Promoting angiogenesis by con-transplantation of bone marrow mesenchymal stem cells and human umbilical vein endothelial cells transfected with vascular endothelial growth factor 165 gene

doi:10.3969/j.issn.2095-4344.0409

Abstract

Abstract:

BACKGROUND: How to promote the early vascularization of large tissue-engineered bone has become the hotspot of current research. Cell co-culture and the addition of bioactive factors to promote angiogenesis are very good methods to promote early vascularization.
OBJECTIVE: To explore the ability of angiogenesis by co-transplantation of bone marrow mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) which were transfected with vascular endothelial growth factor 165 (VEGF165) gene in vivo, to move forward a single step to offer theoretical basis and experimental basis to build vascularized tissue-engineered bone which can be used to repair large segmental bone defects.
METHODS: We built an ischemic skin flap with 4 cm×1.5 cm in the back of Sprague-Dawley rats, and then BMSCs+VEGF165-transfected HUVECs (group A), VEGF165-transfected HUVECs (group B), BMSCs+non-transfected HUVECs (group C), non-transfected HUVECs (group D), DMEM (group E) were respectively transplanted. ELISA method was used to detect peripheral blood VEGF level. Histologically, survival and microvessel density of the flap were observed.
RESULTS AND CONCLUSION: (1) The flap survival quality of group A was better than that in the other groups. VEGF exhibited high expression continuously high expression at 2, 4, 7, 14 days after transplantation, and reached the peak at 7 days, but the expression level at 14 days was obviously lower than that at 2 days postoperatively. The VEGF level of group always exceeded that in group B at different time points (P < 0.05). The flap survival rate and microvessel density of group A was significantly higher than that in the other groups at 11 days postoperatively (both P < 0.05). In summary, co-transplantation of BMSCs and VEGF165-transfected HUVECs can promote survival of an ischemic flap in vivo through pro-angiogenic actions.

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, Tissue Engineering

CLC Number:

R394.2

Xian De-bin, Ming Hua-wei, Xu Rong-sheng, Xia De-lin. Promoting angiogenesis by con-transplantation of bone marrow mesenchymal stem cells and human umbilical vein endothelial cells transfected with vascular endothelial growth factor 165 gene[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(1): 46-52.

Figures/Tables 2

2.1 实验动物数量分析 50只SD大鼠均进入结果分析，中途无脱落。 2.2 大体观察结果见图3。术后第1天：移植细胞4组大鼠背部缺血皮瓣水肿范围较空白对照组小，水肿程度也较空白对照组轻，各组皮瓣均未出现花斑状瘀斑。术后第2天：各组大鼠背部皮瓣开始出现花斑状瘀斑，而移植细胞4组瘀斑明显少于空白对照组。术后第3天：各组大鼠背部缺血皮瓣远蒂端开始发暗变黑，移植细胞4组变黑面积较空白对照组小。术后第4天至第7天：各组大鼠皮瓣发暗变黑部位开始变硬并逐渐收缩，开始转变为黑色硬痂壳。少部分缝线出现松动、脱落现象，空白对照组4只大鼠切口裂开，伤口周围红肿。术后第8天至第11天：各组皮瓣存活区域与坏死区域已明显分界，存活区域皮瓣毛发生长旺盛，去除毛发后见皮肤红润，质地柔软无收缩。坏死区域皮肤毛发停止生长，色泽变暗变黑、皮肤明显皱缩、质地较硬、弹性极差、切割组织后不出血。处死大鼠后取材时发现空白对照组有3只大鼠皮瓣基底部积液(暗红色血性液体)，移植细胞4组皮瓣基底部未见积液；BMSCs+VEGF165-HUVEC组皮瓣成活质量明显较其他组高。 2.3 皮瓣存活率术后第11天，各组大鼠背部皮瓣存活率分别为：BMSCs+VEGF165-HUVEC组(86.4±5.5)%，VEGF165-HUVEC组(62.5±4.7)%，BMSCs+HUVEC组(52.3±4.2)%，HUVEC组(45.8±3.6)%，空白对照组(32.4±1.7)%。除VEGF165-HUVEC组和BMSCs+HUVEC组差异无显著性意义，其余各组之间比较差异有显著性意义(P < 0.05)，见图4，5。 2.4 皮瓣术后各组大鼠外周血血清中VEGF蛋白表达水平 BMSCs+VEGF165-HUVEC组、VEGF165-HUVEC组大鼠术后第2，4，7，14天外周血中VEGF持续高表达且逐渐升高，第7天时达到峰值，第14天时较术后第2天明显下降。不同时期BMSCs+VEGF165-HUVEC组外周血中VEGF水平明显高于VEGF165-HUVEC组，差异有显著性意义(P < 0.05)。BMSCs+HUVEC组、HUVEC组、空白对照组各个时期血清中VEGF水平无明显变化，差异无显著性意义(P > 0.05)。不同时期BMSCs+VEGF165-HUVEC组外周血中VEGF水平明显高于BMSCs+HUVEC组、HUVEC组、空白对照组，差异有显著性意义(P < 0.05)，见表1，图6。 2.5 组织学检测结果术后第11天，各组大鼠皮瓣组织苏木精-伊红染色及内皮细胞CD31免疫组织化学染色见图7，8。5组微血管密度计数(条/mm2)分别为：BMSCs+ VEGF165-HUVEC组63.4±5.7，VEGF165-HUVEC组56.2± 5.2，BMSCs+HUVEC组42.5±3.4，HUVEC组28.3±2.8，空白对照组19.7±1.3。BMSCs+ VEGF165-HUVEC组、BMSCs+HUVEC组、HUVEC组、空白对照组微血管密度计数比较差异有显著性意义(P < 0.05)；BMSCs+ VEGF165-HUVEC组、VEGF165- HUVEC组、HUVEC组、空白对照组微血管密度计数比较，差异有显著性意义(P < 0.05)。BMSCs+VEGF 165-HUVEC组中毛细血管密度远大于其他4组。"

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