Peroxiredoxin Ⅰ(PrxⅠ) is involved in many important cellular progresses such as cell proliferation and differentiation. To further elucidate its roles in ionizing radiation, eukaryotic expression vector containing s...Peroxiredoxin Ⅰ(PrxⅠ) is involved in many important cellular progresses such as cell proliferation and differentiation. To further elucidate its roles in ionizing radiation, eukaryotic expression vector containing siRNA targeting mRNA of peroxiredoxin 1 gene was constructed and transected into NIH3T3 cell line following sequencing.Western blot analysis showed that this siRNA significantly inhibited PrxⅠ expression by 65% at protein level; cell proliferation experiment demonstrated that the double proliferation time of NIH 3T3PrxⅠ cells was postponed for 1.8 hours as compared with control group. MTT results showed that 12 and 48 hours after irradiation the survival rate of NIH 3T3PrxⅠ was dramatically decreased when comparing with sham-irradiated group.These results suggested that peroxiredoxin 1 has radiation-cytoprotection role.展开更多
目的观察过氧化物酶Ⅰ(PrxⅠ)在正常和骨关节炎(OA)膝关节软骨组织中的表达和分布特点,探讨PrxⅠ与活性氧歧化物和凋亡等在OA软骨表层聚集现象之间的关联。方法分别从正常膝关节提取正常软骨(NC,n=21)和接受膝关节表面置换术的OA患者提...目的观察过氧化物酶Ⅰ(PrxⅠ)在正常和骨关节炎(OA)膝关节软骨组织中的表达和分布特点,探讨PrxⅠ与活性氧歧化物和凋亡等在OA软骨表层聚集现象之间的关联。方法分别从正常膝关节提取正常软骨(NC,n=21)和接受膝关节表面置换术的OA患者提取OA软骨(OA,n=21),应用Westernblot技术检测PrxⅠ在正常软骨细胞和OA软骨细胞中表达水平的总体差异,并用免疫组织化学技术观察PrxⅠ蛋白在正常与OA软骨表/中/深各层组织表达和分布的特点。结果 Western blot证实PrxⅠ在OA软骨组织中的表达水平较正常软骨组织显著提高2.89倍(t=18.34,P<0.01)。免疫组织化学显示PrxⅠ在正常软骨组织的表层、中层和深层呈现较均一的表达,但是,在OA软骨组织中,PrxⅠ的表达水平存在显著层间差异。PrxⅠ在软骨组织深层表达水平显著升高,但在浅层细胞中,PrxI的表达水平反而显著减低甚至缺如。结论虽然总体表达水平升高,但PrxⅠ在OA软骨组织表层的表达缺如,可能与OA软骨组织表层活性氧歧化物蓄积和细胞凋亡聚集现象相关。展开更多
AIM: To clone and express mouse peroxiredoxin Ⅰ in IEC-6 cells.METHODS: Total RNAs were isolated from cultured IEC-6 cells, and the coding region of peroxiredoxin I was amplified by RT-PCR. After it was cloned into T...AIM: To clone and express mouse peroxiredoxin Ⅰ in IEC-6 cells.METHODS: Total RNAs were isolated from cultured IEC-6 cells, and the coding region of peroxiredoxin I was amplified by RT-PCR. After it was cloned into T-vector and sequenced,pSG5 was used to transiently express peroxiredoxin I in IEC-6 by liposome-mediated transfection, and the expression of peroxiredoxin I was evaluated by RT-PCRand Western blot. RESULTS: A DNA fragment about 750 bp was amplified from total RNAs of IEC-6 cells using specific primers of peroxiredoxin Ⅰ. The sequencing confirmed the coding region was successfully cloned into T-vector, which was completely coincident with the sequence in GeneBank. After the EcoRI-BamHI fragment of T-vector containing peroxiredoxin I was inserted into pSG5, the recombinant plasmid was transferred to IEC-6 cells. RT-PCR assay showed that a DNA fragment of 930 bp could be amplified,which indicated the transcription of pSG5-Prx. Western blot confirmed the expression of peroxiredoxin Ⅰ in IEC-6 cells.CONCLUSION: Mouse peroxiredoxin I can be successfully expressed in IEC-6 cells.展开更多
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
文摘Peroxiredoxin Ⅰ(PrxⅠ) is involved in many important cellular progresses such as cell proliferation and differentiation. To further elucidate its roles in ionizing radiation, eukaryotic expression vector containing siRNA targeting mRNA of peroxiredoxin 1 gene was constructed and transected into NIH3T3 cell line following sequencing.Western blot analysis showed that this siRNA significantly inhibited PrxⅠ expression by 65% at protein level; cell proliferation experiment demonstrated that the double proliferation time of NIH 3T3PrxⅠ cells was postponed for 1.8 hours as compared with control group. MTT results showed that 12 and 48 hours after irradiation the survival rate of NIH 3T3PrxⅠ was dramatically decreased when comparing with sham-irradiated group.These results suggested that peroxiredoxin 1 has radiation-cytoprotection role.
文摘目的观察过氧化物酶Ⅰ(PrxⅠ)在正常和骨关节炎(OA)膝关节软骨组织中的表达和分布特点,探讨PrxⅠ与活性氧歧化物和凋亡等在OA软骨表层聚集现象之间的关联。方法分别从正常膝关节提取正常软骨(NC,n=21)和接受膝关节表面置换术的OA患者提取OA软骨(OA,n=21),应用Westernblot技术检测PrxⅠ在正常软骨细胞和OA软骨细胞中表达水平的总体差异,并用免疫组织化学技术观察PrxⅠ蛋白在正常与OA软骨表/中/深各层组织表达和分布的特点。结果 Western blot证实PrxⅠ在OA软骨组织中的表达水平较正常软骨组织显著提高2.89倍(t=18.34,P<0.01)。免疫组织化学显示PrxⅠ在正常软骨组织的表层、中层和深层呈现较均一的表达,但是,在OA软骨组织中,PrxⅠ的表达水平存在显著层间差异。PrxⅠ在软骨组织深层表达水平显著升高,但在浅层细胞中,PrxI的表达水平反而显著减低甚至缺如。结论虽然总体表达水平升高,但PrxⅠ在OA软骨组织表层的表达缺如,可能与OA软骨组织表层活性氧歧化物蓄积和细胞凋亡聚集现象相关。
基金Supported by the National Natural Science Foundation of China,No.30230360
文摘AIM: To clone and express mouse peroxiredoxin Ⅰ in IEC-6 cells.METHODS: Total RNAs were isolated from cultured IEC-6 cells, and the coding region of peroxiredoxin I was amplified by RT-PCR. After it was cloned into T-vector and sequenced,pSG5 was used to transiently express peroxiredoxin I in IEC-6 by liposome-mediated transfection, and the expression of peroxiredoxin I was evaluated by RT-PCRand Western blot. RESULTS: A DNA fragment about 750 bp was amplified from total RNAs of IEC-6 cells using specific primers of peroxiredoxin Ⅰ. The sequencing confirmed the coding region was successfully cloned into T-vector, which was completely coincident with the sequence in GeneBank. After the EcoRI-BamHI fragment of T-vector containing peroxiredoxin I was inserted into pSG5, the recombinant plasmid was transferred to IEC-6 cells. RT-PCR assay showed that a DNA fragment of 930 bp could be amplified,which indicated the transcription of pSG5-Prx. Western blot confirmed the expression of peroxiredoxin Ⅰ in IEC-6 cells.CONCLUSION: Mouse peroxiredoxin I can be successfully expressed in IEC-6 cells.
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。
