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

    Next Articles

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

PDF (PC)

250

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
[1] Ullah I, Subbarao RB, Rho GJ. Human mesenchymal stem cells-current trends and future prospective[J]. Biosci Rep, 2015, 35(2):1-18.
[2] 张乐玲, 李府, 吴镇, 等. 再生障碍性贫血患儿骨髓间充质干细胞免疫调节特性和造血支持作用的研究[J]. 中国小儿血液与肿瘤杂志, 2008, 13(4): 145-148.
[3] Wu N, Zhang YL, Wang HT, et al. Overexpression of hepatocyte nuclear factor 4α in human mesenchymal stem cells suppresses hepatocellular carcinoma development through Wnt/β-catenin signaling pathway downregulation[J]. Cancer Biol Ther, 2016, 17(5):558-565.
[4] Davis S, Aldrich TH, Jones PF, et al. Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning[J]. Cell, 1996, 87(7):1161-1169.
[5] Zheng X, Ohsaki E, Ueda K. Mechanism of angiopoietin-1 upregulation in Kaposis sarcoma-associated herpesvirus-infected PEL cell lines[J]. J Virol, 2015, 89(9):4786-4797.
[6] Linares PM, Chaparro M, Gisbert JP. Angiopoietins in inflammation and their implication in the development of inflammatory bowel disease[J]. J Crohns Colitis, 2014, 8(3):183-190.
[7] Cheung AH, Stewart RJ, Marsden PA. Endothelial Tie2/Tek ligands angiopoietin-1(ANGPT1)and angiopoietin-2(ANGPT2): regional localization of the human genes to 8q22.3-q23 and 8p23[J]. Genomics, 1998, 48(3):389-391.
[8] Huang YQ, Li JJ, Karpatkin S. Identification of a family of alternatively spliced mRNA species of angiopoietin-1[J]. Blood, 2000, 95(6):1993-1999.
[9] Mei SH, McCarter SD, Deng Y, et al. Prevention of LPS-induced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin 1[J]. PLoS Med, 2007, 4(9):1525-1537.
[10] Xu J, Qu J, Cao L, et al. Mesenchymal stem cell-based angiopoietin-1 gene therapy for acute lung injury induced by lipopolysaccharide in mice[J]. J Pathol, 2008, 214(4):472-481.
[11] Li Y, Zheng L, Xu X, et al. Mesenchymal stem cells modified with angiopoietin-1 gene promote wound healing[J]. Stem Cell Res Ther, 2013, 4(5):113-123.
[12] 刘小盾, 刘东, 臧传宝, 等. IFN-γ增强人脐带间充质干细胞的免疫抑制能力[J].中国实验血液学杂志, 2014, 229(3): 605-611. LIU Xiaodun, LIU Dong, ZANG Chuanbao,et al. IFN-γ stimulation enhances immunosuppressive capability of human umbilical cord mesenchymal stem cells[J]. Journal of Experimental Hematology, 2014, 22(3): 605-611.
[13] Huang B, Tabata Y, Gao JQ. Mesenchymal stem cells as therapeutic agents and potential targeted gene delivery vehicle for brain diseases[J]. J Control Release, 2012, 162(2):464-473.
[14] Qi H, Chen G, Huang Y, et al. Foxp3-modified bone marrow mesenchymal stem cells promotes liver allograft tolerance through the generation of regulatory T cells in rats[J]. J Transl Med, 2015, 13: 274-286. doi: 10.1186/s12967-015-0638-2.
[15] Koippallil Gopalakrishnan AR, Pandit H, Metkari SM, et al. Adenoviral vector encoding soluble Flt-1 engineered human endometrial mesenchymal stem cells effectively regress endometriotic lesions in NOD/SCID mice[J]. Gene Ther, 2016, 23(7): 580-591.
[16] Li J, Li D, Liu X, et al. Human umbilical cord mesenchymal stem cells reduce systemic inflammation and attenuate LPS-induced acute lung injury in rats[J]. J Inflamm(Lond), 2012, 9(1):33-43.
[17] De Groote P, Grootjans S, Lippens S, et al. Generation of a new Gateway-compatible inducible lentiviral vector platform allowing easy derivation of co-transduced cells[J]. Biotechniques, 2016, 60(5): 252-259.
[1] SHANG Xiao1, CUI Yuan-xiao1, LIU Xiao-yu1, FENG Xiao-ya2. Experimental study of human umbilical cord mesenchymal stem cells combined with minimal hematoma aspiration in the therapy of rats with intracerebral hemorrhage [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2013, 51(10): 10-14.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Copyright 2018 © Editorial office of Journal of Shandong University (Health Sciences)
Supported by Beijing Magtech Co.Ltd