基质细胞衍生因子1对于血管内皮细胞增殖影响及其相关机制研究

发布时间：2018-04-19 14:03

本文选题：基质细胞衍生因子 + 髓核细胞　；参考：《中国修复重建外科杂志》2017年01期

【摘要】：目的探讨人退变髓核细胞(nucelus pulposus cells,NPCs)分泌的基质细胞衍生因子1(stromal cell derived factorl,SDF-1)对血管内皮细胞(vascular endothelial cells,VECs)的增殖影响及其相关分子机制。方法将术中摘取的椎间盘组织分离、培养人退变NPCs,取第1代转染SDF-1过表达慢病毒。取第2代细胞,转染或未转染过表达SDF-1慢病毒后,种植于alvetex?三维培养支架上行三维培养,并与人HMEC-1细胞(即VECs)共培养。共培养72 h扫描电镜观察NPCs在支架上的生长情况;细胞计数试剂盒8(cell counting kit 8,CCK-8)法分别检测12、24、48、72 h转染组与非转染组HMEC-1增殖情况,以及加入Akt特异性抑制剂GSK690693后24 h HMEC-1增殖情况;膜联蛋白Ⅴ/碘化丙啶法分别检测共培养72 h后转染组和非转染组中VECs凋亡率;ELISA法分别检测转染组和非转染组培养基中VEGF含量;Western blot法检测转染组、未转染组和抑制剂组磷酸化Akt(p-Akt)和总Akt蛋白表达。结果扫描电镜观察示NPCs在三维支架中维持了细胞表型,分泌大量细胞外基质;慢病毒成功转染后,Western blot检测示SDF-1在NPCs中表达明显升高。在共培养体系中,NPCs过表达SDF-1后可使HMEC-1增殖上升,凋亡率下降,并在细胞外培养基检测中发现VEGF含量显著上升;而在进一步分子机制检测中发现,SDF-1的过表达使HMEC-1中p-Akt水平升高,采用GSK690693特异性抑制Akt表达后,SDF-1对HMEC-1促细胞增殖作用明显降低。结论人退变NPCs高表达的SDF-1有利于VECs增殖,这一调控机制或许与SDF-1-Akt信号通路有关。
[Abstract]:Objective to investigate the effect of 1(stromal cell derived factorl1 (SDF-1) secreted by nuclear nucleus pulposus cells (NPCs) on the proliferation of vascular endothelial cells (VECs) and its molecular mechanism.Methods the intervertebral disc tissue was isolated and cultured, and the first generation was transfected with SDF-1 overexpression lentivirus.The second passage cells were transfected or not transfected with SDF-1 lentivirus.The three-dimensional culture scaffold was cultured in three-dimensional way and co-cultured with human HMEC-1 cells (VECs).The growth of NPCs on the scaffold was observed by scanning electron microscope at 72 h, and the proliferation of HMEC-1 was detected by 8(cell counting kit 8 CCK-8 (cell count kit) method in the transfected group and non-transfected group at 72 h, and the HMEC-1 proliferation at 24 h after the addition of GSK690693, the specific inhibitor of Akt.The apoptosis rate of VECs in transfected group and non-transfection group was detected by Elisa and VEGF content in culture medium of transfection group and non-transfection group were detected by Western blot method.The expression of phosphorylated Aktna-Akt and total Akt protein in untransfected and inhibitor groups.Results the results of scanning electron microscopy showed that NPCs maintained the phenotype and secreted a large amount of extracellular matrix in the three-dimensional scaffold, and the expression of SDF-1 in NPCs was significantly increased by Western blot after the successful transfection of lentivirus.After overexpression of SDF-1 in co-culture system, the proliferation and apoptosis rate of HMEC-1 were increased, and the content of VEGF was significantly increased in extracellular culture medium, while the level of p-Akt in HMEC-1 was increased by overexpression of SDF-1 in the further molecular mechanism detection.After Akt expression was inhibited by GSK690693, the effect of SDF-1 on the proliferation of HMEC-1 cells was significantly decreased.Conclusion the high expression of SDF-1 in human degenerative NPCs is beneficial to the proliferation of VECs. This regulatory mechanism may be related to the SDF-1-Akt signaling pathway.
【作者单位】：西南医科大学附属中医院骨科;重庆医科大学附属第一医院骨科;
【基金】：国家自然科学基金面上项目资助(81372003)~~
【分类号】：R681.5

【相似文献】