c-Casitas B谱系淋巴瘤原癌基因蛋白参与高糖抑制内皮细胞成管

The c-casitas b-lineage lymphoma protein participates in the impaired tube formation of endothelial cells induced by high glucose

目的探索高糖培养下内皮细胞c-Casitas B谱系淋巴瘤原癌基因蛋白(c-Cbl)表达与活性变化对其成管的影响。方法将人脐静脉内皮细胞(HUVECs)分别以5.5 mmol/L正常浓度葡萄糖(NG)、15.2 mmol/L中等浓度葡萄糖(MG)和25.0 mmol/L高浓度葡萄糖(HG)的培养基处理24 h, 采用锁核酸修饰的反义寡核苷酸间隔物(LNA Gapmers)下调c-Cbl表达。蛋白质印迹法(Western blot)检测c-Cbl表达及磷酸化水平。侵袭小室(Transwell)、细胞计数试剂盒(CCK-8)试验检测细胞迁移、增殖能力, 成管实验判断细胞的成管能力, 并通过t检验评估两组间差异。结果 HG组细胞c-Cbl在Y731位点的相对磷酸化水高于NG组(3.27±0.18比1.00±0.19, t=15.140, P<0.01), HG组细胞迁移数低于NG组[(38.33±6.51)个比(180.67±16.17)个, t=-14.147, P<0.01], HG组CCK-8吸光度值低于NG组(0.45±0.05比1.25±0.05, t=-19.596, P<0.01), HG组细胞成管水平低于NG组[相对总长度(0.42±0.08)比(1.00±0.11);相对结节数(0.36±0.07)比(1.00±0.07);相对网眼结构(0.32±0.08)比(1.00±0.10);t=-7.119、-10.870、-9.114, P<0.01]。另外, 高糖下调c-Cbl(HG+c-Cbl KD)组CCK-8吸光度值高于高糖对照(HG+NC)组(0.81±0.06比0.46±0.06, t=6.906, P<0.01)、其迁移细胞数也高于HG+NC组[(85.00±9.00)个比(43.67±9.30)个, t=5.534, P<0.01], 并且HG+c-Cbl KD组细胞的成管能力高于HG+NC组[相对总长度(0.72±0.05)比(0.42±0.10);相对结节数(0.64±0.08)比(0.33±0.12);相对网眼结构(0.62±0.08)比(0.34±0.08);t=4.874、3.734、4.266, P<0.05]。结论高糖培养下c-Cbl磷酸化激活可抑制血管内皮细胞成管能力。

Objective To explore the effects of c-casitas b-lineage lymphoma protein(c-Cbl)expression and activity changes in endothelial cells induced by high glucose on its tube formation.Methods Human umbilical vein endothelial cells(HUVECs)were respectively cultured in medium containing 5.5 mmol/L normal glucose(NG),15.2 mmol/L medium glucose(MG)and 25 mmol/L high glucose(HG)for 24 h.The c-Cbl level was down-regulated by locked nucleic acid modified antisense oligonucleotide gapmers(LNA Gapmers)transfection.The c-Cbl level and phosphorylation level was tested by Western blotting.Transwell assay and cell counting kit-8(CCK-8)assay were used to test cell migration and proliferation ability.Tube formation ability was tested by tube formation assay.Differences between two groups were evaluated by t test.Results The phosphorylation level of c-Cbl on Y731 site in HG group was higher than in NG group(3.27±0.18 vs.1.00±0.19,t=15.140,P<0.01),and the number of migrating cells in HG group was less than in NG group(38.33±6.51 vs.180.67±16.17,t=-14.147,P<0.01).Also the absorbance value of CCK-8 in HG group was lower than in NG group(0.45±0.05 vs.1.25±0.05,t=-19.596,P<0.01)and the tube formation ability of HG group was lower[relative total length:(0.42±0.08)vs.(1.00±0.11);relative node number:(0.36±0.07)vs.(1.00±0.07);relative mesh number:(0.32±0.08)vs.(1.00±0.10);t=-7.119,-10.870,-9.114,P<0.01].Also,the absorbance value of CCK-8 in cells under HG after knocking-down c-Cbl(HG+c-Cbl KD)was higher than negative control(HG+NC)group(0.81±0.06 vs.0.46±0.06,t=6.906,P<0.01),and the number of migrating cells was greater(85.00±9.00 vs.43.67±9.30,t=5.534,P<0.01).The tube formation ability in HG+c-Cbl KD group was higher than HG+NC group[relative total length:(0.72±0.05)vs.(0.42±0.10);relative node number:(0.64±0.08)vs.(0.33±0.12);relative mesh number:(0.62±0.08)vs.(0.34±0.08);t=4.874,3.734,4.266,P<0.05].Conclusion The phosphorylation of c-Cbl inhibited tube formation ability in HUVECs under HG condition.