[The effect of lipopolysaccharide on the expression and activity of Toll-like receptor 4 in mesenchymal stem cells].
OBJECTIVE
To investigate the effect of lipopolysaccharide(LPS) on the expression and activity of Toll-like receptor 4(TLR-4) in mesenchymal stem cells (MSC).


METHODS
MSCs were harvested from adult rats bone marrow cells by density gradient centrifugation and adhesive culture. The purity of MSC were identified with the cell morphology and osteogenic capacity. The phenotypes were assayed by flow cytometry. Cultured MSCs were treated by LPS with various concentration (1 microg/ml, 10 microg/ml or 100 microg/ml) for 24 hours. The relative expression levels of TLR-4 mRNA were detected by semiquantitative RT-PCR, and costimulatory molecules (CD80, CD86 and MHC-II) expressed on MSC were analyzed by flow cytometry. The levels of TNF-alpha in supernatants were determined by double-antibody sandwich ELISA.


RESULTS
The expression levels of CD80, CD86, MHC-II, TLR-4 mRNA and TNF-alpha in MSC were (9.56 +/- 0.69)%, (22.03 +/- 2.03)%, (2.51 +/- 0.97)%, relative magnitude (0.61 +/- 0.10), (4.97 +/- 2.98) pg/ml, respectively. After incubation with LPS, MSC expressed higher levels of TLR-4 mRNA and costimulatory molecules, and the levels of TNF-alpha were higher than that in untreated group. Among the various concentration of LPS, 10 microg/ml emerged as the most effective in increasing the levels of TLR-4 mRNA (relative magnitude 1.55 +/- 0.02), costimulatory molecules [CD80 (41.70 +/- 2.92)%, CD86 (59.72 +/- 2.00)%, MHC-II (24.56 +/- 2.19)%] and TNF-alpha [(213.12 +/- 69.08) pg/ml] (P < 0.01). The levels of TLR-4 mRNA, costimulatory molecules and TNF-alpha began to decrease when MSC exposed to 100 microg/ml LPS (P < 0.05). Except for the levels of TNF-alpha [(158.05 +/- 28. 05) pg/ml] and MHC-II [(5.62 +/- 2.31)%] (P > 0.05), the levels of CD80, CD86, MHC-II and TLR-4 mRNA were significantly lower than the 10 microg/ml treatment group (P < 0.01).


CONCLUSIONS
MSCs are able to express TLR-4 mRNA. LPS could activate the expression of TLR-4 in MSC obviously, but the activity is dependent on the specific concentration.
Shi
Liang
L
Department of Hematology, Affiliated Hospital of Guiyang Medical College, Guiyang 550004, China.
Liu
Xing-mei
XM
Hu
Xiao-yan
XY
Wang
Ji-shi
JS
Fang
Qin
Q
D000818:Animals; D018122:B7-1 Antigen / Q000378:metabolism; D051940:B7-2 Antigen / Q000378:metabolism; D002478:Cells, Cultured; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D018122:B7-1 Antigen; D051940:B7-2 Antigen; D008070:Lipopolysaccharides; D012333:RNA, Messenger; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha