| Objective To evaluate the angiogenesis ability of extracelluar matrix proteins SRPX2 which from different original locations and species on HUVECs.Methods pc DNA3.1-SRPX2 vector(transfection group) and pc DNA3.1 vector(negative control group) were transfected into HEK293 T cells, SW480 cells(both are used to collect the conditioned medium) and HUVECs respectively, all the experiments are divided into 3 groups,including blank control group(without vector). Western blot was used to detect the express of SRPX2 protein. The proliferation of HUVECs(absorbance, A450) was detected by CCK8 kit. The transmembrane cell number was counted by Transwell migration and wound healing assay to evaluate the migration ability of HUVECs.A three dimensional culture system of cells was constructed on the Matrigel, and tube formation number of HUVECs was assessed.Results The proliferation of HUVECs among transfection group,negative control group and blank control group had no significant difference in the group of conditioned medium(HEK293T cells and SW480 cells) act on HUVECs(p>0.05). The proliferation of HUVECs among transfection group,negative control group and blank control group in 24, 48, 72 h had significant difference in group of directly transfect HUVECs(p<0.05). A signifiant difference was noted in the total branch points of capillary tubes among transfection group, negative control group and blank control group([97±4]/field versus[57±3] and [54±3]/field) in the group of conditioned medium(HEK293T cells) act on HUVECs(p<0.05). A signifiant difference was noted in the total branch points of capillary tubes among transfection group, negative control group and blank control group([86±5]/field versus[52±3] and [51±4]/field) in the group of conditioned medium(SW480 cells) act on HUVECs(p<0.05). A signifiant difference was noted in the total branch points of capillary tubes among transfection group, negative control group and blank control group([34±2]/field versus[15±2] and [13±2]/field) in the group of directly transfect HUVECs(p<0.05). After add pathway inhibitors, the total branch points of capillary tubes of SW480 cells conditioned medium in the blank control group was(12±1) / vision, negative control group was(11±2) / vision, transfection group was(26±3) / vision, transfection group add u PAR antibody was(11±2) / vision, transfection group add FAK inhibitor was(0) / vision,transfection group add PI3 K inhibitor was(10±3) / vision, transfection group add MAPK inhibitor was(15±2) / vision, transfection group add DMSO was(23±1) / vision, the difference was statistically significant(p< 0.05). In wound healing assay, the distance of transfection group compared to 0, 6 and 12 h were both significantly larger than that of negative control group and blank control group([89.72±5.70], [37.14±6.81], [35.87±4.28] μm and [135.38±4.95], [65.00±8.02], [63.11±3.79] μm respectively, both(p<0.05)) in the group of conditioned medium(HEK293T cells) act on HUVECs; the distance of transfection group compared to 0, 6 and 12 h were both significantly larger than that of negative control group and blank control group([86.78±6.81], [38.54±6.32], [34.00±4.28] μm and [129.34±4.43], [63.10±7.62], [57.11±2.99] μm respectively, both(p<0.05)) in the group of conditioned medium(SW480 cells) act on HUVECs; the distance of transfection group compared to 0, 6 and 12 h were both significantly larger than that of negative control group and blank control group([108.32±8.41], [69.97±9.23], [64.87±7.34] μm and [155.87±5.34], [121.19±8.03], [118.89±5.24] μm respectively, both(p<0.05)) in the group of directly transfect HUVECs. In Tranwell assay, the number of migrating cells in the transfection group was significantly more than negative control group and blank control group([549±10]/field vs [334±11]and [329±12]/field(p<0.05)) after co-culture 16 h in the group of conditioned medium(HEK293T cells) act on HUVECs; the number of migrating cells in the transfection group was significantly more than negative control group and blank control group([512±12]/field vs [318±11]and [300±10]/field(p<0.05)) after co-culture 16 h in the group of conditioned medium(SW480 cells) act on HUVECs; the number of migrating cells in the transfection group was significantly more than negative control group and blank control group([586±30]/field vs [300±16]and [290±21]/field(p<0.05)) after culture 16 h in the group of directly transfect HUVECs.Conclusion SRPX2 which from different original locations and species may promote angiogenesis ability by promoting the migration ability and tubing on the Matrigel of HUVECs, elevate endogenous SRPX2 can promote the proliferation ability of HUVECs, but SRPX2 which from HEK293 T and SW480 cells had no obvious effect on the proliferation ability of HUVECs. After adding u PAR antibody, FAK, PI3 K, and MAPK pathway inhibitor, the total branch point of capillary tubes on Matrigel had obvious decreased.SRPX2 promote tumor angiogenesis may be associated with these pathways. |
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