高糖环境下趋化因子8促进间充质干细胞旁分泌功能提高人脐静脉血管内皮细胞归巢

Chemokine(C-X-C motif) ligand 8 enhances the homing ability of human umbilical vein endothelial cells by promoting a paracrine response in mesenchymal stem cells under the high glucose environment

背景:趋化因子具有促进干细胞旁分泌血管活性因子,加速血管新生的重要作用。目的:观察高糖环境下,趋化因子8(CXCL-8)刺激的人骨髓间充质干细胞上清液对人脐静脉血管内皮细胞归巢的影响,并分析Sonic Hedgehog信号通路在CXCL-8刺激骨髓间充质干细胞的机制。方法:(1)细胞体外实验:在细胞高糖环境模型下,将质量浓度100μg/L趋化因子8刺激的间充质干细胞设为高糖CXCL-8组,预先以5μmol/L的shh抑制剂辛基马来酰亚胺处理45 min,再用质量浓度100μg/L CXCL-8刺激间充质干细胞者为高糖Shh抑制剂组,高糖环境下培养的间充质干细胞为高糖对照组。提取各组细胞上清液为条件培养基培养人脐静脉血管内皮细胞,其分组按条件培养基而定。使用CCK8细胞增殖实验、细胞划痕实验或Transwell细胞小室实验分别观察各条件培养基(CM)对人脐静脉血管内皮细胞增殖、凋亡、趋化等能力的影响;(2)动物体内实验:建立C57BL/6J小鼠糖尿病皮肤溃疡模型,分别以各组间充质干细胞条件培养基进行处理,验证CXCL-8刺激的间充质干细胞的条件培养基对人脐静脉血管内皮细胞归巢和溃疡愈合的影响。结果与结论:(1)体外实验结果:与高糖对照条件培养基相比,高糖CXCL-8组条件培养基能够促进人脐静脉血管内皮细胞增殖、降低人脐静脉血管内皮细胞凋亡率,并且细胞划痕面积闭合率、细胞迁移率均明显提高(P<0.01),高糖CXCL-8组条件培养基中血管内皮细胞生长因子、表皮生长因子水平明显提高(P<0.01);与高糖CXCL-8组相比,高糖Shh抑制剂组上述结果指标呈反向变化(P<0.01);(2)体内实验结果:与Shh抑制剂-CM组和间充质干细胞-CM组相比,CXCL-8-CM组糖尿病皮肤溃疡的愈合效果和绿色荧光蛋白标记的人脐静脉血管内皮细胞数均显著升高(P<0.01);(3)结果提示,在高糖环境下,CXCL-8能够通过Shh通路,刺激间充质干细胞旁分泌血管内皮细胞生长因子、表皮生长因子,提高了对脐静脉血管内皮细胞招募的能力。

BACKGROUND： Chemokines can promote（MSCs） the secretion of vasoactive factors from mesenchymal stem cells（MSCs） through paracrine mechanism, which have important role in accelerating angiogenesis. OBJECTIVE： Under the high glucose environment, to the effect of the supernatant of MSCs stimulated by chemokine（C-X-C motif） ligand 8（CXCL-8） on human umbilical vein endothelial cells（HUVECs）, and to analyze the mechanism of Sonic Hedgehog signaling pathway in the stimulation of CXCL-8 on MSCs.METHODS： Under the high glucose environment, the MSCs supplemented with 100 μg/L CXCL-8 were set as CXCL-8 group; the MSCs that were preprocessed with 5 μmol/L octyl maleimide for 45 minutes and then stimulated with 100 μg/L CXCL-8 were as Shh inhibitor group; the MSCs that were routinely cultured in a high-glucose medium were as control group. The cell supernatant of each group was extracted as conditioned medium（CM） to culture HUVECs, respectively, and these cells were referred to as CXCL-8 CM group, Shh inhibitor CM group, and control CM group, respectively. Cell counting kit-8, cell scratch and Transwell chamber tests were used to observe the effect of each CM on HUVEC proliferation, apoptosis and chemotaxis. By establishment of a diabetic skin ulcer model in C57BL/6J mice, the CM of each group was used to treat the mouse model to confirm the effects of CXCL-8 stimulated MSCs CM on HUVEC homing and ulcer healing. RESULTS AND CONCLUSION：（1） The experimental results in vitro： compared with the control CM group, CXCL-8 CM group significantly promoted the proliferation of HUVECs, and decreased the apoptosis of HUVECs, the closure rate and migration rate of HUVECs were significantly increased（P〈0.01 or P〈0.01）, and the levels of vascular endothelial growth factor and epidermal growth factor were significantly increased（P〈0.01 or P〈0.01）. Compared with CXCL-8 CM group, however, the above results in the Shh inhibitor CM group showed reverse changes（P〈0.01）.（2） The experimental results in vivo： compared with the MSCs CM group and Shh inhibitor CM group, the healing effect of diabetic skin ulcer and the number of HUVECs labeled by green fluorescent protein in the CXCL-8 CM group were significantly increased（P〈0.01）. To conclude, these findings indicate that CXCL-8 stimulated MSCs secrete paracrine factors, vascular endothelial growth factor and epidermal growth factor, through the Sonic Hedgehog signaling pathway under the high glucose environment, which enhance the homing ability of HUVECs.