Transfection with vascular endothelial growth factor 165, neurotrophin 3 and angiopoietin 1 induces the differentiation of bone marrow mesenchymal stem cells into neurons and vascular endothelial cells

doi:10.3969/j.issn.2095-4344.0909

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are a kind of multifunctional mesenchymal stem cells that have multi-directional differentiation potential. These cells can be genetically modified by methods such as adenovirus transfection, to produce a variety of growth factors such as neurotrophin-3 (NT-3), angiopoietin-1 (Ang-1), and vascular endothelial growth factor (VEGF). BMSCs have been applied as seed cells in tissue engineering research.
OBJECTIVE: To transfect VEGF165, NT-3 and Ang-1 gene into rat BMSCs and to induce them to differentiate into neuronal cells and vascular endothelial cells.
METHODS: BMSCs were isolated using differentia velocity adherent method and identified by flow cytometry. Differentiation potential of BMSCs into osteogenesis and adipogenesis was also identified. We constructed two adenoviral vectors with cytomegalovirus (CMV) as a promoter. One of the two vectors was a bicistronic vector (Adv-Bic), carrying hVEGF165 and Ang-1 gene; the other vector was an AdvNT-3 vector, carrying NT-3 gene. Adv-Bic (green fluorescence) and AdvNT-3 (red fluorescence) were simultaneously transferred into rat BMSCs at various multiplicities of infection (MOIs). Two days after the transfection, the optimal MOI value was determined based on the expression of green and red fluorescent proteins under a fluorescence microscope. Then, well-grown BMSCs at passage 3 were divided into two groups. The experimental group was transfected with Adv-Bic (without fluorescence) and AdvNT-3 (without fluorescence) at the optimal MOI. The control group was transfected with blank control virus at the same MOI. The two groups were cultured in vitro for 7 days and then were detected with immunofluorescence and qPCR.
RESULTS AND CONCLUSION: The expression of CD29 and CD44, which are identifications of BMSCs, was positive, while the expression of CD34 and CD45, which are identifications of hematopoietic stem cells, was negative. BMSCs could be differentiated into osteoblasts and adipocytes. After BMSCs were transfected with adenovirus for 48 hours, the expression of green and red fluorescent proteins was observed under a fluorescence microscope. With the increase of MOIs, the intensity of fluorescence gradually increased, and peaked when MOI was equal to 100. Immunofluorescence and qPCR results showed that the expression of VEGF165, NT-3 and Ang-1 in the experimental group was higher than that in the control group. The expression level of CD31 and CD34, the markers of vascular endothelium, in the experimental group was higher than that in the control group. The expression level of neuron-specific markers NSE and Nestin in the experimental group was higher than that in the control group. These results suggested that VEGF165, NT-3 and Ang-1 genes were successfully transfected into BMSCs. This transfection made BMSCs overexpress growth factors of VEGF165, NT-3 and Ang-1, which could induce BMSCs to differentiate into neuronal cells and vascular endothelial cells.

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, Vascular Endothelial Growth Factors, Neurotrophin 3, Angiopoietin-1, Tissue Engineering

CLC Number:

R394.2

Jiang Xing-hai, Zhao Biao, Wu Kai, Xiao Ren-shun, Wang Xiao-mei. Transfection with vascular endothelial growth factor 165, neurotrophin 3 and angiopoietin 1 induces the differentiation of bone marrow mesenchymal stem cells into neurons and vascular endothelial cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 3956-3962.

Figures/Tables 2

2.1 大鼠骨髓间充质干细胞形态及鉴定结果细胞接种培养48 h后，倒置显微镜下可见少量梭形细胞开始贴壁生长，接种第3天显微镜下观察可见由大量长梭形细胞组成的多个细胞集落散在分布于培养皿底(图1A)，培养7 d后细胞大量增殖并融合，呈梭形、多角形或扁平形(图1B)。传至第2，3代的单个细胞形态呈典型的长梭形或纺锤状，融合后呈放射状或漩涡状排列(图1C，D)。大鼠骨髓间充质干细胞免疫表型鉴定：见图2。流式细胞术检测结果显示间充质干细胞表面分子CD29、CD44阳性表达率为97.1%，99.8%(图2C，D)，造血干细胞表面分子CD34、CD45阳性表达率为1.61%，1.71%(图2E，F)。大鼠骨髓间充质干细胞成骨、成脂诱导分化能力：骨髓间充质干细胞在成骨诱导2周时进行茜素红染色，倒置显微镜下可观察到大量钙结节(图3A)；成脂诱导2周时进行油红O染色，倒置显微镜下可观察到细胞出现脂滴(图3B)。 2.2 病毒转染最佳MOI值以不同MOI值将Adv-Bic(绿色荧光)及AdvNT-3(红色荧光)同时转染大鼠骨髓间充质干细胞，培养2 d后，荧光显微镜下观察。随着MOI值增高，绿色及红色荧光表达量均相应增多，当MOI=100时两种荧光到达高峰(图4A-H)。MOI为300时部分细胞死亡，MOI为1 000时大量细胞死亡。因此，MOI=100时能达到最佳的转染效果。 2.3 免疫荧光检测结果转染VEGF165、NT-3、Ang-1基因的骨髓间充质干细胞在体外培养7 d后，通过免疫荧光检测VEGF165、NT-3、Ang-1均阳性表达(图5A-C)。血管内皮标记物CD31，CD34均阳性表达(图5D，E，H)；神经元相关标记物NSE、Nestin均阳性表达(图5F-H)。 2.4 实时荧光定量PCR(qRT-PCR)检测结果实验组中转染的目的基因VEGF165、NT-3、Ang-1表达水平均明显高于对照组(P < 0.05)；实验组血管内皮细胞相关基因CD31、CD34表达水平明显高于对照组(P < 0.05)；实验组神经元相关基因NSE、Nestin表达水平明显高于对照组(P < 0.05)，见图6。"

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