Receptor pathways of glycated basic fibroblast growth factor on proliferation and vascularization of human dermal microvascular endothelial cells
10.3760/cma.j.cn501120-20200916-00412
- VernacularTitle:糖化碱性成纤维细胞生长因子影响人真皮微血管内皮细胞增殖和血管化效应的受体途径
- Author:
Xiaozan CAO
;
Ting XIE
;
Shuliang LU
- From:
Chinese Journal of Burns
2020;37(1):E020-E020
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the receptor pathways of glycated basic fibroblast growth factor (bFGF) on proliferation and vascularization of human dermal microvascular endothelial cells (HDMECs).Methods:The experimental research method was used. Glycated bFGF stimulating solution was prepared with glucose and bFGF. Cells of the third to sixth passages were subcultured from the HDMEC primary cell line. The cells were divided into normal control group, glycated bFGF alone group, small interfering RNAs (siRNA)-advanced glycation end product receptor (RAGE) alone group, and siRNA-RAGE+glycated bFGF group, and seeded into 96-well plate with 6 wells in each group, and seeded into 6-well plate with 3 wells in each group. Cells in siRNA-RAGE alone group and siRNA-RAGE+glycated bFGF group were transfected with siRNA-RAGE and then were added into HDMEC culture medium for routine culture. After two days, the original HDMEC culture medium was discarded, and cells in siRNA-RAGE alone group were routinely cultured in HDMEC culture medium, cells in siRNA-RAGE+glycated bFGF group were routinely cultured in glycated bFGF stimulating solution. Cells in normal control group were routinely cultured in HDMEC culture medium, and cells in glycated bFGF alone group were routinely cultured in glycated bFGF stimulating solution. After transfection with siRNA-RAGE, cells were seeded into 48-well plate and divided into siRNA-RAGE alone group and siRNA-RAGE+glycated bFGF group, with 4 wells in each group. Another cells were directly seeded into 48-well plate without transfection and divided into normal control group and glycated bFGF alone group, with 4 wells in each group. Cells in normal control group and siRNA-RAGE group were routinely cultured in HDMEC culture medium. Cells in siRNA-RAGE alone group and siRNA-RAGE+glycated bFGF group were routinely cultured in glycated bFGF stimulating solution. Cells were divided into normal control group, glycated bFGF alone group, siRNA-fibroblast growth factor receptor (FGFR) alone group, and siRNA-FGFR+glycated bFGF group and seeded into 96-, 6- and 48-well plates. respectively. Cell density, sample number, and corresponding treatment were the same as before. Only siRNA-RAGE was replaced by siRNA-FGFR. Cells were divided into siRNA-positive control group, siRNA-negative control group, siRNA-RAGE group, and siRNA-FGFR group and transfected with siRNA-glyceraldehyde-3-phosphate dehydrogenase positive control, siRNA-negative control, siRNA-RAGE, and siRNA-FGFR for 4 to 6 hours, and then were added into HDMEC culture medium for routine culture. The transfection effect of siRNA was identified by reverse transcription PCR. Cell counting kit 8 method was used to determine the proliferation of cells after 2 days of culture (sample number was 6), flow cytometry was used to detect the apoptosis of cells after 2 days of culture (sample number was 3), tube forming test was used to detect the angiogenesis of cells cultured after 6 hours of culture (sample number was 4). Data were statistically analyzed with one-way analysis of variance and least significant difference t test. Results:At the 200 bp band, there were no complementary DNA (cDNA) bands in siRNA-positive control group, siRNA-RAGE group, and siRNA-FGFR group, but cDNA bands were detected in siRNA-negative control group, indicating the success of siRNA transfection. After 2 days of culture, the absorbance value of cells in glycated bFGF alone group was significantly lower than that of normal control group ( t=2.359, P<0.05); absorbance value of cells in siRNA-RAGE+glycated bFGF group was significantly higher than that of glycated bFGF alone group ( t=3.858, P<0.01), which was similar to that of siRNA-RAGE alone group ( t=2.148, P>0.05). The absorbance value of cells in siRNA-FGFR+glycated bFGF group was similar to that of glycated bFGF alone group ( t=0.805, P>0.05), but significantly lower than that of siRNA-FGFR alone group ( t=4.201, P<0.01). After 2 days of culture, the apoptotic rate of cells in glycated bFGF alone group was significantly higher than that of normal control group ( t=2.416, P<0.05). The apoptotic rate of cells in siRNA-RAGE+glycated bFGF group was significantly lower than the rates in glycated bFGF alone group and siRNA-RAGE alone group ( t=3.861, 2.724, P<0.05 or P<0.01). There were no significant differences in apoptosis rate of cells among normal control group, glycated bFGF alone group, siRNA-FGFR alone group, and siRNA-FGFR+glycated bFGF group ( F=2.218, P>0.05). After 6 hours of culture, the number of tubules of cells in normal control group (636±5) was significantly more than that of glycated bFGF alone group [580±8, t=10.825, P<0.01], and the number of tubules of cells in siRNA-RAGE+glycated bFGF group (647±10) was significantly more than those of glycated bFGF alone group and siRNA-RAGE alone group (628±4, t=13.040, 3.641, P<0.01). After 6 hours of culture, the number of tubules of cells in siRNA-FGFR+glycated bFGF group (619±5) was significantly more than that of glycated bFGF alone group ( t=9.000, P<0.01), but less than that of siRNA-FGFR alone group (632±3, t=2.814, P<0.05). Conclusions:Glycated bFGF affects the angiogenesis of HDMEC through RAGE pathway, which may be one of the reasons for the impaired wound healing of diabetic skin.
