ABSTRACT
Objective: To investigate the effects of TFPI-2 (tissue factor pathway inhibitor 2) on the growth and angiogenesis of subcutaneous tumor xenografts in nude mice established from human liver cancer cells. Methods: The Hep3B cells stably expressing TFPI-2 (Hep3B-TFPI-2 group) and the Hep3B cells transfected with empty vector PCDNA3.1 (Hep3B-V group) or without transfection (Hep3B-P group) were subcutaneously transplanted into nude mice respectively to generate subcutaneous tumor xenografts. The volume of tumor xenograft was measured every three days, and the growth curve of tumor xenograft was drawn when the subcutaneous tumor xenograft was visible. The nude mice were killed three weeks after transplant, the volume of tumor xenograft was measured, and the total RNAs and proteins in tumor xenografts were extracted. The mRNA and protein expressions of TFPI-2 and VEGF (vascular endothelial growth factor) in tumor xenografts were analyzed by RFQ-PCR (real-time fluorescence quantitative PCR) and Western blotting, respectively. The expression of TFPI-2 protein and the MVD (microvessel density) in tumor xenografts were observed by immunohistochemistry. Results: The eventual tumor volume of tumor xenografts in Hep3B-TFPI-2 group was apparently smaller than those in Hep3B-V group and Hep3B-P group (both P < 0.05). The expression of mRNA and abundance of protein of TFPI-2 in Hep3B-TFPI-2 group were significantly higher than those in the other two groups (P < 0.05); while the expression of mRNA and abundance of protein of VEGF in Hep3B-TFPI-2 group were apparently lower than those in the other two groups. Compared with Hep3B-V group and Hep3B-P group, the inhibitory rates of VEGF protein expression in Hep3B-TFPI-2 group were 19.8% and 23.5%, respectively (P < 0.05). The MVD in Hep3B-TFPI-2 group was apparently lower than those in the other two groups (P < 0.05). Conclusion: TFPI-2 can significantly inhibit the growth and angiogenesis of subcutaneous tumor xenografts in nude mice established from hepatocarcinoma Hep3B cells. Copyright © 2013 by TUMOR.
ABSTRACT
Objective: To establish a molecule binding model of mouse PD-1/PD-L1 protein in vitro, so as to lay a foundation for studying the biological activities of the recombinant protein and to establish a high throughput drug screening model. Methods: Prokaryotic expression plasmid pET28 a (+) /mPDL-1 and pGEX-4T-1/mPD-1 were transformed to E. coli BL21 (DE3), which was then induced with IPTG. The expression products were purified by fast affinity chromatography with FPLC Protein Purification Instrument or gel separation. ELISA and GST pull-down were used to detect the interaction between mPD-1 and mPD-L1. In addition, Alamar blue was used to test the role of protein mixtures in the mixed lymphocyte proliferation. Results: SDS-PAGE and Western blotting analysis showed that the recombinant proteins were successfully expressed. Then purified mPD-1 and mPD-L1 protein was obtained by affinity chromatography, Gel seperation and refolding, etc. ELISA and GST pull-down showed that mPD-1 and mPD-L1 protein had a specific binding activity in vitro. The mPD-1/ mPD-L1 protein had a significantly decreased influence on the proliferation of lymphocytes(P<0. 05). Conclusion: Mouse PD-1/PD-L1 recombinant protein model has been successfully established using purified mPD-1 and mPD-L1 protein expressed prokaryotically, which lays a foundation for high-throughput drug screening.