Construction Of Recombinant Secretory Type STRAIL Adenovirus And Its Induction Of Apoptosis In Brain Glioma U251 Cells

Objective To construct a secreting recombinant adenovirus vector with the extracellular region of the human TRAIL gene (sTRAIL) and to investigate the antitumor activities of Ad-sTRAIL on malignant glioma cell line U251.Methods The TRAIL gene fragment encoding114-281amino acids from human liver cDNA library was amplified by PCR. The pAdTrack-CMV-IL-2-TRAIL was constructed by inserting TRAIL into the adenoviral shuttle vector pAdTrack-CMV-IL-2with secreting peptides IL-2. Pmel-digested pAdTrack-CMV-IL-2-TRAIL was transformed into E.coli BJ5183harboring pAdEasy-1by electrotransformation. Recombinant plasmid pAd-sTRAIL was selected. Packing in the HEK293cells, the replication-deficient adenovirus Ad-sTRAIL particles was purified by cesium chloride density gradient centrifugation and detected the titer. Through RT-PCR and wetern blotting, the expression of target gene was detected. Cell line U251was infected by Ad-sTRAIL. The expression of sTRAIL was detected by immunofluorescence. Cell viability was determined by MTT. Tumor cells apoptosis was demonstrated by morphologic changes, flow cytometry. Cells of sub-phases and cell cycle were detected by flow cytometry with PI staining.Results The gene fragment of104bp was amplified by RT-PCR. The expression of target gene in U251cells infected by Ad-sTRAIL was shown by western blotting and immunofluorescence. The higher rate of apoptosis was demonstrated by MTT method, flow cytometry. The rate of Ad-sTRAIL induced U251cell apoptosis increased with the dosage and time increase.The apoptosis of G0/G1, S phase cells were more significant than the control groups.Conclusion The secreting recombinant adenoviral vector with the extracellular region of human TRAIL gene has been constructed successfully. The growth and proliferation of U251cell line was inhibited after the infection of Ad-sTRAIL. It is concluded that apoptosis coiled the cell cycle progression and present a cell cycle specificity, implying that apoptosis induced by sTRAIL is a cell cycle event.