基质金属蛋白酶在肺纤维化发病机制中的作用及其调节的研究
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摘要
第一部分MT1-MMP在SiO2作用的巨噬细胞的中表达的研究
目的观察SiO2刺激下的巨噬细胞在不同时间点膜型基质金属蛋白酶Ⅰ(MT1-MMP)表达的变化。方法取小鼠原代腹腔巨噬细胞体外培养并以SiO2刺激,倒置显微镜下观察作用不同的时间点巨噬细胞形态的变化,用RT-PCR及wetern blot方法分别检测SiO2作用Oh、2h、6h、12h、18h、24h时巨噬细胞MT1-MMPmRNA及蛋白表达的变化。
结果巨噬细胞与100μg/mlSiO2共育后细胞形态无显著变化,细胞内MT1-MMPmRNA及蛋白表达呈同步改变:在SiO2刺激后2h开始升高,mRNA相对量较正常对照组增加0.92倍(p<0.05),蛋白表达升高1.52%(p<0.01),到第12h-18h时达到高峰,随后开始下降。
结论SiO2可上调AM内MT1-MMPmRNA及蛋白的表达,可能由此影响细胞外基质的代谢最终导致纤维化的形成。
第二部分EGR-1在SiO2刺激的巨噬细胞产生MT1-MMP中的作用的研究
目的探讨SiO2刺激巨噬细胞生成膜型基质金属蛋白酶Ⅰ(MT1-MMP)增多的分子机制及核转录因子早期反应生长因子1(Egr-1)介导的信号通路在其中的重要作用。
方法以Egr-1抗体及Egr-1靶向性寡核苷酸(ODN)圈套策略(decoy strategy)分别处理SiO2刺激的巨噬细胞后,用RT-PCR和western blot的方法分别检测细胞中MT1-MMPmRNA及MT1-MMP蛋白的表达。
结果与正常对照组相比较,SiO2刺激组MT1-MMP蛋白表达明显升高(p<0.01),其mRNA表达也相应升高(p<0.01),与非抗体处理组比较,经Egr-1抗体处理的巨噬细胞在SiO2作用后MT1-MMP蛋白及mRNA的表达均有所下降(p<0.05);与未转染Egr-1"decoy"ODN和转染对照ODN的巨噬细胞相比,Egr-1"decoy"ODN转染巨噬细胞后,在SiO2刺激下巨噬细胞生成MT1-MMP蛋白减少,其mRNA表达也相应下降(p<0.01)。
结论MT1-MMP在SiO2刺激的巨噬细胞中表达明显增加,它可能在矽肺的发生发展过程中具有重要的作用,Egr-1作为其重要的转录因子参与了对MT1-MMP基因表达的调控。Egr-1抗体和Egr-1"decoy"ODN的应用可能会在一定程度上抑制MT1-MMP的表达及活性从而延缓矽肺的进展。
第三部分己酮可可碱对肺纤维化大鼠MMP-2和TIMP-1的表达的影响
目的观察己酮可可碱对博来霉素致肺纤维化大鼠MMP-2和TIMP-1表达的影响,初步探讨其抗肺纤维化的作用机制。
方法SD大鼠30只,随机分为模型组、治疗组和空白组。模型组和治疗组气管内注射博来霉素诱导成肺纤维化,空白组在相同条件下给予生理盐水。第二天起治疗组大鼠腹腔给予己酮可可碱6mg/kg.d,其余的组相同条件下给予生理盐水。治疗的第7天和28天,用RT-PCR和免疫组化ABC法观察各组鼠肺组织中MMP-2和TIMP-1表达的变化。
结果己酮可可碱作用的第7天和28天肺组织中MMP-2和TIMP-1mRNA的基因转录均较模型组减少,免疫组化也显示出同样的结果(前者P<0.001、后者P<0.05)。
结论己酮可可碱对肺纤维化不同时期肺组织中MMP-2和TIMP-1的表达均有一定程度的降低作用,其可能通过调整MMP-2和TIMP-1比值使其趋于平衡,从而延缓甚至抑制纤维化的进程。
Part I Effect of silicon dioxide on the expreeion of MT1-MMP in
macrophage
Objective To investigate the expression of MT1-MMP under the stimulation of
silicon dioxide in the primary macrophages in vitro.
Methods Celiac macrophage were primary cultured, western-blot and RT-PCR
analysis were used to detect the expressions of MT1-MMP protein and the dynamic
change of MT1-MMPmRNA in the primary macrophages which were treated with
silicon dioxide within 0h、 2h、 6h、 12h、 18h、 24h.
Results In the cells which were stimulated by silicon dioxide, there were increased
expressions of MT1-MMP proteins and transcriptions of MT1-MMP during 2 to 24
hours. The expression levels peaked at the 12th hour. Both the mRNAs and the
proteins appeared to be the same tendency.
Conclusions Silicon dioxide induced the expression of MT1-MMPmRNA and
proteins, which might contribute to the mechanism of pulmonary fibrosis.
Part II EGR-1 mediate SiO2 driven transcription of membrane
type I matrix metalloproteinase in macrophage
Objective To study the up-regulation mechanism of membrane type I matrix metalloproteinase (MT1-MMP) in macrophage stimulated by silica in vitro, and the contribution of early growth response 1 (Egr-1) transcription factor in the gene expression pathway.
Methods Macrophages stimulated by silica were treated with Egr-1 antibody or Egr-1 decoy oligodeoxynucleotides(ODN). The levels of MT1-MMP proteins were determined by western blot and the expression of MT1-MMP mRNAs was detected by RT-PCR. Results Compared with control macrophages, silica stimulated group showed up-regulated gene expression of MT1-MMP via Egr-1(p<0.01). Compared with silica-stimulated macrophages untreated with antibody, the cells treated with 5μg/ml Egr-1 antibody were associated with reduced expression of MT1-MMP proteins(p<0.01) and mRNAs(p<0.05). Compared with silica-stimulated untransfected group, the Egr-1 "decoy" ODN group was associated with reduction in expression of MT1-MMP proteins and mRNAs(p<0.01).
Conclusions Gene expression of MT1-MMP which may play a critical role in silicosis was up-regulated by silica in macrophage. Egr-1 participated in the expression of MT1-MMP and positively regulated the expression. Both Egr-1 antibody and Egr-1 decoy ODN suppressed the expression of MT1-MMP through the Egr-1 pathway and may become a potential therapeutic tool in the management of silicosis in the future.
PartIII Study on the treatment mechanism of Pentoxifylline to the
pulmonary fibrosis
Objective To investigate the treatment Pentoxifylline to the mouse with pulmonary fibrosis and to study the mechanism.
Methods Thirty Spague-Dawley rats were randomly divided into three groups. The model group and the treatment group were injected with bleomycin endotracheally, while empty group with normal saline instead in the same condition. From the second day the treatment groups were injected with Pentoxifylline endoceliacly with 6mg/kg.d. while the other groups with normal saline instead. On the seventh day and the twenty-eighth day the lungs were isolated. The mRNA expression of MMP-2 and TIMP-1 were evaluated by RT-PCR quantity analysis also the immunohistochemical method was used to investigate the change of MMP-2 and TIMP-1. Results The MMP-2 and TIMP-1 gene expression of the Pentoxifylline treatment groups decreased on the 7th and 28th day and the immunohistochemical showed the same result.
Conclusions The treatment mechanism of Pentoxifylline to pulmonary fibrosis is probably that it can adjust the ratio of MMP-2 /TIMP-1 and make the ratio to a state of balance, to make the process of pulmonary fibrosis go slowly and even block it.
目的观察SiO2刺激下的巨噬细胞在不同时间点膜型基质金属蛋白酶Ⅰ(MT1-MMP)表达的变化。方法取小鼠原代腹腔巨噬细胞体外培养并以SiO2刺激,倒置显微镜下观察作用不同的时间点巨噬细胞形态的变化,用RT-PCR及wetern blot方法分别检测SiO2作用Oh、2h、6h、12h、18h、24h时巨噬细胞MT1-MMPmRNA及蛋白表达的变化。
结果巨噬细胞与100μg/mlSiO2共育后细胞形态无显著变化,细胞内MT1-MMPmRNA及蛋白表达呈同步改变:在SiO2刺激后2h开始升高,mRNA相对量较正常对照组增加0.92倍(p<0.05),蛋白表达升高1.52%(p<0.01),到第12h-18h时达到高峰,随后开始下降。
结论SiO2可上调AM内MT1-MMPmRNA及蛋白的表达,可能由此影响细胞外基质的代谢最终导致纤维化的形成。
第二部分EGR-1在SiO2刺激的巨噬细胞产生MT1-MMP中的作用的研究
目的探讨SiO2刺激巨噬细胞生成膜型基质金属蛋白酶Ⅰ(MT1-MMP)增多的分子机制及核转录因子早期反应生长因子1(Egr-1)介导的信号通路在其中的重要作用。
方法以Egr-1抗体及Egr-1靶向性寡核苷酸(ODN)圈套策略(decoy strategy)分别处理SiO2刺激的巨噬细胞后,用RT-PCR和western blot的方法分别检测细胞中MT1-MMPmRNA及MT1-MMP蛋白的表达。
结果与正常对照组相比较,SiO2刺激组MT1-MMP蛋白表达明显升高(p<0.01),其mRNA表达也相应升高(p<0.01),与非抗体处理组比较,经Egr-1抗体处理的巨噬细胞在SiO2作用后MT1-MMP蛋白及mRNA的表达均有所下降(p<0.05);与未转染Egr-1"decoy"ODN和转染对照ODN的巨噬细胞相比,Egr-1"decoy"ODN转染巨噬细胞后,在SiO2刺激下巨噬细胞生成MT1-MMP蛋白减少,其mRNA表达也相应下降(p<0.01)。
结论MT1-MMP在SiO2刺激的巨噬细胞中表达明显增加,它可能在矽肺的发生发展过程中具有重要的作用,Egr-1作为其重要的转录因子参与了对MT1-MMP基因表达的调控。Egr-1抗体和Egr-1"decoy"ODN的应用可能会在一定程度上抑制MT1-MMP的表达及活性从而延缓矽肺的进展。
第三部分己酮可可碱对肺纤维化大鼠MMP-2和TIMP-1的表达的影响
目的观察己酮可可碱对博来霉素致肺纤维化大鼠MMP-2和TIMP-1表达的影响,初步探讨其抗肺纤维化的作用机制。
方法SD大鼠30只,随机分为模型组、治疗组和空白组。模型组和治疗组气管内注射博来霉素诱导成肺纤维化,空白组在相同条件下给予生理盐水。第二天起治疗组大鼠腹腔给予己酮可可碱6mg/kg.d,其余的组相同条件下给予生理盐水。治疗的第7天和28天,用RT-PCR和免疫组化ABC法观察各组鼠肺组织中MMP-2和TIMP-1表达的变化。
结果己酮可可碱作用的第7天和28天肺组织中MMP-2和TIMP-1mRNA的基因转录均较模型组减少,免疫组化也显示出同样的结果(前者P<0.001、后者P<0.05)。
结论己酮可可碱对肺纤维化不同时期肺组织中MMP-2和TIMP-1的表达均有一定程度的降低作用,其可能通过调整MMP-2和TIMP-1比值使其趋于平衡,从而延缓甚至抑制纤维化的进程。
Part I Effect of silicon dioxide on the expreeion of MT1-MMP in
macrophage
Objective To investigate the expression of MT1-MMP under the stimulation of
silicon dioxide in the primary macrophages in vitro.
Methods Celiac macrophage were primary cultured, western-blot and RT-PCR
analysis were used to detect the expressions of MT1-MMP protein and the dynamic
change of MT1-MMPmRNA in the primary macrophages which were treated with
silicon dioxide within 0h、 2h、 6h、 12h、 18h、 24h.
Results In the cells which were stimulated by silicon dioxide, there were increased
expressions of MT1-MMP proteins and transcriptions of MT1-MMP during 2 to 24
hours. The expression levels peaked at the 12th hour. Both the mRNAs and the
proteins appeared to be the same tendency.
Conclusions Silicon dioxide induced the expression of MT1-MMPmRNA and
proteins, which might contribute to the mechanism of pulmonary fibrosis.
Part II EGR-1 mediate SiO2 driven transcription of membrane
type I matrix metalloproteinase in macrophage
Objective To study the up-regulation mechanism of membrane type I matrix metalloproteinase (MT1-MMP) in macrophage stimulated by silica in vitro, and the contribution of early growth response 1 (Egr-1) transcription factor in the gene expression pathway.
Methods Macrophages stimulated by silica were treated with Egr-1 antibody or Egr-1 decoy oligodeoxynucleotides(ODN). The levels of MT1-MMP proteins were determined by western blot and the expression of MT1-MMP mRNAs was detected by RT-PCR. Results Compared with control macrophages, silica stimulated group showed up-regulated gene expression of MT1-MMP via Egr-1(p<0.01). Compared with silica-stimulated macrophages untreated with antibody, the cells treated with 5μg/ml Egr-1 antibody were associated with reduced expression of MT1-MMP proteins(p<0.01) and mRNAs(p<0.05). Compared with silica-stimulated untransfected group, the Egr-1 "decoy" ODN group was associated with reduction in expression of MT1-MMP proteins and mRNAs(p<0.01).
Conclusions Gene expression of MT1-MMP which may play a critical role in silicosis was up-regulated by silica in macrophage. Egr-1 participated in the expression of MT1-MMP and positively regulated the expression. Both Egr-1 antibody and Egr-1 decoy ODN suppressed the expression of MT1-MMP through the Egr-1 pathway and may become a potential therapeutic tool in the management of silicosis in the future.
PartIII Study on the treatment mechanism of Pentoxifylline to the
pulmonary fibrosis
Objective To investigate the treatment Pentoxifylline to the mouse with pulmonary fibrosis and to study the mechanism.
Methods Thirty Spague-Dawley rats were randomly divided into three groups. The model group and the treatment group were injected with bleomycin endotracheally, while empty group with normal saline instead in the same condition. From the second day the treatment groups were injected with Pentoxifylline endoceliacly with 6mg/kg.d. while the other groups with normal saline instead. On the seventh day and the twenty-eighth day the lungs were isolated. The mRNA expression of MMP-2 and TIMP-1 were evaluated by RT-PCR quantity analysis also the immunohistochemical method was used to investigate the change of MMP-2 and TIMP-1. Results The MMP-2 and TIMP-1 gene expression of the Pentoxifylline treatment groups decreased on the 7th and 28th day and the immunohistochemical showed the same result.
Conclusions The treatment mechanism of Pentoxifylline to pulmonary fibrosis is probably that it can adjust the ratio of MMP-2 /TIMP-1 and make the ratio to a state of balance, to make the process of pulmonary fibrosis go slowly and even block it.
引文
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