[Secretory expression vector V-pLNCX-s-hri inhibits the growth of mouse B16 melanoma].
OBJECTIVE
Human ribonuclease inhibitor (hRI) extracted and purified from human placenta has been shown to remarkably inhibit some solid tumors in mice. This study was to construct V-pLNCX-s-hri, a secretory expression vector, and explore its inhibition effects on the growth of mouse B16 melanoma cells.


METHODS
The hRI gene sequence conjugated with the synthesized signal peptide of mouse IgG was cloned into the retroviral vector V-pLNCX to construct V-pLNCX-s-hri. The PA317 cells were used for viral package and NIH3T3 cells were employed to determine the viral titer. The expression of hRI gene was detected by RT-PCR and Western blot. The content of RI was determined by enzyme-linked immunoabsorption assay (ELISA). The model of B16 melanoma-carrying mouse was established and received different treatments. The tumor weight and microvessle density (MVD) were assessed. Normal saline (NS), V-pLNCX, and V-pLNCX-hri were used as controls.


RESULTS
The infection efficiency of V-pLNCX-s-hri on cultured B16 cells reached 38.5%. mRNA and protein levels of hRI were detected in B16 cells infected by V-pLNCX-s-hri. The hRI content in the supernatant of infected B16 cells reached 0.228 microg/mL. The hRI content in the peripheral blood of experimental mice was significantly higher in the V-pLNCX-s-hri group (0.249 microg/mL) than in the NS group (0.035 microg/mL), V-pLNCX group (0.028 microg/mL) and V-pLNCX-hri group (0.169 microg/mL) (P<0.01). The tumor weight and MVD were significantly lower in the V-pLNCX-s-hri group compared with those in the NS, V-pLNCX and V-pLNCX-hri groups (P>0.01).


CONCLUSIONS
V-pLNCX-s-hri can effectively infect B16 cells and induce high expression of hRI. V-pLNCX-s-hri is superior to V-pLNCX-hri in inhibiting the growth of B16 cells.
Sheng
Fu-Xiu
FX
Department of Biochemistry & Molecular Biology, Dalian Medical University, Dalian, Liaoning, P. R. China.
Fan
Jian-Hui
JH
Wang
Dong-Mei
DM
Zhao
Bao-Chang
BC
Cui
Xiu-Yun
XY
Tian
Yu-Xiang
YX
D000818:Animals; D049109:Cell Proliferation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D007074:Immunoglobulin G / Q000235:genetics; D008297:Male; D008546:Melanoma, Experimental / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D055806:Microvessels / Q000473:pathology; D041681:NIH 3T3 Cells; D009389:Neovascularization, Pathologic / Q000517:prevention & control*; D010926:Placental Hormones / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D011897:Random Allocation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D012260:Ribonucleases / Q000037:antagonists & inhibitors*; D014162:Transfection
D007074:Immunoglobulin G; D010926:Placental Hormones; D012333:RNA, Messenger; D011994:Recombinant Proteins; C022052:placental ribonuclease inhibitor; D012260:Ribonucleases