脂肪干细胞诱导分化为淋巴管内皮细胞信号通路的探究  

Signaling pathway in the differentiation process from HADSCs into LECs

作　　者：豆倩影 薛斌[1] 

机构地区：[1]重庆医科大学附属第一医院烧伤整形外科,重庆400016

出　　处：《重庆医科大学学报》2017年第1期108-113,共6页Journal of Chongqing Medical University

基　　金：重庆市基础与前沿研究计划项目(编号:CSTC2013jcyja10062);重庆市卫生局重点课题资助项目(编号:2013-1-014)

摘　　要：目的:探讨血管内皮细胞生长因子C(vascular endothelial growth factor C,VEGF-C156s)诱导脂肪间充质干细胞(adipose-derived stem cells,ADSCs)向淋巴管内皮细胞(lymphatic endothelial cells,LECs)早期分化的调控通路。方法:取人脂肪间充质干细胞(human adipose-derived stem cells,HADSCs)随机分为5组:空白对照组(常规DMEM/F12)、VEGF-C诱导组(含100ng/m L VEGF-C)、VEGF-C联合抑制剂a组(含100 ng/m L VEGF-C+5μmol/L的VEGFR3磷酸化酶抑制剂MAZ51)、VEGF-C联合抑制剂b组(含100 ng/m L VEGF-C+10μg/m L的Anti-Integrinα9功能封闭性抗体Y9A2)、VEGF-C联合抑制剂a+b组,连续培养10 d后,Western blot和细胞免疫荧光(IF)检测LECs标志性分子VEGFR3、Integrinα9、LYVE-1的相对表达量;VEGF-C诱导分化后的细胞用Anti-Integrinα9封闭性抗体和MAZ51作用后,Transwell小室检测各组细胞迁移数量的变化。结果:与诱导组相比,分别阻断VEGFR3和Integrinα9通路都会相应减弱LYVE-1的表达(PWestern=0.000,PIF=0.000),且在上述溶度下两抑制剂对LYVE-1的减弱作用无统计学差异(PWestern=0.164,PIF=0.927);而分别阻断任一通路对另一通路的表达量都没有影响(PWestern VEGFR3(BVSD)=0.804,PIF VEGFR3(BVSD)=0.528,PWestern Integrinα9(BVSC)=0.914,PIF Integrinα9(BVSC)=0.593)。与抑制剂作用之前相比,分别阻断Integrinα9和VEGFR3会使迁移细胞数量相应减少(P=0.000),而同时阻断Integrinα9和VEGFR3会使迁移细胞数量进一步减少(P=0.000)。结论:VEGF-C诱导HADSCs向LECs分化过程中有VEGF-C/VEGFR3和VEGF-C/Integrinα9两条独立信号通路,且Integrinα9和VEGFR3在诱导后的细胞的迁移中均发挥重要作用。Objective：To explore the regulatory pathways from human adipose-derived stem cells（HADSCs）differentiate into lymphatic endothelial cells（LECs）by vascular endothelial growth factor C（VEGF-C156s）in the early lymphangiogenesis. Methods：HADSCs were randomly divided into 5 groups：blank control group,VEGF-C inducing group（containing 100 ng/m L VEGF-C）,VEGF-C unite inhibitor ＂a＂group（containing 100 ng/m L VEGF-C＋5 μmol/L VEGFR3 phosphorylase inhibitor MAZ51）,VEGF-C unite inhibitor ＂b＂group（containing 100 ng/m L VEGF-C＋10 μg/m L Anti-Integrinα9 function blocking antibody Y9A2）,VEGF-C unite inhibitor ＂a＂and ＂b＂group. After 10 d culturing,the relative expression level of LECs markers VEGFR3,Integrinα9,LYVE-1 were detected by Western blot and immunofluorescence. The induced cells with VEGF-C were incubated by anti-Integrinα9 function blocking antibody and MAZ51; quantity of cell migration in all groups were calculated using Transwell. Results：Compared with that in induction group,the expression level of LYVE-1 was modestly weakened with blocking VEGFR3 and Integrinα9 pathways（PWestern=0.000,PIF=0.000）,and there was no statistical difference in LYVE-1 level between the two inhibitors at the concentration above（PWestern=0.164,PIF=0.927）.Blocking one of the two pathways,the expression of another had no change（PWestern VEGFR3（BVSD）=0.804,PIF VEGFR3（BVSD）=0.528,PWestern Integrinα9（BVSC）=0.914,PIF Integrinα9（BVSC）=O.593）. Compared with that of no inhibitors,the quantity of cell migration suffered a modest decrease with blocking VEGFR3 and Integrinα9（P=0.000）,while the numbers of cell migration was further reduced when blocking VEGFR3 and Integrinα9 at the same time（P=0.000）. Conclusion：There are two independent pathways：VEGF-C/VEGFR3 and VEGF-C/Integrinα9 in the differentiation process from stem cells into LECs. VEGFR3 and Integrinα9 play major role in the migration of induced cells.

关 键 词：脂肪干细胞 淋巴管内皮细胞 血管内皮生长因子C 人整合素α9 血管内皮生长因子受体3 

分 类 号：R619.9[医药卫生—外科学]