中国生物工程杂志

Objective To construct recombinant retrovirus expressing human bone morphogenetic protein-2 gene BMP7 and to discuss its apoptosis-inducing activities and the mechanism in liver cancer cell line HepG2. Methods BMP7 gene was amplified and reconstructed with retroviral plasmid pLP-LNCX by loxP homologous recombination, and then the plasmid pLP-LNCX-BMP7 (pLLBMP7) was transferred into packing cell line PT67 and the supernatant was collected to assay viral titer. MTT assay was adopted to observe HepG2 cells amplification. 48h after pLLBMP7 infection agarose electrophoresis and flow cytometry were used to verify apoptosis of tumor cells, and then the expression of BMP7, caspase-3 and bcl-2proteins were detected by Western blotting. Results Recombinant retrovirus pLLBMP7 was justified and transformed into PT67 package cell with supernatant viral titer amounted to 5×109 pfu/ml. In MTT assay retrovirus group had no evident difference from controls in cellular inhibition 72h later (35.1% vs. 5.3%，68.5% vs.18.3%, p<0.05). 48h after transfection, agarose electrophoresis of genomic DNA showed typical ladder-like pattern and flow cytometry analysis showed obvious apoptosis peaks with the highest percentage rate of apoptotic cells present and cellular caspase-3 expression could be seen in pLLBMP7 group without any change of bcl-2. Conclusion BMP7 retrovirus vector was reconstructed and could express BMP7 protein correctly in vitro and it could induce apoptosis in HepG2 cell line in vivo by activating caspase-3 expression.