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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 investigate the early differentiation of adipose-derived stem cells (ADSCs) into lymphatic endothelial cells (LECs) induced by vascular endothelial growth factor C (VEGF-C156s) Regulatory channels. Methods: Human adipose-derived stem cells (HADSCs) were randomly divided into 5 groups: blank control group (conventional DMEM / F12), VEGF-C induction group (containing 100ng / , VEGF-C combined inhibitor a group (containing 100 ng / m L VEGF-C + 5μmol / L VEGFR3 phosphorylase MAZ51), VEGF-C combined inhibitor group b C + 10μg / m L Anti-Integrinα9 functional blocking antibody Y9A2), VEGF-C combined inhibitor a + b group after 10 days of continuous culture, Western blot and immunofluorescence (IF) detection of LECs landmark VEGFR3, Integrinα9, LYVE-1 relative expression levels; After VEGF-C-induced differentiation of cells with Anti-Integrinα9 blocking antibody and MAZ51 effect, Transwell chamber detection of changes in the number of cell migration. Results: Compared with the induction group, blocking the VEGFR3 and Integrinα9 pathways respectively decreased the expression of LYVE-1 (PWestern = 0.000, PIF = 0.000), and the attenuated effect of the two inhibitors on LYVE-1 (PWestern = 0.164, PIF = 0.927) .While blocking each pathway had no effect on the expression of the other pathway (PWSD = 0.804, PFS VEGFR3 (BVSD) = 0.528, PWestern Integrinα9 BVSC) = 0.914, PIF Integrin α9 (BVSC) = 0.593). Blocking integrinα9 and VEGFR3 decreased the number of migrating cells (P = 0.000), respectively, compared with that before inhibitor treatment, while blocking integrinα9 and VEGFR3 resulted in a further decrease in the number of migrating cells (P = 0.000). Conclusion: There are two independent signaling pathways of VEGF-C / VEGFR3 and VEGF-C / Integrinα9 during the differentiation of HADSCs into LECs induced by VEGF-C, and both Integrinα9 and VEGFR3 play an important role in the induction of cell migration.