摘要
目的:使用血管内皮细胞生长因子C(vascular endothelial growth factor C,VEGF-C)与肝细胞生长因子(hepatocyte growth factor,HGF)联合诱导大鼠骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)向淋巴管内皮细胞(lymphatic endothelial cells,LECs)方向分化,寻求最适HGF浓度,探索HGF的促分化机制。方法:原代培养获得BMSCs,流式细胞仪检测相关表面抗原以及向成骨诱导分化鉴定干细胞特性,取原代培养第3代细胞进行诱导实验。设置50 ng/ml VEGF-C分别联合10、20、40、60、80、100 ng/ml HGF配置诱导培养基的6个联合诱导组以及同时设置50 ng/ml VEGF-C诱导组、50 ng/ml HGF诱导组和空白对照组,诱导10 d后利用real-time PCR检测各组LECs标志性分子淋巴管内皮细胞透明质酸受体-1(lymphatic endothelial hyaluronan receptor 1,LYVE-1),同源异形盒蛋白1(prospero homeobox protein 1,Prox1)的表达,得出HGF最适浓度。Western blot、real-time PCR检测HGF诱导组、空白对照组、VEGF-C诱导组的LECs标志性分子LYVE-1、Prox1的表达,以及检测空白对照组、VEGF-C诱导组、HGF联合VEGF-C诱导组的integrinα9的表达。结果:成功分离获得原代培养大鼠BMSCs,80 ng/ml HGF联合50 ng/ml VEGF-C诱导组的LYVE-1、Prox1的m RNA表达量最高(PProx1=0.000,PLYVE-1=0.000),同时HGF诱导组未检测到相关LECs标志性分子的表达,HGF联合VEGF-C诱导组的integrinα9的表达较VEGF-C诱导组明显提高(PWestern blot=0.001,Preal-time PCR=0.000)。结论:当HGF浓度为80 ng/ml时,此时HGF联合VEGF-C可更好地促进大鼠BMSCs向LECs分化。同时,在该浓度条件下HGF不具有单独诱导大鼠BMSCs分化为LECs的能力,但能在诱导分化过程中明显提高integrinα9的表达,证明在促分化过程中HGF可能是通过间接上调integrinα9表达发挥作用。
Objective：To explore the optimal concentration of hepatocyte growth factor（HGF）and the mechanism of marrow stem cells（BMSCs）differentiate into lymphatic endothelial cells（LECs）by HGF synergizeing with vascular endothelial growth factor C（VEGFC）. Methods：BMSCs were isolated and cultured. Flow cytometry（FCM）was used to detect the relative surface antigen of rat BMSCs and the ability of multipotent differentiation was identified. The 3th generation of rat BMSCs were used in the experiment and were divided into 6 synergizeing groups including 50 ng/ml VEGF-C synergizeing with 10,20,40,60,80,100 ng/ml HGF and 50 ng/ml VEGF-C inducing group,50 ng/ml HGF induing group,blank control inducing group were established. After 10 d inducing,lymphatic endothelial hyaluronan receptor 1（LYVE-1）and prospero homeobox protein 1（Prox1）,the cell makers of LECs were detected using real-time PCR. Prox1 and LYVE-1 in blank control group,VEGF-C inducing group and HGF inducing group after10 d inducing were detected by using Western blot and real-time PCR.Expression of integrinα9 in blank control group,VEGF-C inducing group and HGF synergizeing with VEGF-C group was detected by Western blot and real-time PCR. Results：Purification of rat bone marrow mesenchymal stem cells was successfully isolated and cultured. The expressions of LYVE-1 and Prox1 were the highest（PProx1=0.000,PLYVE-1=0.000）in 80 ng/ml HGF synergizeing with VEGF-C group. Meanwhile,LECs markers were not detected in HGF inducing group.Higher expression of integrinα9 was observed in HGF synergizeing with VEGF-C group than in VEGF-C inducing group（PWestern blot=0.001,Preal-time PCR=0.000）. Conclusion：HGF synergizeing with VEGF-C can better enable rat BMSCs differentiate into LECs when the concentration of HGF is 80 ng/ml,but HGF alone could not induce rat BMSCs differentiate into LECs at this concentration. HGF can up-regulate integrinα9 expression indirectly in the process of differentiation,indicating that HGF may promote the diffe
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2015年第6期850-855,共6页
Journal of Chongqing Medical University
基金
重庆市科委基础与前沿研究计划资助项目(编号:CSTC2013jcyja10062)
重庆市卫生局重点课题资助项目(编号:2013-1-014)
