RESUMO
OBJECTIVE: To investigate the apoptosis-inducing effect of caspase-3 on human nasopharyngeal carcinoma cells (CNE2). METHODS: Recombinant caspase-3 was subcloned into the eukaryotic expression vector PEGFP-C1 containing the reporter gene using DNA recombinant technique. CNE2 cells were transfected with the recombinant caspase-3 gene via lipofectamine 2000 and the expression of caspase-3 mRNA was detected by reverse transcription-polymerase chain reaction. The cell morphological changes were observed under fluorescence microscope and electron microscope and the cell survival rate after the transfection was assessed by MTT assay. RESULTS: Transfection with the recombinant caspase-3 gene induced significant apoptosis in CNE2 cells, which exhibited obvious morphological changes typical of apoptotic cells. CONCLUSION: The recombinant caspase-3 gene can inhibit the growth and effectively induce apoptosis of CNE2 cells.
Assuntos
Apoptose/genética , Caspase 3/biossíntese , Neoplasias Nasofaríngeas/genética , Transfecção , Caspase 3/genética , Vetores Genéticos , Humanos , Neoplasias Nasofaríngeas/patologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Células Tumorais CultivadasRESUMO
OBJECTIVE: To construct a recombinant adenovirus vector carrying antisense heat shock protein 70 (HSP70) cDNA and observe its effect on inhibiting the growth of laryngeal carcinoma Hep-2 cells. METHODS: The HSP70 gene fragment encoding the 5' region was cloned reversely into the shuttle plasmid PAdTrack-CMV, and the resultant plasmid was recombined with the backbone plasmid PadEasy-1 in the E.coli Bj5183 cells to generate the recombinant adenovirus vector. The adenovirus were then packaged and amplified in 293 cells, and the viral titer was determined using GFP. RESULTS: The recombinant adenovirus vector carrying antisense HSP70 cDNA was constructed successfully with a viral titer of 8 x 10(9). HSP70 expression of Hep-2 cells was obviously blocked by antisense HSP70 RNA, and Western blotting and immuohistochemistry demonstrated that cells transfected with antisense HSP70 did not express or express HSP70 at low levels. Flow cytometry presented apoptotic peak in the antisense HSP70-transfected cells, but not in the control cells. CONCLUSION: The recombinant adenovirus vector containing antisense HSP70 cDNA can effectively deliver antisense HSP70 gene into Hep-2 cells, suggesting the great potential of this gene therapy strategy in management of human laryngeal carcinoma.
