的机制。 方法：利用LPS诱导肺成纤维细胞增殖模型，首先观察LPS刺激原代培养的Thy-1(＋)肺成纤维细胞0-72小时细胞增殖及Thy-1基因表达的动态变化过程。随后深入研究LPS诱导Thy-1(＋)肺成纤维细胞表型转变的表观遗传学机制，重点探讨LPS通过TLR4的活化对肺成纤维细胞Thy-1基因启动区组蛋白乙酰化水平以及细胞Thy-1基因表达的影响，并尝试通过RNAi技术抑制肺成纤维细胞TLR4的表达从而抑制由LPS引起的Thy-1基因沉默现象。 结果：BrdU检测发现：以1μg/ml浓度LPS刺激细胞最初24小时内未见细胞明显增殖，48-72小时后细胞增殖速率显著增加。real-time PCR及Western-blot检测发现：原代培养的小鼠肺成纤维细胞Thy-1基因呈阳性表达；以LPS刺激细胞最初24小时内Thy-1基因及蛋白表达量开始下降；LPS刺激48-72小时后Thy-1基因及蛋白表达量显著下降。real-time PCR及Western-blot检测发现：以LPS刺激肺成纤维细胞72小时可引起细胞Thy-1基因及蛋白表达量显著下降，但是细胞在转染TLR4siRNA慢病毒载体抑制TLR4表达后，以上过程被抑制。Western-blot检测发现：以LPS刺激肺成纤维细胞72小时可引起细胞乙酰化组蛋白H3及H4（Ace-H3, Ace-H4）表达量显著下降，提示组蛋白乙酰化程度降低。但是细胞在转染TLR4siRNA慢病毒载体抑制TLR4表达后，以上过程被抑制。 结论：LPS可以在表观遗传学水平改变Thy-1(＋)肺成纤维细胞的表型，通过其特异性受体TLR4降低Thy-1基因启动区组蛋白的乙酰化水平而抑制Thy-1基因的表达，使肺成纤维细胞发生表型转变，从而获得了异常的增殖能力，在LPS刺激下发生异常增殖，最终引起了肺纤维化的病理过程。
[Abstract]:Pulmonary fibrosis is an important pathological stage in the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). It is characterized by abnormal proliferation of pulmonary fibroblasts, activation and secretion of collagen, and formation of diffuse fibrosis in pulmonary interstitium and alveoli. Lipopolysaccharide (LPS) can act on Toll-like receptor 4 (TLR4), a specific body of lung fibroblasts, and directly induce the activation and proliferation of lung fibroblasts, which is closely related to the development of pulmonary fibrosis. However, the results of LPS on the proliferation of fibroblasts are consistent, suggesting that different sources of formation. Fibroblast subtypes may be heterogeneous due to different phenotypes, which may be an important reason for different biological effects of cells on LPS stimulation.
Thy-1 is a glycoprotein anchored by glycophosphatidylinositol on the surface of fibroblasts. According to the expression of thymocyte differentiation antigen-1 (Thy-1) on the surface of fibroblasts, we can divide the pulmonary stem cells into two different phenotypes: Thy-1 (+) and Thy-1 (-). In idiopathic pulmonary fibrosis (IPF) patients, a large number of Thy-1 (-) pulmonary fibroblasts with negative Thy-1 expression are present in the lung tissue. Thy-1 (-) pulmonary fibroblasts have more active responsiveness to various fibrogenic factors and are closely related to the occurrence and development of pulmonary fibrosis. Gene expression is regulated by epigenetic mechanisms such as histone deacetylation, and some studies have shown that LPS can regulate the expression of many genes through epigenetic mechanisms such as histone deacetylation. Type 1, the proliferation of lung fibroblasts.
The purpose of this study is to establish a model of LPS-induced proliferation of lung fibroblasts. First, the effect of LPS on the phenotypic transformation and abnormal proliferation of primary cultured Thy-1 (+) lung fibroblasts was clarified. Then, the expression of TLR4 in lung fibroblasts was inhibited by RNAi, and the Thy-1 gene expression and cell proliferation were studied. Epigenetic regulation of phenotypic transformation and abnormal proliferation.
AIM: To investigate the effect of lipopolysaccharide (LPS) on proliferation and phenotypic transformation of pulmonary fibroblasts in the early stage of pulmonary fibrosis after acute lung injury.
METHODS: The primary cultured mouse lung fibroblasts were stimulated with LPS at the final concentration of 0.01 ug/ml, 0.1 ug/ml and 1 ug/ml, and negative control was set up. After LPS treatment for 0 h, 6 h, 24 h, 48 h and 72 h, cell counting kit-8 kit was used to detect cell proliferation and real-time PCR was used to detect the dynamic expression of Thy-1 mRNA. Condition.
Results: Thy-1 gene expression was positive in primary cultured mouse pulmonary fibroblasts. No cell proliferation was observed in the first 24 hours after LPS stimulation at different concentrations, and the proliferation rate increased significantly after 48-72 hours, accompanied by a significant decrease in Thy-1 gene expression. The difference was statistically significant (P 0.05) compared with 0.01. LPS at concentrations of 0.1 ug/ml and 0.1 ug/ml stimulated cell proliferation and down-regulation of Thy-1 gene expression.
Conclusion: LPS can induce phenotypic change and abnormal proliferation of lung fibroblasts by inhibiting Thy-1 gene expression.
Objective: to design, construct and identify TLR4-RNAi lentiviral vector (Lentivirus-TLR4-siRNA) for subsequent experiments.
METHODS: Three siRNA plasmids targeting TLR4 gene were designed and prepared and packaged with lentiviruses.
Results: Three siRNA plasmids targeting TLR4 gene were successfully constructed and packaged into Lentivirus-TLR4-siRNA by PCR identification and sequencing, and the best TLR4-RNAi lentivirus vector (Target2) was screened by Real-time PCR. The titer of TLR4-RNAi lentivirus was 8, and the inhibition efficiency was about 80%.
CONCLUSION: Lentivirus-TLR4-siRNA targeting TLR4 gene can effectively inhibit the expression of TLR4 gene in primary cultured mouse lung fibroblasts, and can be used in subsequent experiments.
Objective: to clarify the mechanism of lipopolysaccharide regulating Thy-1 (+) lung fibroblast phenotype transformation and abnormal proliferation.
METHODS: LPS-induced proliferation of lung fibroblasts was used to observe the dynamic changes of proliferation and Thy-1 gene expression of primary cultured Thy-1(+) lung fibroblasts stimulated by LPS for 0-72 hours. Activation of TLR4 inhibited histone acetylation in the Thy-1 gene promoter region and Thy-1 gene expression in lung fibroblasts, and attempted to suppress the Thy-1 gene silencing induced by LPS by RNAi.
Results: BrdU assay showed that no cell proliferation was observed within the first 24 hours after LPS stimulation at 1 ug/ml concentration, and the proliferation rate increased significantly after 48-72 hours. The expression of Thy-1 gene and protein decreased significantly after LPS stimulation for 48-72 hours. Real-time PCR and Western-blot analysis showed that the expression of Thy-1 gene and protein decreased significantly after LPS stimulation for 72 hours, but the expression of TLR4 was inhibited by transfected TLR4 siRNA lentiviral vector. Western-blot analysis showed that LPS stimulated lung fibroblasts for 72 hours resulted in a significant decrease in the expression of acetylated histones H3 and H4 (Ace-H3, Ace-H4), suggesting a decrease in histone acetylation.
CONCLUSION: LPS can alter the phenotype of Thy-1 (+) lung fibroblasts at the epigenetic level, and inhibit the expression of Thy-1 gene by decreasing the acetylation level of Thy-1 gene promoter region histone through its specific receptor TLR4, thus resulting in phenotypic changes of lung fibroblasts, resulting in abnormal proliferative capacity, which can be stimulated by LPS. Chang Zengzhi eventually caused the pathological process of pulmonary fibrosis.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R563.9
[Abstract]:Pulmonary fibrosis is an important pathological stage in the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). It is characterized by abnormal proliferation of pulmonary fibroblasts, activation and secretion of collagen, and formation of diffuse fibrosis in pulmonary interstitium and alveoli. Lipopolysaccharide (LPS) can act on Toll-like receptor 4 (TLR4), a specific body of lung fibroblasts, and directly induce the activation and proliferation of lung fibroblasts, which is closely related to the development of pulmonary fibrosis. However, the results of LPS on the proliferation of fibroblasts are consistent, suggesting that different sources of formation. Fibroblast subtypes may be heterogeneous due to different phenotypes, which may be an important reason for different biological effects of cells on LPS stimulation.
Thy-1 is a glycoprotein anchored by glycophosphatidylinositol on the surface of fibroblasts. According to the expression of thymocyte differentiation antigen-1 (Thy-1) on the surface of fibroblasts, we can divide the pulmonary stem cells into two different phenotypes: Thy-1 (+) and Thy-1 (-). In idiopathic pulmonary fibrosis (IPF) patients, a large number of Thy-1 (-) pulmonary fibroblasts with negative Thy-1 expression are present in the lung tissue. Thy-1 (-) pulmonary fibroblasts have more active responsiveness to various fibrogenic factors and are closely related to the occurrence and development of pulmonary fibrosis. Gene expression is regulated by epigenetic mechanisms such as histone deacetylation, and some studies have shown that LPS can regulate the expression of many genes through epigenetic mechanisms such as histone deacetylation. Type 1, the proliferation of lung fibroblasts.
The purpose of this study is to establish a model of LPS-induced proliferation of lung fibroblasts. First, the effect of LPS on the phenotypic transformation and abnormal proliferation of primary cultured Thy-1 (+) lung fibroblasts was clarified. Then, the expression of TLR4 in lung fibroblasts was inhibited by RNAi, and the Thy-1 gene expression and cell proliferation were studied. Epigenetic regulation of phenotypic transformation and abnormal proliferation.
AIM: To investigate the effect of lipopolysaccharide (LPS) on proliferation and phenotypic transformation of pulmonary fibroblasts in the early stage of pulmonary fibrosis after acute lung injury.
METHODS: The primary cultured mouse lung fibroblasts were stimulated with LPS at the final concentration of 0.01 ug/ml, 0.1 ug/ml and 1 ug/ml, and negative control was set up. After LPS treatment for 0 h, 6 h, 24 h, 48 h and 72 h, cell counting kit-8 kit was used to detect cell proliferation and real-time PCR was used to detect the dynamic expression of Thy-1 mRNA. Condition.
Results: Thy-1 gene expression was positive in primary cultured mouse pulmonary fibroblasts. No cell proliferation was observed in the first 24 hours after LPS stimulation at different concentrations, and the proliferation rate increased significantly after 48-72 hours, accompanied by a significant decrease in Thy-1 gene expression. The difference was statistically significant (P 0.05) compared with 0.01. LPS at concentrations of 0.1 ug/ml and 0.1 ug/ml stimulated cell proliferation and down-regulation of Thy-1 gene expression.
Conclusion: LPS can induce phenotypic change and abnormal proliferation of lung fibroblasts by inhibiting Thy-1 gene expression.
Objective: to design, construct and identify TLR4-RNAi lentiviral vector (Lentivirus-TLR4-siRNA) for subsequent experiments.
METHODS: Three siRNA plasmids targeting TLR4 gene were designed and prepared and packaged with lentiviruses.
Results: Three siRNA plasmids targeting TLR4 gene were successfully constructed and packaged into Lentivirus-TLR4-siRNA by PCR identification and sequencing, and the best TLR4-RNAi lentivirus vector (Target2) was screened by Real-time PCR. The titer of TLR4-RNAi lentivirus was 8, and the inhibition efficiency was about 80%.
CONCLUSION: Lentivirus-TLR4-siRNA targeting TLR4 gene can effectively inhibit the expression of TLR4 gene in primary cultured mouse lung fibroblasts, and can be used in subsequent experiments.
Objective: to clarify the mechanism of lipopolysaccharide regulating Thy-1 (+) lung fibroblast phenotype transformation and abnormal proliferation.
METHODS: LPS-induced proliferation of lung fibroblasts was used to observe the dynamic changes of proliferation and Thy-1 gene expression of primary cultured Thy-1(+) lung fibroblasts stimulated by LPS for 0-72 hours. Activation of TLR4 inhibited histone acetylation in the Thy-1 gene promoter region and Thy-1 gene expression in lung fibroblasts, and attempted to suppress the Thy-1 gene silencing induced by LPS by RNAi.
Results: BrdU assay showed that no cell proliferation was observed within the first 24 hours after LPS stimulation at 1 ug/ml concentration, and the proliferation rate increased significantly after 48-72 hours. The expression of Thy-1 gene and protein decreased significantly after LPS stimulation for 48-72 hours. Real-time PCR and Western-blot analysis showed that the expression of Thy-1 gene and protein decreased significantly after LPS stimulation for 72 hours, but the expression of TLR4 was inhibited by transfected TLR4 siRNA lentiviral vector. Western-blot analysis showed that LPS stimulated lung fibroblasts for 72 hours resulted in a significant decrease in the expression of acetylated histones H3 and H4 (Ace-H3, Ace-H4), suggesting a decrease in histone acetylation.
CONCLUSION: LPS can alter the phenotype of Thy-1 (+) lung fibroblasts at the epigenetic level, and inhibit the expression of Thy-1 gene by decreasing the acetylation level of Thy-1 gene promoter region histone through its specific receptor TLR4, thus resulting in phenotypic changes of lung fibroblasts, resulting in abnormal proliferative capacity, which can be stimulated by LPS. Chang Zengzhi eventually caused the pathological process of pulmonary fibrosis.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R563.9
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