Dou Qianying, Xue Bin. Signaling pathway in the differentiation process from HADSCs into LECs. [J]. Journal of Chongqing Medical University 42(1):108-113(2017)
Dou Qianying, Xue Bin. Signaling pathway in the differentiation process from HADSCs into LECs. [J]. Journal of Chongqing Medical University 42(1):108-113(2017) DOI: 10.13406/j.cnki.cyxb.001004.
Signaling pathway in the differentiation process from HADSCs into LECs
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
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.