The Effection Of CCR2 In Monocyte Chemotactic Protein-1 Induce Apoptosis Of HECs

Objective: To study the chemokine (C-C motif) receptor 2 (CCR2) in monocyte chemotactic protein- 1(MCP-1) induced human umbilical vein endothelial cell apoptosis (CRL-1730) and discuss the influence of protein kinase C (PKC) signaling pathways on MCP-1 inducing CRL-1730 apoptosis .Methods: The first part: CRL-1730 cell line was cultured in vitro and identified immunofluorescently with VIII antiboday. CRL-1730 cells were stimulated with different concentrations of MCP-1 at 0.1ng/ml, 1.0ng/ml, 10ng/ml and 100ng/ml for 24 hours, the expressions of CCR2 protein were detected by Western bloting. The CCR2 positive-sense oligonucleotide, antisense oligonucleotide and nonhomology of oligonucleotide sequence with human gene were designed and synthesized. After embedded by lipofectin , the different oligonucleotides were transfected into CRL-1730 cells , CCR2 protein expression was detected by Western Blot, while the cell apoptosis rates were detected by flow cytometry(FCM) . The second part: After treated with the antagonist (RS102895) of CCR2 and MCP-1, the cells of apoptosis morphologically were observed under laser scanning confocal microscope, Annexin V/PI double staining kits were applied in FCM to detect the early apoptotic events. The third Part: After treated with different concentrations of PMA and chelerythrine respectively , CRL-1730 cells were induced by MCP-1 at 10ng/ml and then the apoptosis rates of cells were detected by using FCM .Results: CRL-1730 cells were identified by immunofluorescence. The Protein expression of CCR2 increased with the increase of MCP-1 concentration. The best efficiency of CCR2 antisense oligonucleotides labeled with FITC transfecting to CRL-1730 cells was determined by inverted fluorescence microscope and FCM. After transfected with CCR2 antisense oligonucleotides, the CCR2 protein expression in CRL-1730 cells was inhibited (P<0.05) and the apoptosis rates of CRL-1730 cells induced by MCP-1 was also inhibited (P<0.05). Observing by laser scanning confocal microscope, the cell apoptosis rates were reduced after transfection of CCR2 comparing with the cells only treated with MCP-1. The cell apoptosis rates gradually were reduced with the increase of RS102895 (P<0.01, P<0.01, P<0.01). Different concentrations ofPMA (1x10^-4 mmol/L, 1x10^-3 mmol/L) enhanced the cell apoptosis induced by MCP-1 in a dose-dependent manner (P<0.05, P<0.01). Different concentrations of PKC inhibitor chelerythrine did not inhibit the effect of cell apoptosis induced by MCP-1, while the apoptosis effect of chelerythrine at 1x10^-6 mmol after treatment for 12 hours was the weakest (P < 0.05).Conclusion: CCR2 mediated the CRL-1730 cell apoptosis induced by MCP-1. Combining with CCR2 and sharing same binding site when exerting the effect of chemokine, MCP-1 induces CRL-1730 cell apoptosis. PKC signaling pathways is involved in CRL-1730 cell apoptosis induced by MCP-1.