目的探讨细胞外信号调节激酶1/2(ERK1/2)在转化生长因子-β1(TGF-β1)诱导的肺成纤维细胞合成Ⅰ、Ⅲ型胶原蛋白中的作用,及新型过氧化物酶Peroxiredoxin-1(Prx-1)对该作用的影响。方法体外培养肺成纤维细胞随机分为4组:对照组(0.4%血清)...目的探讨细胞外信号调节激酶1/2(ERK1/2)在转化生长因子-β1(TGF-β1)诱导的肺成纤维细胞合成Ⅰ、Ⅲ型胶原蛋白中的作用,及新型过氧化物酶Peroxiredoxin-1(Prx-1)对该作用的影响。方法体外培养肺成纤维细胞随机分为4组:对照组(0.4%血清)、TGF-β1组(5μg/L)、阴性转染组(TGF-β1+阴性对照si RNA)和Prx-1 si RNA转染组(TGF-β1+Prx-1 si RNA)。采用脂质体转染法转染si RNA,实时定量逆转录-聚合酶链反应(RT-PCR)检测转染后Prx-1 m RNA表达;Western blot检测Ⅰ和Ⅲ型胶原蛋白、ERK1/2及Prx-1表达;2,7-二氯荧光素二乙酸(DCFH-DA)检测活性氧(ROS)水平。结果 Prx-1 si RNA转染肺成纤维细胞后,Prx-1 m RNA表达明显降低,最大抑制率为92%。与对照组比较,TGF-β1组的Ⅰ和Ⅲ型胶原蛋白、ROS、磷酸化ERK1/2(p-ERK1/2)及Prx-1蛋白的表达水平均明显提高。与TGF-β1组比较,阴性转染组中的上述观察指标无明显变化,但Prx-1转染组的Ⅰ和Ⅲ型胶原蛋白、ROS、p-ERK1/2水平进一步提高,而Prx-1蛋白的表达被抑制。结论 TGF-β1能够诱导肺成纤维细胞生成ROS,并促进ERK1/2通路的激活,导致Ⅰ、Ⅲ型胶原蛋白合成增加,而Prx-1 si RNA可通过提高ROS水平进一步促进TGF-β1该作用。展开更多
BACKGROUND Recently,the exclusive use of mesenchymal stem cell(MSC)-secreted molecules,named as the secretome,have been evaluated for overcoming the limitations of cell-based therapy while maintaining its advantages.A...BACKGROUND Recently,the exclusive use of mesenchymal stem cell(MSC)-secreted molecules,named as the secretome,have been evaluated for overcoming the limitations of cell-based therapy while maintaining its advantages.AIM To improve cell-free therapy by adding disease-specificity through stimulation of MSCs using disease-causing materials.METHODS We collected the secretory materials(named as inducers)released from AML12 hepatocytes that had been pretreated with thioacetamide(TAA)and generated the TAA-induced secretome(TAA-isecretome)after stimulating adipose-derived stem cells with the inducers.The TAA-isecretome was intravenously administered to mice with TAA-induced hepatic failure and those with partial hepatectomy.RESULTS TAA-isecretome infusion showed higher therapeutic potential in terms of(1)restoring disorganized hepatic tissue to normal tissue;(2)inhibiting proinflammatory cytokines(interleukin-6 and tumor necrosis factor-α);and(3)reducing abnormally elevated liver enzymes(aspartate aminotransferase and alanine aminotransferase)compared to the naïve secretome infusion in mice with TAA-induced hepatic failure.However,the TAA-isecretome showed inferior therapeutic potential for restoring hepatic function in partially hepatectomized mice.Proteomic analysis of TAA-isecretome identified that antioxidant processes were the most predominant enriched biological networks of the proteins exclusively identified in the TAA-isecretome.In addition,peroxiredoxin-1,a potent antioxidant protein,was found to be one of representative components of TAA-isecretome and played a central role in the protection of TAA-induced hepatic injury.CONCLUSION Appropriate stimulation of adipose-derived stem cells with TAA led to the production of a secretome enriched with proteins,especially peroxiredoxin-1,with higher antioxidant activity.Our results suggest that appropriate stimulation of MSCs with pathogenic agents can lead to the production of a secretome specialized for protecting against the pathogen.This approach is expected to op展开更多
文摘目的探讨细胞外信号调节激酶1/2(ERK1/2)在转化生长因子-β1(TGF-β1)诱导的肺成纤维细胞合成Ⅰ、Ⅲ型胶原蛋白中的作用,及新型过氧化物酶Peroxiredoxin-1(Prx-1)对该作用的影响。方法体外培养肺成纤维细胞随机分为4组:对照组(0.4%血清)、TGF-β1组(5μg/L)、阴性转染组(TGF-β1+阴性对照si RNA)和Prx-1 si RNA转染组(TGF-β1+Prx-1 si RNA)。采用脂质体转染法转染si RNA,实时定量逆转录-聚合酶链反应(RT-PCR)检测转染后Prx-1 m RNA表达;Western blot检测Ⅰ和Ⅲ型胶原蛋白、ERK1/2及Prx-1表达;2,7-二氯荧光素二乙酸(DCFH-DA)检测活性氧(ROS)水平。结果 Prx-1 si RNA转染肺成纤维细胞后,Prx-1 m RNA表达明显降低,最大抑制率为92%。与对照组比较,TGF-β1组的Ⅰ和Ⅲ型胶原蛋白、ROS、磷酸化ERK1/2(p-ERK1/2)及Prx-1蛋白的表达水平均明显提高。与TGF-β1组比较,阴性转染组中的上述观察指标无明显变化,但Prx-1转染组的Ⅰ和Ⅲ型胶原蛋白、ROS、p-ERK1/2水平进一步提高,而Prx-1蛋白的表达被抑制。结论 TGF-β1能够诱导肺成纤维细胞生成ROS,并促进ERK1/2通路的激活,导致Ⅰ、Ⅲ型胶原蛋白合成增加,而Prx-1 si RNA可通过提高ROS水平进一步促进TGF-β1该作用。
基金Supported by National Research Foundation of Korea,No.NRF-2015R1C1A1A02036931
文摘BACKGROUND Recently,the exclusive use of mesenchymal stem cell(MSC)-secreted molecules,named as the secretome,have been evaluated for overcoming the limitations of cell-based therapy while maintaining its advantages.AIM To improve cell-free therapy by adding disease-specificity through stimulation of MSCs using disease-causing materials.METHODS We collected the secretory materials(named as inducers)released from AML12 hepatocytes that had been pretreated with thioacetamide(TAA)and generated the TAA-induced secretome(TAA-isecretome)after stimulating adipose-derived stem cells with the inducers.The TAA-isecretome was intravenously administered to mice with TAA-induced hepatic failure and those with partial hepatectomy.RESULTS TAA-isecretome infusion showed higher therapeutic potential in terms of(1)restoring disorganized hepatic tissue to normal tissue;(2)inhibiting proinflammatory cytokines(interleukin-6 and tumor necrosis factor-α);and(3)reducing abnormally elevated liver enzymes(aspartate aminotransferase and alanine aminotransferase)compared to the naïve secretome infusion in mice with TAA-induced hepatic failure.However,the TAA-isecretome showed inferior therapeutic potential for restoring hepatic function in partially hepatectomized mice.Proteomic analysis of TAA-isecretome identified that antioxidant processes were the most predominant enriched biological networks of the proteins exclusively identified in the TAA-isecretome.In addition,peroxiredoxin-1,a potent antioxidant protein,was found to be one of representative components of TAA-isecretome and played a central role in the protection of TAA-induced hepatic injury.CONCLUSION Appropriate stimulation of adipose-derived stem cells with TAA led to the production of a secretome enriched with proteins,especially peroxiredoxin-1,with higher antioxidant activity.Our results suggest that appropriate stimulation of MSCs with pathogenic agents can lead to the production of a secretome specialized for protecting against the pathogen.This approach is expected to op
