脂多糖誘導(dǎo)肺成纖維細(xì)胞表型轉(zhuǎn)變及異常增殖的機(jī)制研究
發(fā)布時(shí)間：2018-09-06 15:45
【摘要】：肺纖維化是急性肺損傷（acute lung injury, ALI）和急性呼吸窘迫綜合征（acuteespiratory distress syndrome, ARDS）發(fā)展的重要病理階段，以肺成纖維細(xì)胞異常增、活化并分泌膠原蛋白，形成肺間質(zhì)和肺泡內(nèi)彌散性纖維化為特點(diǎn)。革蘭氏陰性菌細(xì)胞壁的脂多糖（lipopolysaccharide, LPS）可以作用于肺成纖維細(xì)胞的特異性體—Toll樣受體4（Toll-like receptor4, TLR4），并直接引起肺成纖維細(xì)胞的活化增殖，與肺纖維化發(fā)生密切相關(guān)。然而LPS對(duì)成纖維細(xì)胞增殖作用的研究結(jié)果很一致，提示不同來(lái)源的成纖維細(xì)胞亞型可能因存在著不同的表型而產(chǎn)生異質(zhì)性heterogeneity），其可能是細(xì)胞對(duì)LPS刺激產(chǎn)生不同生物學(xué)效應(yīng)的重要原因。 Thy-1是成纖維細(xì)胞表面由糖磷脂酰肌醇錨定的糖蛋白，，根據(jù)細(xì)胞表面胸腺細(xì)分化抗原-1（thymocyte differentiation antigen1, Thy-1）表達(dá)的情況，可以將肺成維細(xì)胞分為T(mén)hy-1(＋)及Thy-1(－)兩種不同的表型。Thy-1在正常肺成纖維細(xì)胞面大量表達(dá)，但是在特發(fā)性肺纖維化（idiopathic pulmonary fibrosis, IPF）患者肺織存在著大量Thy-1呈陰性表達(dá)的Thy-1(－)肺成纖維細(xì)胞。Thy-1(－)的肺成纖細(xì)胞在各種致纖維化因子刺激下具有更活躍的反應(yīng)能力，與肺纖維化的發(fā)生和發(fā)密切相關(guān)。有研究表明Thy-1基因表達(dá)受組蛋白去乙�；缺碛^遺傳學(xué)機(jī)制的調(diào)，也有研究表明LPS可以通過(guò)組蛋白去乙酰化等表觀遺傳學(xué)機(jī)制調(diào)控多種基因的達(dá)。但是，目前尚未明確LPS是否通過(guò)表觀遺傳調(diào)控作用抑制肺成纖維細(xì)胞Thy-基因的表達(dá)而改變細(xì)胞的表型，引起肺成纖維細(xì)胞的增殖。 本課題擬利用LPS誘導(dǎo)肺成纖維細(xì)胞增殖的模型，首先明確LPS直接誘導(dǎo)原代養(yǎng)的Thy-1(＋)肺成纖維細(xì)胞發(fā)生表型轉(zhuǎn)變的和異常增殖的作用；隨后通過(guò)RNAi術(shù)抑制肺成纖維細(xì)胞TLR4的表達(dá)，研究LPS通過(guò)TLR4對(duì)肺成纖維細(xì)胞Thy-1因表達(dá)以及細(xì)胞表型轉(zhuǎn)變和異常增殖的表觀遺傳學(xué)調(diào)控作用。 目的：明確在急性肺損傷肺纖維化早期，脂多糖（LPS）誘導(dǎo)肺成纖維細(xì)胞增殖及表型轉(zhuǎn)變的作用。 方法：原代培養(yǎng)小鼠肺成纖維細(xì)胞，以終濃度為0.01μg/ml、0.1μg/ml和1μg/ml的LPS刺激細(xì)胞，并設(shè)陰性對(duì)照；LPS作用0h、6h、24h、48h和72h后以CCK-8（cell counting kit-8）細(xì)胞計(jì)數(shù)試劑盒檢測(cè)細(xì)胞增殖情況，以real-time PCR檢測(cè)各組細(xì)胞Thy-1mRNA的動(dòng)態(tài)表達(dá)情況。 結(jié)果：原代培養(yǎng)的小鼠肺成纖維細(xì)胞Thy-1基因呈陽(yáng)性表達(dá)；以不同濃度LPS刺激細(xì)胞最初24小時(shí)內(nèi)未見(jiàn)細(xì)胞明顯增殖，48-72小時(shí)后細(xì)胞增殖速率顯著增加，同時(shí)伴有Thy-1基因表達(dá)量顯著下降，經(jīng)統(tǒng)計(jì)學(xué)檢驗(yàn)差異存在顯著性（P0.05）。相對(duì)于0.01μg/ml和0.1μg/ml兩個(gè)濃度，以1μg/ml的LPS刺激細(xì)胞產(chǎn)生的細(xì)胞增殖及Thy-1基因表達(dá)下調(diào)的反應(yīng)更為顯著。 結(jié)論：LPS可通過(guò)抑制肺成纖維細(xì)胞Thy-1基因的表達(dá)而使細(xì)胞發(fā)生表型轉(zhuǎn)變并引起異常增殖。 目的：設(shè)計(jì)、構(gòu)建和鑒定TLR4-RNAi慢病毒載體（Lentivirus-TLR4-siRNA）用于后續(xù)實(shí)驗(yàn)。 方法：設(shè)計(jì)、制備3個(gè)針對(duì)TLR4基因的siRNA質(zhì)粒并進(jìn)行慢病毒包裝。轉(zhuǎn)染原代培養(yǎng)的小鼠肺成纖維細(xì)胞后通過(guò)Real-time PCR篩選RNA干擾最佳靶點(diǎn)。 結(jié)果：經(jīng)PCR鑒定和測(cè)序驗(yàn)證，成功構(gòu)建了3個(gè)針對(duì)的TLR4基因的siRNA質(zhì)粒并進(jìn)行慢病毒包裝，形成TLR4-RNAi慢病毒載體（Lentivirus-TLR4-siRNA）；經(jīng)Real-time PCR篩選出最佳TLR4-RNAi慢病毒載體（Target2＃），其病毒滴度測(cè)定結(jié)果為8×107TU/ml，抑制效率約80%。 結(jié)論：針對(duì)TLR4基因的Lentivirus-TLR4-siRNA能有效抑制原代培養(yǎng)的小鼠肺成纖維細(xì)胞TLR4基因的表達(dá)，用于后續(xù)實(shí)驗(yàn)。 目的：明確脂多糖調(diào)控Thy-1(＋)肺成纖維細(xì)胞表型轉(zhuǎn)變及異常增殖的機(jī)制。 方法：利用LPS誘導(dǎo)肺成纖維細(xì)胞增殖模型，首先觀察LPS刺激原代培養(yǎng)的Thy-1(＋)肺成纖維細(xì)胞0-72小時(shí)細(xì)胞增殖及Thy-1基因表達(dá)的動(dòng)態(tài)變化過(guò)程。隨后深入研究LPS誘導(dǎo)Thy-1(＋)肺成纖維細(xì)胞表型轉(zhuǎn)變的表觀遺傳學(xué)機(jī)制，重點(diǎn)探討LPS通過(guò)TLR4的活化對(duì)肺成纖維細(xì)胞Thy-1基因啟動(dòng)區(qū)組蛋白乙�；揭约凹�(xì)胞Thy-1基因表達(dá)的影響，并嘗試通過(guò)RNAi技術(shù)抑制肺成纖維細(xì)胞TLR4的表達(dá)從而抑制由LPS引起的Thy-1基因沉默現(xiàn)象。 結(jié)果：BrdU檢測(cè)發(fā)現(xiàn)：以1μg/ml濃度LPS刺激細(xì)胞最初24小時(shí)內(nèi)未見(jiàn)細(xì)胞明顯增殖，48-72小時(shí)后細(xì)胞增殖速率顯著增加。real-time PCR及Western-blot檢測(cè)發(fā)現(xiàn)：原代培養(yǎng)的小鼠肺成纖維細(xì)胞Thy-1基因呈陽(yáng)性表達(dá)；以LPS刺激細(xì)胞最初24小時(shí)內(nèi)Thy-1基因及蛋白表達(dá)量開(kāi)始下降；LPS刺激48-72小時(shí)后Thy-1基因及蛋白表達(dá)量顯著下降。real-time PCR及Western-blot檢測(cè)發(fā)現(xiàn)：以LPS刺激肺成纖維細(xì)胞72小時(shí)可引起細(xì)胞Thy-1基因及蛋白表達(dá)量顯著下降，但是細(xì)胞在轉(zhuǎn)染TLR4siRNA慢病毒載體抑制TLR4表達(dá)后，以上過(guò)程被抑制。Western-blot檢測(cè)發(fā)現(xiàn)：以LPS刺激肺成纖維細(xì)胞72小時(shí)可引起細(xì)胞乙�；M蛋白H3及H4（Ace-H3, Ace-H4）表達(dá)量顯著下降，提示組蛋白乙�；潭冉档汀５羌�(xì)胞在轉(zhuǎn)染TLR4siRNA慢病毒載體抑制TLR4表達(dá)后，以上過(guò)程被抑制。 結(jié)論：LPS可以在表觀遺傳學(xué)水平改變Thy-1(＋)肺成纖維細(xì)胞的表型，通過(guò)其特異性受體TLR4降低Thy-1基因啟動(dòng)區(qū)組蛋白的乙酰化水平而抑制Thy-1基因的表達(dá)，使肺成纖維細(xì)胞發(fā)生表型轉(zhuǎn)變，從而獲得了異常的增殖能力，在LPS刺激下發(fā)生異常增殖，最終引起了肺纖維化的病理過(guò)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R563.9
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