The Changes Of Gene Expression Of Toll Like Receptors And The Antioxidant Capacitiesin Long-term Cultured Human Umbilical Cord Mesenchymal Stem Cells

AIM：To investigate the changes of gene expressions of TLRs and theantioxidant capacities which occur in human umbilical cord mesenchymalstem cells （hUC-MSCs） during long-term culture in vitro, and to obtain theexperimental parameters and theory bases for the proliferation,differentiation,migration, damages repairing and immunoregulation of hUC-MSCs.METHODS：Human umbilical cords from neonates via caesarean sectionwere collected, hUC-MSCs were isolated and purified by collagenaseⅡdigestion and adherence screening methods, and then hUC-MSCs weresubcultivated. The3rdand28thpassages cells surface antigens wereidentified by flow cytometry, adipogenic and osteogenic ability weredetermined by specially inducing culture. The oxygen radical absorbancecapacities in different passages of hUC-MSCs were detected by ORAC.TLR1¹0mRNA levels in the3rdand28thpassages of hUC-MSCs wereevaluated by RT-PCR. Gene expressions of TLR9in3/8/13/18/23/28passagesof hUC-MSCs were investigated by qRT-PCR.RESULTS: The3rdand28thpassages of hUC-MSCs phenotypes wereCD₂₉⁺/CD₄₄⁺/CD₁₀₅⁺/CD₃₁^-/CD₃₄^-/CD₄₀^-/CD₄₅^-/CD₁₀₆^-/HLA-DR^-, and couldbe induced toward to osteoblast and adipocyte differentiation in appropriateinduction conditions. The oxygen radical absorbance capacities ofhUC-MSCs reduced gradually along with the passages, the8^th passage cellsis highest with values of4.97μmol Trolox/ml. When compared the28^thpassage with the8^th passage of MSCs, the oxygen radical absorbancecapacities decreased29.3%, the differences were statistically significant（P<0.01）. All of TLR1¹0mRNA were found in the3^rd passage ofhUC-MSCs, with high expression levels of TLR1/TLR2/TLR3/TLR6/TLR9 and low expression levels of TLR3/TLR4/TLR7/TLR8/TLR10. Whencompared the28^th passage with the3^rd passage of MSCs, the mRNAexpression of TLR1/2/3/4/6/9decreased and TLR5/7/8/10were not detected.qRT-PCR results showed that the mRNA expressions of TLR9reduced alongwith the passages. Conpared with those of the3^rd passage, the mRNAexpressions of the18^th,23^th and28^th passages decreased by89.5%,95.2%and89%, respectively. The differences were statistically significant（P<0.01）.CONCLUSION: hUC-MSCs maintained the basic biologicalcharacteristics after long-term culture in vitro. The oxygen radicalabsorbance capacities of hUC-MSCs reduced gradually along with thepassages. The TLR1¹0mRNA expression of hUC-MSCs reduced or notdetected gradually along with the passages. The TLR9mRNA expressionreduced gradually along with the passages suggested that long-term culturemight have no effect on the proliferation, differentiation, migration, damagesrepairing and immunoregulation of hUC-MSCs which remains to be furtherconfirmed. The gene expressions of TLRs in hUC-MSCs changed along withthe passages whether suggested their related functions changed whichremains to be further confirmed.