Experimental study of recombinant adenoviral-mediated PDGF gene delivery of human umbilical cord derived mesenchymal stem cells

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Author:
DONG Chun-lan(Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College; State Key Laboratory of Experimental Hematology, Tianjin 300020, China)
GE Jing(Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College; State Key Laboratory of Experimental Hematology, Tianjin 300020, China)
LIANG Lu(Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College; State Key Laboratory of Experimental Hematology, Tianjin 300020, China)
CHEN Xiao-jun(Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College; State Key Laboratory of Experimental Hematology, Tianjin 300020, China)
XUE Feng(Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College; State Key Laboratory of Experimental Hematology, Tianjin 300020, China)
HAN Zhong-chao(Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College; State Key Laboratory of Experimental Hematology, Tianjin 300020, China)
Journal Title:
JOURNAL OF LEUKEMIA & LYMPHOMA
Issue:
Volume 20, Issue 06, 2011
DOI:
10.3760/cma.j.issn.1009-9921.2011.06.002
Key Word:
Adenoviridae;Mesenchymal stem cells;Platelet-derived growth factor Gene therapy

Abstract: Objective To investigate the infect capability of recombinant adenovirus mediated PDGF to human umbilical cord derived mesenchymal stem cells (MSCs). Methods The PDGF cDNA sequence was amplified with RT-PCR and constructed recombinant adenovirus vector AdPDGF. The human umbilical cord derived MSCs were isolated and cultured. In vitro AdPDGF infected MSCs with various MOI,and determine the efficiency using FACS and fluorescence microscope. Trypan blue and MTT assay was used to detect cell viability and proliferation. ELISA was used to quantify PDGF secretion. Results Recombinant adenovirus AdPDGF was successfully and constructed,in which infection efficiency to MSCs was dose dependent. The highest efficiency was around 87.36 % when MOI =50,at which cell viability and proliferation wasn' t impaired. Cell viability of uninfected,AdPDGF infected and control virus infected MSCs was (97.8 ±2.3) %,(91.9±4.0) % and (92.8±4.0) %,separately. The cell proliferation was (100±16.8) %,(95.9±12.0) % and (87.5±9.7) %,separately. There was no statistic significance among AdPDGF infected,control virus infected and uninfected MSCs. 48 hrs post infection,PDGF secretion can be measured from infectious MSCs' supernatant. The secretion level was (1.53±0.37),(3.03±0.68) and (5.25±0.92) ng/ml,separately,when MOI= 10,30,50. No PDGF can be detected from MSC-GFP and control MSC group. Conclusion Recombinant adenovirus AdPDGF was constructed and can infect human umbilical cord derived MSC at high efficiency.

  • [1]Charbord P.Bone marrow mesenchymal stem cells:historical overview and concepts.Hum Gene Ther,2010,21:1045-1056.
  • [2]唐晶晶,林艳娟.间充质干细胞在移植中的作用.白血病·淋巴瘤,2009,18:373-376.
  • [3]Kinnaird T,Stabile E,Burnett MS,et al.Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms.Circ Res,2004,94:678-685.
  • [4]Luo J,Deng ZL,Luo X,et al.A protocol for rapid generation of recombinant adenoviruses using the AdEasy system.Nat Protoc,2007,2:1236-1247.
  • [5]Lu LL,Liu YJ,Yang SG,et al.Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials.Haematologica,2006,91:1017-1026.
  • [6]Song L,Chau L,Sakamato Y,et al.Electric field-induced molecular vibration for noninvasive,high-efficiency DNA transfection.Mol Ther,2004,9:607-616.
  • [7]Bergelson JM,Cunningham JA,Droguett G,et al.Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5.Science,1997,275:1320-1323.
  • [8]Conget PA,Minguell JJ.Adenoviral-mediated gene transfer into ex vivo expanded human bone marrow mesenchymal progenitor cells.Exp Hematol,2000,28:382-390.
  • [9]Hung SC,Lu CY,Shyue SK,et al.Lineage differentiation-associated loss of adenoviral susceptibility and Coxsackie-aHenovirus receptor expression in human mesenchymal stem cells.Stem Cells,2004,22:1321-1329.
  • [10]Tanii M,Yonemitsu Y,Fujii T,et al.Diabetic microangiopathy in ischemic limb is a disease of disturbance of the platelet-derived growth factor-BB/protein kinase C axis but not of impaired expression of angiogenic factors.Circ Res,2006,98:55-62.
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