An expected 276bp fragment of the gene precursor encoding the signal peptide and mature protein of human β chemokine RANTES was amplified by reverse transcription\|polymerase chain reaction (RT\|PCR) from RNA of PHA\...An expected 276bp fragment of the gene precursor encoding the signal peptide and mature protein of human β chemokine RANTES was amplified by reverse transcription\|polymerase chain reaction (RT\|PCR) from RNA of PHA\|activated human peripheral blood lymphocytes.This putative interested gene was inserted directly into a T\|vector and the ligation was confirmed by restriction enzyme digestion.The sequence data of the cloned fragment showed that it was almost identical with published sequences of RANTES gene,except for only one nucleotide substitution within the signal peptide region.The \%in vitro\% expressed recombinant RANTES protein was detected by the chemiluminescence enzyme\|linked immune Dot blotting assay after combining the recombinant plasmid with the \%in vitro\% SP6/T7 transcription and translation system.The successful cloning and expression of RANTES gene should shed light on future′s gene therapy of AIDS.展开更多
目的:探讨高糖环境中人近端肾小管上皮细胞(HK-2)RANTES(regulated upon activation normal Tcell expressed and secreted)和转化生长因子β1(TGF-β1)表达变化及辛伐他汀(Sim)的干预作用。方法:原代培养HK-2分为正常糖对照组(NG)、甘...目的:探讨高糖环境中人近端肾小管上皮细胞(HK-2)RANTES(regulated upon activation normal Tcell expressed and secreted)和转化生长因子β1(TGF-β1)表达变化及辛伐他汀(Sim)的干预作用。方法:原代培养HK-2分为正常糖对照组(NG)、甘露醇组(MG)、高糖组(HG)、HG+Sim2μmol/L干预组(HG+Sim2)、HG+Sim4μmol/L干预组(HG+Sim4),RT-PCR法测定细胞RANTES和TGF-β1mRNA表达,ELISA法检测细胞上清液中RANTES、TGF-β1蛋白含量。结果:RANTES、TGF-β1的mRNA和蛋白在NG及MG仅有少量表达,而在HG则明显升高(P<0.05);加入Sim后,上述表达均较前下降(P<0.05),具有浓度依赖性。各组HK-2表达的RANTES和TGF-β1无论是mRNA还是蛋白之间均存在显著正相关(P<0.01)。结论:高糖促进HK-2RANTES、TGF-β1的表达,而Sim明显抑制高糖环境中上述因子的过度表达,发挥肾脏保护作用。展开更多
文摘An expected 276bp fragment of the gene precursor encoding the signal peptide and mature protein of human β chemokine RANTES was amplified by reverse transcription\|polymerase chain reaction (RT\|PCR) from RNA of PHA\|activated human peripheral blood lymphocytes.This putative interested gene was inserted directly into a T\|vector and the ligation was confirmed by restriction enzyme digestion.The sequence data of the cloned fragment showed that it was almost identical with published sequences of RANTES gene,except for only one nucleotide substitution within the signal peptide region.The \%in vitro\% expressed recombinant RANTES protein was detected by the chemiluminescence enzyme\|linked immune Dot blotting assay after combining the recombinant plasmid with the \%in vitro\% SP6/T7 transcription and translation system.The successful cloning and expression of RANTES gene should shed light on future′s gene therapy of AIDS.
文摘目的:探讨高糖环境中人近端肾小管上皮细胞(HK-2)RANTES(regulated upon activation normal Tcell expressed and secreted)和转化生长因子β1(TGF-β1)表达变化及辛伐他汀(Sim)的干预作用。方法:原代培养HK-2分为正常糖对照组(NG)、甘露醇组(MG)、高糖组(HG)、HG+Sim2μmol/L干预组(HG+Sim2)、HG+Sim4μmol/L干预组(HG+Sim4),RT-PCR法测定细胞RANTES和TGF-β1mRNA表达,ELISA法检测细胞上清液中RANTES、TGF-β1蛋白含量。结果:RANTES、TGF-β1的mRNA和蛋白在NG及MG仅有少量表达,而在HG则明显升高(P<0.05);加入Sim后,上述表达均较前下降(P<0.05),具有浓度依赖性。各组HK-2表达的RANTES和TGF-β1无论是mRNA还是蛋白之间均存在显著正相关(P<0.01)。结论:高糖促进HK-2RANTES、TGF-β1的表达,而Sim明显抑制高糖环境中上述因子的过度表达,发挥肾脏保护作用。
