三种诱导因子在骨髓间充质干细胞向淋巴管内皮细胞分化中的作用  

Effects of three inducing factors on differentiation of bone marrow derived mesenchymal stem cells into lymphatic endothelial cells

作　　者：王远航 薛斌[1] Wang Yuanhang;Xue Bin(Department of Burns and Plastic Surgery,the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016,China)

机构地区：[1]重庆医科大学附属第一医院烧伤整形外科,400016

出　　处：《中华烧伤杂志》2019年第2期125-133,共9页Chinese Journal of Burns

基　　金：重庆市基础与前沿研究计划;重庆市卫生局重点课题(2013-1-014).

摘　　要：目的 观察碱性成纤维细胞生长因子(bFGF)、肝细胞生长因子(HGF)、血管内皮生长因子C(VEGF-C)在骨髓间充质干细胞(BMSC)向淋巴管内皮细胞(LEC)分化中的作用. 方法 取第3~5代大鼠BMSC进行实验.(1)取大鼠BMSC,采用随机数字表法(分组方法下同)分为阴性对照组、CD90组、CD44组、CD34组,每组样本数为3,阴性对照组细胞加入磷酸盐缓冲液5μL,余3组细胞分别加入相应抗体5 μL,流式细胞仪检测细胞表面抗原阳性情况.(2)取3个批次大鼠BMSC,均分为空白对照组、VEGF-C组、HGF组、bFGF组、VEGF-C+HGF组、VEGF-C+bFGF组、HGF+bFGF组、VEGF-C+ HGF+ bFGF组,每组样本数为3.空白对照组细胞加入2 mL完全培养基,VEGF-C组细胞中加入2 mL完全培养基和10 μg/mL VEGF-C 10 μL,HGF组细胞加入2 mL完全培养基和10 μg/mL HGF 16 μL,bFGF组细胞加入2 mL完全培养基和1μg/mL bFGF 20 μL,VEGF-C+ HGF组、VEGF-C +bFGF组、HGF+ bFGF组、VEGF-C+ HGF+ bFGF组细胞加入2 mL完全培养基及同前相应浓度和量的诱导因子.培养10d,倒置相差显微镜下观察细胞形态,蛋白质印迹法和实时荧光定量反转录PCR法分别检测淋巴管内皮透明质酸受体1(LYVE-1)、VEGF受体3(VEGFR3)及整合素α9蛋白和mRNA表达.(3)取大鼠BMSC,分为空白对照组、HGF+ VEGF-C+ bFGF组、bFGF+ VEGF-C+HGF组、VEGF-C+ HGF+ bFGF组,每组样本数为3.空白对照组细胞加入2 mL完全培养基;HGF+VEGF-C +bFGF组细胞加入2 mL完全培养基、10 μg/mL HGF 16 μL、10 μg/mL VEGF-C 10μL,6h后加入1 μg/mL bFGF 20 μL.bFGF+VEGF-C+HGF组细胞加入2 mL完全培养基以及1μg/mLbFGF 20 μL和10 μg/mL VEGF-C 10 μL,6h后加入10 μg/mL HGF 16 μL;VEGF-C+ HGF+ bFGF组细胞同时加入2 mL完全培养基及同前浓度及量的3种诱导因子.另取2个批次大鼠BMSC,同前进行分组,除将HGF+ VEGF-C+ bFGF组、bFGF+ VEGF-C+ HGF组6h的间隔时间调整为12、24 h外,其余处理方法同前.培养10 d,蛋白质印迹法检测LYVE-1、VEGFR3及整合�Objective To observe the effects of basic fibroblast growth factor (bFGF),hepatocyte growth factor (HGF),and vascular endothelial growth factor C (VEGF-C) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into lymphatic endothelial cells (LECs).Methods The third to the fifth passage of BMSCs of rats were collected for the following experiments.(1) BMSCs of rats were collected and divided into negative control group,CD90 group,CD44 group,and CD34 group according to the random number table (the same grouping method below),with 3 samples in each group.Phosphate buffer of 5 μL was added to cells in negative control group,and cells in the other 3 groups were added with 5 μL corresponding antibodies respectively.The positive expression of cell surface antigen was detected by flow cytometer.(2) BMSCs of rats in 3 batches were collected and divided into blank control group,VEGF-C group,HGF group,bFGF group,VEGF-C + HGF group,VEGF-C + bFGF group,HGF + bFGF group,and VEGF-C + HGF + bFGF group,with 3 samples in each group.Cells in blank control group were added with 2 mL complete medium,cells in VEGF-C group were added with 2 mL complete medium and 10 μL VEGF-C of 10 μg/mL,cells in HGF group were added with 2 mL complete medium and 16 μL HGF of 10 μg/mL,and cells in bFGF group were added with 2 mL complete medium and 20 μL bFGF of 1 μg/mL.Cells in VEGF-C + HGF group,VEGF-C + bFGF group,HGF + bFGF group,and VEGF-C + HGF + bFGF group were added with 2 mL complete medium and induction factors with corresponding concentration and volume as above.On 10 d of culture,the morphology of the cells was observed by the inverted phase contrast microscope,and the protein and mRNA expressions of lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1),VEGF receptor 3 (VEGFR3),and integrin α9 were detected by Western blotting and real-time fluorescent quantitative reverse transcription polymerase chain reaction respectively.(3) BMSCs of rats were collected and divided into blank control group,HGF + VEGF-

关 键 词：间充质干细胞 血管内皮生长因子C 肝细胞生长因子 细胞分化 碱性成纤维细胞生长因子 淋巴管内皮细胞 

分 类 号：R551.2[医药卫生—血液循环系统疾病]