JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES) ›› 2016, Vol. 54 ›› Issue (12): 1-7.doi: 10.6040/j.issn.1671-7554.0.2016.730

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Construction and identification of lentiviral vector for angiopoietin-1 gene and its expression in human umbilical cord mesenchymal stem cells

LIU Ning1, L(¨overU)Xin1, LI Dong2, HUNAG Zhiwei3, LIU Yi1, ZHANG Leling3   

  1. 1. Institute of Pediatric Research, Qilu Childrens Hospital of Shandong University, Jinan 250022, Shandong, China;
    2. Cryomedicine Laboratory, Qilu Hospital of Shandong University, Jinan 250012, Shandong, China;
    3. Department of Hematologyoncology, Qilu Childrens Hospital of Shandong University, Jinan 250022, Shandong, China
  • Received:2016-06-22 Online:2016-12-10 Published:2016-12-10

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Abstract: Objective To construct the lentivirus vector carrying angiopoietin-1 gene(Ang-1), and to investigate its expression in human umbilical cord mesenchymal stem cells(hUC-MSCs)and the immunomodulatory effect on Ang-1-modified hUC-MSCs. Methods The cDNA encoding full-length human Ang-1 was amplified by RT-PCR from the total RNA of hUC-MSCs, and then subcloned to lentiviral vector backbone plasmid GV287. The recombinant plasmid GV287-Ang-1 was co-transfected with packaging and enveloping plasmids pHelper 1.0 and pHelper 2.0 into 293T cells. Vector titer was quantified using cell fluorescence. The transfection was detected by fluorescence microscopy, and protein expression of Ang-1 was detected by Western blotting. T lymphocytes proliferation was assessed by cell counting kit 8(CCK8). Results The recombinant lentiviral vector GV287-Ang-1 was verified correctly by PCR and DNA sequencing. The titer of concentrated virus was 2×108 TU/mL, and the optimal MOI for transfecting hUC-MSCs was 8. Western blotting results showed that the Ang-1 expression of the GV287-Ang-1 transfected group was significantly higher 山 东 大 学 学 报 (医 学 版)54卷12期 -刘宁,等.人血管生成素1基因慢病毒表达载体的构建及其在脐带间充质干细胞的表达 \=-than those of the non-transfected group and GV287 group. The suppression ratio of T lymphocytes in Ang-1-UC-MSCs group was significantly increased as compared to that in the UC-MSCs group. Conclusion The lentivirus vector carrying human Ang-1 was constructed successfully and overexpressed effectively in hUC-MSCs. Ang-1-modified hUC-MSCs can enhance the immunosuppressive capability, which suggests a potential clinical application.

Key words: Angiopoietin-1, 293T cells, Lentiviral vector, Overexpression, Human umbilical cord mesenchymal stem cells

CLC Number: 

  • Q78
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