Co-transplantation of bone marrow mesenchymal stem cells and human umbilical vein endothelial cells transfected with vascular endothelial growth factor 165 gene promotes vascularization

doi:10.3969/j.issn.2095-4344.2017.33.004

Abstract

Abstract:

BACKGROUND: Vascularized bone tissue engineering is a hotspot of current research and early vascularization of tissue-engineered bone also becomes an urgent problem to be solved. Addition of bioactive factors and cell co-culture method both contribute to promoting the early vascularization.
OBJECTIVE: To investigate the pro-angiogenic effect of human umbilical vein endothelial cells (HUVECs) transfected with vascular endothelial growth factor 165 (VEGF165) co-cultured with bone marrow mesenchymal stem cells (BMSCs).
METHODS: HUVECs were transfected with VEGF165 and then co-cultured with BMSCs which were purified from mouse femoral bone marrow. There were six groups in this experiment: (1) AdVEGF165-HUVECs+BMSCs, (2) AdVEGF165-HUVECs, (3) AdGFP-HUVECs+BMSCs, (4) AdGFP-HUVECs, (5) HUVECs+BMSCs, and (6) HUVECs (blank control). Cell counting kit-8 detection was applied to analyze the proliferative ability of HUVECs. The abilities of HUVECs migration and vascularization were then detected by crystal violet staining and matrigel determination, respectively.
RESULTS AND CONCLUSION: Compared with the blank control group, the proliferation of HUVECs was significantly increased in the other groups except for AdGFP-HUVECs group (P < 0.05). Compared with the blank control group, the migration and vascularization abilities of HUVECs were significantly stronger in the group of AdVEGF-HUVECs+BMSCs (P < 0.05). To conclude, the co-culture of HUVECs transfected with VEGF165 and BMSCs could promote early vascularization effectively.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Umbilical Veins, Endothelial Cells, Vascular Endothelial Growth Factors, Transfection, Coculture Techniques, Tissue Engineering

CLC Number:

R394.2

Xu Rong-sheng, He Yun, Li Biao, Xian De-bin, Xia De-lin. Co-transplantation of bone marrow mesenchymal stem cells and human umbilical vein endothelial cells transfected with vascular endothelial growth factor 165 gene promotes vascularization[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(33): 5268-5273.

Figures/Tables 1

2.1 腺病毒转染结果及细胞生长曲线荧光显微镜下可见pHBAd-MCMV-GFP-VEGF165-HUVECs组及pHBAd- MCMV-GFP-HUVECs组大部分细胞显示绿色荧光(图1)。 Ad-VEGF-HUVECs、Ad-GFP-HUVECs及HUVECs在不同时间点的增殖情况见图2，腺病毒转染后细胞数量逐渐增多，与空白对照组相比，无论是Ad-GFP还是Ad-VEGF，腺病毒转染组增殖数目差异无显著意义(P > 0.05)。 2.2 BMSCs形态接种于培养皿后，镜下可见大量圆形大小不一的细胞悬浮于培养液中，24 h后细胞贴壁，呈多边形、梭形，培养14 d后细胞排列整齐，融合成片生长(图3A)。将细胞传代培养，可见细胞形态基本一致，呈梭形(图3B)。 2.3 HUVECs/BMSCs共培养后HUVECs增殖能力变化随着时间的增加，各组细胞吸光度值均增加，与空白对照组相比，转染共培养组、转染非共培养组、空载体共培养组和未转染共培养组的吸光度值在各时间点均明显增加 (P < 0.05)。其中转染共培养组吸光度值增加最为明显，其他3组次之。而空载体组与空白对照组相比，在各时间点吸光度值差异无显著性意义(P > 0.05)，见图4。 2.4 HUVECs/BMSCs共培养后HUVECs迁移能力变化转染共培养组HUVECs数量最多，其他3组次之，与空白对照组相比差异有显著性意义(P < 0.05)，而空载体组与空白对照组相比差异无显著性意义(P > 0.05)，见图5。与空载体共培养组和未转染共培养组相比，转染共培养组HUVECs迁移能力均明显增强，差异有显著性意义(P < 0.05)。 2.5 HUVECs/BMSCs共培养后HUVECs成管能力变化 Matrigel基质中，3 h左右开始逐渐形成管腔结构，在培养18 h可见Matrigel基质中管腔样结构基本形成，之后管腔结构逐渐解聚，36-48 h后管腔样结构基本消失。转染共培养组比空白对照组形成的小管数目更多形成时间更早，更稳定(P < 0.05)。转染非共培养组、空载体共培养组和未转染共培养组次之。空载体组与空白对照组相比小管数量差异无显著性意义(P > 0.05)，见图6。"

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