ABSTRACT
Objective To investigate the suppression of mrp1 and MRP1 induced by small interfering RNA and the restoration of sensitivity to chemotherapeutic drugs in the multidrug-resistant hepatocellular carcinoma cell lines HepG2/mrp1. Methods mrp1-targeted small interfering RNA duplexes were designed and composed and introduced into multidrug-resistant hepatocellular carcinoma cell lines HepG2/mrp1. The suppression of mrp1 mRNA and its gene product MRP1 was examined by RT-PCR and flow cytometry (FCM), respectively. MTT assay was performed to measure the reverse effect of small interfering RNA based on the results of ICs0. Results The overexpression of mrp1 mRNA and MRP1 was effectively suppressed by small interfering RNAs. The level of mrp1 mRNA in the transfected HepG2/mrp1 cells was reduced to (86.36±2.76)% and MRP1 to (89.38±3.76)%compared with those of the controls. The resistance to ADR was reversed five-fold, which indicated the restoration of sensitivity to drugs. Conclusion Small interfering RNA can inhibit mrp1 expression effectively and reverse the multidrug resistance mediated by MRP1.
ABSTRACT
The fragment of MDR1 gene obtained from the plasmid pHaMDR1-1 carrying the whole human MDR1 cDNA, was cloned reversely into the shuttle plasmid pAdTrack-CMV. With the resultant plasmid and the backbone plasmid pAdEasy-1, the homologous recombination took place in the Escherichia coli BJ5183 and the recombinant adenoviral plasmid was generated. The adenoviruses were packaged in the 293 cells. The recombinant adenovirus MDR1 vector would introduce the antisense MDR1 gene into the human multidrug resistance hepatocellular cell line effectively, which would provide an experimental basis for studies on the multidrug resistance in human hepatocellular carcinoma.
Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Genetics , Adenoviridae , Genetics , Metabolism , Carcinoma, Hepatocellular , Drug Therapy , Genetics , Cell Line, Tumor , Drug Resistance, Neoplasm , Genetics , Escherichia coli , Genetics , Gene Transfer Techniques , Genes, MDR , Genetics , Genetic Vectors , Liver Neoplasms , Drug Therapy , Genetics , Multidrug Resistance-Associated Proteins , Genetics , Oligonucleotides, Antisense , Pharmacology , Plasmids , Recombinant Proteins , Genetics , Recombination, GeneticABSTRACT
Objective To construct recombinant adenovirus vector containing mouse CD40Ig fusion gene for the study of induction of donor-specific tolerance. Methods CD40Ig fusion gene was constructed by PCR overlapping technique, and was cloned into the shuttle plasmid pAdTtrack-CMV. The linearized shuttle plasmid was co-transfected into the E.coli strain BJ5183 with bone plasmid pAdeasy1, then the recombinant adenovirus plasmid was generated. The adenovirus was packaged and amplified in Cells 293. Results The recombinant virus AdCD40Ig was successfully constructed and its titer was 5?109 efu/ml. Conclusion AdCD40Ig can be used as an agent in experiment to induce donor-specific tolerance.