Objective To construct and identify the Gpx1-Klk1 vector which contains kidney-specific promoter (Ksp-cadherin). Methods Through PCR amplification, the human Gpx1, Klk1, and Ksp-cadherin cDNA were obtained by taking G...Objective To construct and identify the Gpx1-Klk1 vector which contains kidney-specific promoter (Ksp-cadherin). Methods Through PCR amplification, the human Gpx1, Klk1, and Ksp-cadherin cDNA were obtained by taking Gpx1 cDNA, Klk1 cDNA, and Ksp-cadherin BAC as templates. After being testified, the PCR products were inserted into the expressive vector pIRES-EGFP step-by-step to produce a recombinant vector Ksp-cadherin-Gpx1-Klk1. This vector was examined by restriction enzyme digestion and sequence analysis. Results The recombinant expressive vector Ksp-cadherin-Gpx1-Klk1 was successfully constructed. Conclusion The construction of the recombinant vector Ksp-cadherin-Gpx1-Klk1 laid foundations for investigations in establishing transgenic animal models, the over-expression of Gpx1 and Klk1 in mammal kidney, and gene therapy for ischemia-reperfusion injury during kidney transplantation.展开更多
目的:探讨谷胱甘肽过氧化物酶1(glutathione peroxidase 1,GPX1)在结直肠癌(colorectal cancer,CRC)组织中的表达水平及其对CRC细胞株HT29和LOVO细胞增殖、迁移及侵袭能力的影响。方法:收集2015年6月至2016年3月间海口市人民医院滨江分...目的:探讨谷胱甘肽过氧化物酶1(glutathione peroxidase 1,GPX1)在结直肠癌(colorectal cancer,CRC)组织中的表达水平及其对CRC细胞株HT29和LOVO细胞增殖、迁移及侵袭能力的影响。方法:收集2015年6月至2016年3月间海口市人民医院滨江分院普外科手术切除的60例临床CRC组织及癌旁组织,应用实时荧光定量PCR(q PCR)技术检测CRC组织及癌旁组织中GPX1 m RNA水平。在高表达GPX1 m RNA的HT29细胞株用慢病毒携带sh RNA感染的方法构建敲除GPX1的细胞株,瞬时转染法在低表达GPX1 m RNA的LOVO细胞系构建过表达GPX1的细胞株,并在GPX1 m RNA及蛋白水平进行验证。通过MTS法检测CRC细胞的增殖能力的变化,用划痕实验、Transwell侵袭实验分别检测CRC细胞迁移侵袭能力的变化,同时用Western blotting检测E-钙黏蛋白和波形蛋白表达的变化。结果:CRC组织中GPX1 m RNA表达水平显著低于癌旁组织(0.051±0.024 vs 0.142±0.051,P<0.01)。敲除GPX1后,HT29细胞的增殖能力显著增强(12.901±2.790 vs 6.617±2.462,P<0.01)、侵袭能力显著增强[(384.7±37.9)vs(209.2±31.2)个,P<0.01]、迁移能力显著增强[(0.139±0.025)vs(0.251±0.038)mm,P<0.01]、E-钙黏蛋白表达下调(P<0.01)、波形蛋白表达上调(P<0.05)。过表达GPX1的LOVO细胞的增殖能力显著降低(P<0.01)、迁移和侵袭能力下降(均P<0.05)、E-钙黏蛋白上调(P<0.01)、波形蛋白表达下调(P<0.01)。结论:GPX1 m RNA在人CRC组织中低表达,GPX1负向调控CRC细胞的增殖、迁移和侵袭,其在CRC中可能发挥抑癌作用。展开更多
基金supported by the National Natural Science Foundation of China (No.30471640)
文摘Objective To construct and identify the Gpx1-Klk1 vector which contains kidney-specific promoter (Ksp-cadherin). Methods Through PCR amplification, the human Gpx1, Klk1, and Ksp-cadherin cDNA were obtained by taking Gpx1 cDNA, Klk1 cDNA, and Ksp-cadherin BAC as templates. After being testified, the PCR products were inserted into the expressive vector pIRES-EGFP step-by-step to produce a recombinant vector Ksp-cadherin-Gpx1-Klk1. This vector was examined by restriction enzyme digestion and sequence analysis. Results The recombinant expressive vector Ksp-cadherin-Gpx1-Klk1 was successfully constructed. Conclusion The construction of the recombinant vector Ksp-cadherin-Gpx1-Klk1 laid foundations for investigations in establishing transgenic animal models, the over-expression of Gpx1 and Klk1 in mammal kidney, and gene therapy for ischemia-reperfusion injury during kidney transplantation.
文摘目的:探讨谷胱甘肽过氧化物酶1(glutathione peroxidase 1,GPX1)在结直肠癌(colorectal cancer,CRC)组织中的表达水平及其对CRC细胞株HT29和LOVO细胞增殖、迁移及侵袭能力的影响。方法:收集2015年6月至2016年3月间海口市人民医院滨江分院普外科手术切除的60例临床CRC组织及癌旁组织,应用实时荧光定量PCR(q PCR)技术检测CRC组织及癌旁组织中GPX1 m RNA水平。在高表达GPX1 m RNA的HT29细胞株用慢病毒携带sh RNA感染的方法构建敲除GPX1的细胞株,瞬时转染法在低表达GPX1 m RNA的LOVO细胞系构建过表达GPX1的细胞株,并在GPX1 m RNA及蛋白水平进行验证。通过MTS法检测CRC细胞的增殖能力的变化,用划痕实验、Transwell侵袭实验分别检测CRC细胞迁移侵袭能力的变化,同时用Western blotting检测E-钙黏蛋白和波形蛋白表达的变化。结果:CRC组织中GPX1 m RNA表达水平显著低于癌旁组织(0.051±0.024 vs 0.142±0.051,P<0.01)。敲除GPX1后,HT29细胞的增殖能力显著增强(12.901±2.790 vs 6.617±2.462,P<0.01)、侵袭能力显著增强[(384.7±37.9)vs(209.2±31.2)个,P<0.01]、迁移能力显著增强[(0.139±0.025)vs(0.251±0.038)mm,P<0.01]、E-钙黏蛋白表达下调(P<0.01)、波形蛋白表达上调(P<0.05)。过表达GPX1的LOVO细胞的增殖能力显著降低(P<0.01)、迁移和侵袭能力下降(均P<0.05)、E-钙黏蛋白上调(P<0.01)、波形蛋白表达下调(P<0.01)。结论:GPX1 m RNA在人CRC组织中低表达,GPX1负向调控CRC细胞的增殖、迁移和侵袭,其在CRC中可能发挥抑癌作用。
