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

曹晓赞; 谢挺; 陆树良

doi:10.3760/cma.j.cn501120-20200916-00412

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

doi: 10.3760/cma.j.cn501120-20200916-00412

1. 上海交通大学医学院附属瑞金医院灼伤研究所 200025;
2. 上海交通大学医学院附属第九人民医院急诊科 200011

基金项目:

国家自然科学基金（81071568、81272111）

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出版历程
- 收稿日期: 2020-09-16
- 网络出版日期: 2021-10-28
- 刊出日期: 2021-01-20

Receptor pathways of glycated basic fibroblast growth factor affecting the proliferation and vascularization of human dermal microvascular endothelial cells

1. Department of Burns Institute, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China;
2. Emergency Department, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China

摘要

摘要:
目的探讨糖化碱性成纤维细胞生长因子(bFGF)影响人真皮微血管内皮细胞(HDMEC)增殖及血管化效应的受体途径。方法采用实验研究方法。采用葡萄糖、bFGF制备糖化bFGF刺激液。取第3～6代HDMEC进行实验。取细胞，分为小干扰RNA(siRNA)-阳性对照组、siRNA-阴性对照组、siRNA-晚期糖基化终末产物受体(RAGE)组和siRNA-成纤维细胞生长因子受体(FGFR)组，分别转染siRNA-阳性对照3-磷酸甘油醛脱氢酶、siRNA-阴性对照、siRNA-RAGE和siRNA-FGFR 4～6 h，之后加入HDMEC培养基常规培养，反转录PCR法鉴定siRNA转染效果。取细胞，分为正常对照组、单纯糖化bFGF组、单纯siRNA-RAGE组、siRNA-RAGE＋糖化bFGF组，接种于96孔板和6孔板。单纯siRNA-RAGE组、siRNA-RAGE＋糖化bFGF组细胞转染siRNA-RAGE之后，加入HDMEC培养基常规培养，2 d后，弃去原HDMEC培养基，单纯siRNA-RAGE组细胞加入HDMEC培养基常规培养，siRNA-RAGE＋糖化bFGF组细胞加入糖化bFGF刺激液常规培养；正常对照组细胞采用HDMEC培养基常规培养；单纯糖化bFGF组细胞采用糖化bFGF刺激液常规培养。取细胞，同前转染siRNA-RAGE后，接种于48孔板，分为单纯siRNA-RAGE组和siRNA-RAGE＋糖化bFGF组；另取细胞不转染直接同前接种到48孔板，分为正常对照组及单纯糖化bFGF组，4组分别同前处理。取细胞，分为正常对照组、单纯糖化bFGF组、单纯siRNA-FGFR组、siRNA-FGFR＋糖化bFGF组，分别接种于96、6、48孔板中，相应处理同前，仅siRNA-RAGE换为siRNA-FGFR。采用细胞计数试剂盒8法测定培养2 d细胞增殖活性(样本数为6)，流式细胞术检测培养2 d细胞凋亡情况(样本数为3)，成管实验检测培养6 h细胞成管能力(样本数为4)。对数据行单因素方差分析、LSD-
t
检验。结果 200 bp条带处，未见siRNA-阳性对照组、siRNA-RAGE组和siRNA-FGFR组表达目的基因，可见siRNA-阴性对照组表达目的基因，说明siRNA转染成功。培养2 d后，单纯糖化bFGF组细胞吸光度值明显低于正常对照组(
t
＝2.359，
P
<0.05)；siRNA-RAGE＋糖化bFGF组细胞吸光度值明显高于单纯糖化bFGF组(
t
＝3.858，
P
<0.01)，与单纯siRNA-RAGE组相近(
t
＝2.148，
P
>0.05)。培养2 d后，siRNA-FGFR＋糖化bFGF组细胞吸光度值与单纯糖化bFGF组相近(
t
＝0.805，
P
>0.05)，但明显低于单纯siRNA-FGFR组(
t
＝4.201，
P
<0.01)。培养2 d后，单纯糖化bFGF组细胞凋亡率明显高于正常对照组(
t
＝2.416，
P
<0.05)，siRNA-RAGE＋糖化bFGF组细胞凋亡率明显低于单纯糖化bFGF组和单纯siRNA-RAGE组(
t
＝3.861、2.724，
P
<0.05或
P
<0.01)。培养2 d后，正常对照组、单纯糖化bFGF组、单纯siRNA-FGFR组、siRNA-FGFR＋糖化bFGF组细胞凋亡率组间总体比较，差异无统计学意义(
F
＝2.218，
P
>0.05)。培养6 h后，正常对照组细胞小管成环数[(636±5)个]明显多于单纯糖化bFGF组[(580±8)个，
t
＝10.825，
P
<0.01]，siRNA-RAGE＋糖化bFGF组细胞小管成环数[(647±10)个]明显多于单纯糖化bFGF组和单纯siRNA-RAGE组[(628±4)个，
t
＝13.040、3.641，
P
<0.01]。培养6 h后，siRNA-FGFR＋糖化bFGF组细胞小管成环数[(619±5)个]明显多于单纯糖化bFGF组(
t
＝9.000，
P
<0.01)，但少于单纯siRNA-FGFR组[(632±3)个，
t
＝2.814，
P
<0.05]。结论糖化bFGF通过RAGE途径影响HDMEC的增殖和血管生成，可能是造成糖尿病皮肤创面难愈的原因之一。
- 新生血管化，生理性 /
- 成纤维细胞生长因子2 /
- 伤口愈合 /
- 晚期糖基化终末产物受体
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. HDMECs of the third to sixth passages were used in the experiment. Cells were divided into small interfering RNA (siRNA)-positive control group, siRNA-negative control group, siRNA-receptor for advanced glycation end product (RAGE) group, and siRNA-receptor for fibroblast growth factor (FGFR) group and transfected with siRNA-positive control glyceraldehyde-3-phosphate dehydrogenase, 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 polymerase chain reaction. The cells were divided into normal control group, glycated bFGF alone group, siRNA-RAGE alone group, and siRNA-RAGE+ glycated bFGF group, and seeded into 96-well plate and 6-well plate. 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. Another cells were directly seeded into 48-well plate without transfection and divided into normal control group and glycated bFGF alone group. Cells in the 4 groups were conducted with the corresponding treatment as above. Cells were divided into normal control group, glycated bFGF alone group, siRNA-FGFR alone group, and siRNA-FGFR+ glycated bFGF group and seeded into 96-, 6-, and 48-well plates, respectively, with the corresponding treatment the same as above. Only siRNA-RAGE was replaced by siRNA-FGFR. 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 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 target genes in siRNA-positive control group, siRNA-RAGE group, or siRNA-FGFR group, but target genes 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 statistically 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 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 proliferation and angiogenesis of HDMEC through RAGE pathway, which may be one of the reasons for impaired wound healing of diabetic skin.
- Neovascularization, physiologic /
- Fibroblast growth factor 2 /
- Wound healing /
- Receptor for advanced glycation end product