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

王远航; 薛斌

doi:10.3760/cma.j.issn.1009-2587.2019.02.008

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

doi: 10.3760/cma.j.issn.1009-2587.2019.02.008

王远航,
薛斌

重庆医科大学附属第一医院烧伤整形外科 400016

基金项目:

重庆市基础与前沿研究计划

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出版历程
- 收稿日期: 2018-01-14
- 网络出版日期: 2021-10-28
- 刊出日期: 2019-02-20

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

Department of Burns and Plastic Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China

摘要

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5 μL，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1 μg/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10 d，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 10 μL，6 h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1 μg/mL bFGF 20 μL和10 μg/mL VEGF-C 10 μL，6 h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6 h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及整合素α9的蛋白表达。对数据行析因设计方差分析、单因素方差分析、LSD-t检验、Bonferroni校正。结果 (1)阴性对照组、CD90组、CD44组、CD34组细胞表面抗原阳性表达率分别为0.39%、99.84%、99.90%、0.57%。(2)培养10 d，空白对照组、HGF组、bFGF组、HGF＋bFGF组细胞呈长梭形，其余各组细胞呈多边形。(3)培养10 d，空白对照组、HGF组、bFGF组、HGF＋bFGF组细胞无LYVE-1、VEGFR3和整合素α9蛋白表达。培养10 d，VEGF-C＋HGF＋bFGF组细胞LYVE-1、VEGFR3和整合素α9的蛋白表达量均明显高于VEGF-C组(t＝24.21、11.04、15.43，P<0.01)、VEGF-C＋HGF组(t＝10.81、9.93、10.20，P<0.01)、VEGF-C＋bFGF组(t＝11.67、6.32、19.00，P<0.01)。VEGF-C＋HGF组及VEGF-C＋bFGF组细胞LYVE-1的蛋白表达量明显高于VEGF-C组(t＝8.69、15.20，P<0.01)；VEGF-C＋bFGF组细胞VEGFR3的蛋白表达量明显高于VEGF-C组及VEGF-C＋HGF组(t＝8.67、7.21，P<0.01)；VEGF-C＋HGF组细胞整合素α9的蛋白表达量明显高于VEGF-C组及VEGF-C＋bFGF组(t＝8.80、8.83，P<0.01)。(4)培养10 d，空白对照组、HGF组、bFGF组、HGF＋bFGF组细胞均无LYVE-1、VEGFR3和整合素α9 mRNA表达。培养10 d，VEGF-C组细胞LYVE-1、VEGFR3的mRNA表达量明显低于VEGF-C＋bFGF组、VEGF-C＋HGF＋bFGF组(t_LYVE-1＝6.22、18.01，t_VEGFR3＝8.49、15.34，P<0.01)，整合素α9的mRNA表达量明显低于VEGF-C＋HGF组、VEGF-C＋HGF＋bFGF组(t＝13.24、9.65，P<0.01)。VEGF-C＋HGF＋bFGF组细胞LYVE-1、VEGFR3和整合素α9的mRNA表达量明显高于VEGF-C＋HGF组、VEGF-C＋bFGF组(t＝13.92、11.95，13.72、5.27，5.64、9.10，P<0.01)。VEGF-C＋HGF组细胞VEGFR3 mRNA表达量明显低于VEGF-C＋bFGF组(t＝6.91，P<0.01)，整合素α9 mRNA表达量明显高于VEGF-C＋bFGF组(t＝11.69，P<0.01)。(5)间隔6、12、24 h培养10 d，空白对照组细胞均无LYVE-1、VEGFR3及整合素α9蛋白表达。间隔6、12、24 h培养10 d，HGF＋VEGF-C＋bFGF组、bFGF＋VEGF-C＋HGF组、VEGF-C＋HGF＋bFGF组细胞LYVE-1、VEGFR3及整合素α9蛋白表达量相近(F_{6 h}＝2.25、2.47、2.19，F_{12 h}＝2.93、1.47、3.25，F_{24 h}＝0.28、0.20、1.01，P>0.05)。结论 VEGF-C是诱导BMSC向LEC分化必需的因子，HGF和bFGF可能分别是通过上调整合素α9和VEGFR3的表达来促进分化的，但二者的诱导作用可能是各自独立的。3种诱导因子联合作用诱导分化效果最佳。
- 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
Abstract: 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-C+ bFGF group, bFGF+ VEGF-C+ HGF 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 HGF+ VEGF-C+ bFGF group were added with 2 mL complete medium, 16 μL HGF of 10 μg/mL, and 10 μL VEGF-C of 10 μg/mL, after 6 hours, 20 μL bFGF of 1 μg/mL was added. Cells in bFGF+ VEGF-C+ HGF group were added with 2 mL complete medium, 20 μL bFGF of 1 μg/mL, and 10 μL VEGF-C of 10 μg/mL, after 6 hours, 16 μL HGF of 10 μg/mL was added. Cells in VEGF-C+ HGF+ bFGF group were simultaneously added with 2 mL complete medium and the same concentration and volume of three inducing factors as above. In addition, BMSCs of rats in another 2 batches were collected and grouped, and they were dealt with the same methods as above except that the interval time of 6 hours in HGF+ VEGF-C+ bFGF group and bFGF+ VEGF-C+ HGF group was adjusted to 12 and 24 hours. On 10 d of culture, protein expressions of LYVE-1, VEGFR3, and integrin α9 were detected by Western blotting. Data were processed with analysis of variance of factorial design, one-way analysis of variance, and least significant difference t test, and Bonferroni correction. Results (1) The positive expression rates of surface antigen of cells in negative control group, CD90 group, CD44 group, and CD34 group were 0.39%, 99.84%, 99.90%, and 0.57%, respectively. (2) On 10 d of culture, cells in blank control group, HGF group, bFGF group, and HGF+ bFGF group presented long fusiform, while cells in the other groups presented polygonal shape. (3) On 10 d of culture, there were no protein expressions of LYVE-1, VEGFR3, and integrin α9 in cells of blank control group, HGF group, bFGF group, and HGF+ bFGF group. On 10 d of culture, protein expressions of LYVE-1, VEGFR3, and integrin α9 in cells of VEGF-C+ HGF+ bFGF group were significantly higher than those in VEGF-C group (t=24.21, 11.04, 15.43, P<0.01), VEGF-C+ HGF group (t=10.81, 9.93, 10.20, P<0.01), and VEGF-C+ bFGF group (t=11.67, 6.32, 19.00, P<0.01). Protein expressions of LYVE-1 in cells of VEGF-C+ HGF group and VEGF-C+ bFGF group were significantly higher than the protein expression in VEGF-C group (t=8.69, 15.20, P<0.01). Protein expression of VEGFR3 in cells of VEGF-C+ bFGF group was obviously higher than the protein expressions in VEGF-C group and VEGF-C+ HGF group (t=8.67, 7.21, P<0.01). Protein expression of integrin α9 in cells of VEGF-C+ HGF group was obviously higher than the protein expressions in VEGF-C group and VEGF-C+ bFGF group (t=8.80, 8.83, P<0.01). (4) On 10 d of culture, there were no mRNA expressions of LYVE-1, VEGFR3, and integrin α9 in cells of blank control group, HGF group, bFGF group, and HGF+ bFGF group. On 10 d of culture, mRNA expressions of LYVE-1 and VEGFR3 in cells of VEGF-C group were significantly lower than those in VEGF-C+ bFGF group and VEGF-C+ HGF+ bFGF group (t_LYVE-1=6.22, 18.01, t_VEGFR3=8.49, 15.34, P<0.01), and mRNA expression of integrin α9 were significantly lower than that in VEGF-C+ HGF group and VEGF-C+ HGF+ bFGF group (t=13.24, 9.65, P<0.01). The mRNA expressions of LYVE-1, VEGFR3, and integrin α9 in cells of VEGF-C+ HGF+ bFGF group were obviously higher than those in VEGF-C+ HGF group and VEGF-C+ bFGF group (t=13.92, 11.95, 13.72, 5.27, 5.64, 9.10, P<0.01). Compared with those of VEGF-C+ bFGF group, the mRNA expression of VEGFR3 of cells in VEGF-C+ HGF group was significantly lower (t=6.91, P<0.01), while the mRNA expression of integrin α9 of cells in VEGF-C+ HGF group was significantly higher (t=11.69, P<0.01). (5) On 10 d of culture at interval time of 6, 12, 24 h, there were no protein expressions of LYVE-1, VEGFR3, or integrin α9 in cells of blank control group. On 10 d of culture at interval time of 6, 12, 24 h, the protein expressions of LYVE-1, VEGFR3, and integrin α9 in cells of HGF+ VEGF-C+ bFGF group, bFGF+ VEGF-C+ HGF group, and VEGF-C+ HGF+ bFGF group were close (F_{6 h}=2.25, 2.47, 2.19, F_{12 h}=2.93, 1.47, 3.25, F_{24 h}=0.28, 0.20, 1.01, P>0.05). Conclusions VEGF-C is a necessary factor for inducing BMSCs to differentiate into LECs. HGF and bFGF may promote the differentiation by up-regulating the expressions of integrin α9 and VEGFR3 respectively. But the induction effects of the two factors may be independent. The combination of VEGF-C, HGF, and bFGF have the best effects of promoting differentiation.
- Mesenchymal stem cells /
- Vascular endothelial growth factor C /
- Hepatocyte growth factor /
- Cell differentiation /
- Basic fibroblast growth factor /
- Lymphatic endothelial cells