| Objective:To explore the roles of platelet factor 4 (PF4) and p17-70 peptide in the pathological process of multiple myeloma (MM).Methods:Full-length cDNA and 17-70cDNA of PF4 were acquired and connected into shuttle plasmids respectively. Then the shuttle plasmid genes were sequenced and compared to correct sequence. MM cells (U266, RPMI8226, LP-1) were transduction by the empty lentiviral vector and lentiviral expression vectors (PF4 or sPF4) which made from the shuttle plasmid. The MM cells which had stably expressed the platelet factor 4 protein and 17-70 peptides were identified and screened. Vascular endothelial growth factor (VEGF) levels in the supernatants of these MM cells were detected by ELISA. The mononuclear cells of healthy adult's peripheral blood were obtained by Ficoll density gradient centrifugation. The isolated cells were suspended in Medium 199 with 20% New-Born Calf Serum (NBCS),30ng/ml recombinated human vascular endothelial growth factor (rh-VEGF) and 6ng/ml recombinated human basic fibroblast growth factor (rh-b-FGF) for culturing, differentiating and proliferating. Cells'morphous, emergence time of cell colonies and fusiform attached cells were observed. The expressions of specific antigens on cell surface were analyzed by immunohistochemistry, immunocytochemistry and fluorescence activated cell sorting (FACS). The acquired EPCs and MM cells were co-cultured in a transwell system in vitro. The proliferations and migrations of the EPCs were measured. The apoptosis of the EPCs were measured by FACS. SCID-rab mice models of MM were established by injecting three sorts of MM cells which transduction by platelet factor 4 or 17-70 cDNA or neo by lentiviral vectors. The human light chain proteins in serums of mice were detected every 2 weeks. The VEGF in serums of mice were detected every 4 weeks. The volumes and density of vascular of tumors and overall survival times of mice were observed.Results:Full-length sequences of shuttle plasmid vectors were shown that each shuttle plasmid vector contained the correct PF4 and 17-70cDNA sequences. The MM cells which expressed the transduction genes stablely were identified and screened. There were significant difference between each groups of VEGF levels (P<0.01). The VEGF levels in the supernatants of PF4 positive MM cells groups were less than that of neo MM cells groups and more than that of p17-70 peptide positive MM cells groups (P<0.01). Part of the culture cells attached to the wall after 24 hours, cell colonies formed after 3 days and most of the cells showed fusiform shape at 8 days.95% of the culture cells could be shown to endocytose 1, 1'-dioctadecy1-3,3,3',3'-tetra-methylindocarbocyanine perchlorate acetylated-low-density lipoprotein (DiI-ac-LDL) and combine FITC labeled Ulex europaeus agglutinin I (FITC-UEA-I) by fluorescence microscope and LSCM. The culture cells which positively expressed CD34, CD133, and VEGFR2 were 0.862±0.062, 0.835±0.053,0.862±0.082 by FACS. The proliferative and migratory capacities of the EPCs decreased after co-culture 48h with the MM cells which expressed the full-length or fragment of PF4 (P<0.05). The apoptosis of the EPCs had no significant difference (P>0.05). The SCID-rab models of MM were established successfully. It had been demonstrated that the tumors in the sections of injection were plasma cells malignancy by pathological methods. The results showed that there had significant difference in light chains proteins and VEGF in serums between three groups (P<0.05). The light chains proteins and VEGF in serums of mice in 17-70 cDNA groups were less than in PF4 groups. The light chains proteins and VEGF in serums of mice in PF4 groups were less than in control groups. The results also showed that there had significant difference in the overall survival times in three different groups in three sorts of SCID-rab models of MM. The overall survival times in control groups were the shortest than in other groups.Conclusion:The MM cells which transduction by the lentiviral vectors can be identified and screened successfully in vitro. The VEGF levels in the supernatants of p17-70 peptide of PF4 positive MM cells were less than that of PF4 positive MM cells, the VEGF levels in the supernatants of PF4 positive MM cells were less than that of MM cells. EPCs can be obtained from mononuclear cells in adult's peripheral blood and be differentiated into endothelial cells in vitro. It is concluded that transduction of platelet factor 4 or 17-70 cDNA into multiple myeloma cells can inhibit growth of MM cells and prolong the overall survival times in vivo. In summary, the PF4 and p17-70 peptide can inhibit the growth of MM. One of the mechanisms of the inhibition is to reduce the VEGF level and angiogenesis.
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