[Enhanced green fluorescence protein reporter gene labeling of mesenchymal stem cells mediated by lentivirus].
OBJECTIVE
To explore the effect of enhanced green fluorescence protein (EGFP) labeling mediated by lentivirus on the biophysical properties of mesenchymal stem cells (MSC), and whether the EGFP gene expression is permanent and stable.


METHODS
MSC were infected with EGFP lentivirus at different virus multiplicity of infection (MOI). EGFP positive rate was measured with fluorescent-activated cell scanning (FACS) analysis, and EGFP expression in MSC was investigated under a fluorescence microscope. Cell viability, proliferation, apoptosis and cell cycle were detected with trypan blue stain, MTT colorimetric assay, Hoechst stain and FACS analysis respectively. To evaluate the stability of EGFP expression, EGFP lentivirus infected MSC were harvested after cultured continuously in vitro for 2, 4, 8 or 16 weeks, and EGFP positive rate and fluorescence strength were detected with FACS analysis.


RESULTS
After infected with EGFP lentivirus (MOI = 20) for 96 h, EGFP positive rate of MSC was 97.39% +/- 0.68%. Cell viability, proliferation, apoptosis and cell cycle of MSC infected with EGFP lentivirus were unaffected, as compared with control MSC (P > 0.05). When cultured in vitro continuously for 2, 4, 8 or 16 weeks, EGFP positive rates of EGFP-MSC were 97.50% +/- 0.54%, 97.32% +/- 0.51%, 97.39% +/- 0.11%, and 97.48% +/- 0.13% respectively, while EGFP fluorescence strength were 440 +/- 13, 445 +/- 12, 458 +/- 13 and 456 +/- 16 respectively. Both EGFP positive rate and fluorescence strength kept in a stable level.


CONCLUSIONS
EGFP lentivirus can efficiently label MSC and has no significant effect on the biophysical properties of MSC. EGFP gene expression in MSC is permanent and stable. EGFP-MSC can be used for further cell tracing research.
Li
Dan
D
Laboratory of Molecular Imaging, Department of Radiology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China.
Zhu
Kang-shun
KS
Zhou
Bin
B
Wang
Jin
J
Yan
Rong-hua
RH
Li
Zheng-ran
ZR
Meng
Xiao-chun
XC
Huang
Ming-sheng
MS
Jiang
Zai-bo
ZB
Shan
Hong
H
D000818:Animals; D015415:Biomarkers; D002478:Cells, Cultured; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D015415:Biomarkers; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins