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

发布时间：2018-03-08 17:42

本文选题：组织工程　切入点：干细胞　出处：《中国组织工程研究》2017年05期 　论文类型：期刊论文

【摘要】：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(P0.01),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(P0.01);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(P0.01);(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(P0.01);(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。
[Abstract]:Background: chemokines play an important role in promoting the paracrine vasoactive factors of stem cells and accelerating angiogenesis. The effect of supernatant of human bone marrow mesenchymal stem cells stimulated by chemokine CXCL-8 on homing of human umbilical vein endothelial cells. The mechanism of Sonic Hedgehog signaling pathway in stimulating bone marrow mesenchymal stem cells by CXCL-8 was analyzed. Methods: under the model of high glucose environment, mesenchymal stem cells stimulated by 100 渭 g / L chemokine 8 were divided into high glucose CXCL-8 group. 5 渭 mol/L shh inhibitor octyl maleimide was pretreated for 45 min, then stimulated mesenchymal stem cells (MSCs) with 100 渭 g / L CXCL-8 for 45 min. The cells were treated with high glucose Shh inhibitor group. Mesenchymal stem cells cultured in high glucose environment were treated with high glucose control group. Human umbilical vein endothelial cells were cultured on conditioned medium with supernatant of each group of cells. The cells were divided into two groups according to conditioned medium. CCK8 cell proliferation test was used. Cell scratch test or Transwell cell chamber test were used to observe the effects of different conditioned media on the proliferation, apoptosis and chemotaxis of human umbilical vein endothelial cells. In vivo experiments, C57BL / 6J mice model of diabetic skin ulcer was established, and the effect of C57BL / 6J on the proliferation, apoptosis and chemotaxis of human umbilical vein endothelial cells was observed. The mesenchymal stem cells in each group were treated on conditioned medium. To verify the effect of conditioned medium of mesenchymal stem cells stimulated by CXCL-8 on homing and ulcer healing of human umbilical vein endothelial cells. The conditioned medium of high glucose CXCL-8 group could promote the proliferation of human umbilical vein endothelial cells, reduce the apoptosis rate of human umbilical vein endothelial cells, and close the area of cell scratches. The cell mobility was significantly increased in both groups, and the levels of vascular endothelial cell growth factor and epidermal growth factor in the conditioned medium of high glucose CXCL-8 group were significantly higher than those in high glucose CXCL-8 group, while those in high glucose CXCL-8 group were significantly higher than those in high glucose CXCL-8 group. In vivo experimental results: compared with Shh inhibitor -CM group and mesenchymal stem cell -CM group, the healing effect of diabetic skin ulcer and the human umbilical cord labeled by green fluorescent protein in CXCL-8-CM group were compared with those of Shh inhibitor -CM group and mesenchymal stem cell -CM group. The number of venous endothelial cells increased significantly (P 0.01). CXCL-8 can stimulate the paracrine secretion of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) from mesenchymal stem cells (MSCs) through Shh pathway in high glucose environment.
【作者单位】：齐齐哈尔医学院解剖教研室;齐齐哈尔医学院组织胚胎学教研室;
【基金】：国家自然科学基金项目(81541137)~~
【分类号】：R587.2

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