[An experimental study on the role of keratin in angiogenesis in vitro].

ABSTRACT

OBJECTIVE: To investigate the effect of keratin 17 (K-17) on the migration, proliferation and tube formation of human umbilical vein endothelial cell (HUVEC), and to realize the role of K-17 in angiogenesis. METHODS: After HUVEC were cultured in DMEM medium supplemented with 10%FBS overnight, K-17-siRNA-mixture (experimental group) and control-siRNA-mixture (negative control group) were added into HUVEC, respectively, by Lipofectamine 2000 transfection assay, and the final concentration of the siRNA was 50 nmol/L. Lipofectamine 2000 alone was used as the control. After the cells were cultured for 36 hours, the cell proliferation ability was detected by cell counting. After 30-hour culture, the cell's abilities of migration and differentiation to tube were detected by 24-well Millicell units and the collagen gel assay, respectively. In addition, non-siRNA-treated HUVEC were cultured for 24 hours in DMEM medium supplemented with 10%FBS (group A), 2%FBS (group B) and 2%FBS+10 ng/mL bFGF (group C), respectively, and then the expression of K-17 in HUVEC was detected by RT-PCR and Western blot. RESULTS: After the treatment with K-17-siRNA for 36 hours, HUVEC exhibited no significant difference in the proliferation, compared with both control and negative control groups (P > 0.05). After transfected with K-17-siRNA for 30 hours, the number of HUVEC in the experimental group which migrated from the upper chamber to the lower chamber of Millicell wells within 24 hours (3719.0 +/- 319.0) was smaller than both control (7 437.5 +/- 212.0) and negative control (7 356.3 +/- 795.7) groups, with significant difference (P < 0.01). However, there was no significant difference between the control group and the negative control group (P > 0.05). After HUVEC were transfected with K-17-siRNA for 30 hours, the number of tubes in the experimental group, the negative control group and the control group in 24 hours was (1.1 +/- 0.5), (3.6 +/- 0.5) and (3.2 +/- 0.6) per field, respectively. The experimental group was significantly different from both control and negative control groups (P < 0.01), and there was no significant difference between the negative control group and the control group (P > 0.05). The expression of K-17 protein in HUVEC in groups A, B and C was 0.25 +/- 0.02, 0.08 +/- 0.01 and 0.72 +/- 0.03, respectively. There was significant difference among these three groups (P < 0.01). CONCLUSION: K-17 has no impact on cell proliferation, but may augment endothelial cell migration, which may facilitate angiogenesis.