ABSTRACT
In the application of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) as clinical therapeutics, long term cells ex vivo expansion results in decline in their function. It has been widely concerned that cellular senescence is associated with UC-MSCs immunomodulatory ability. In this study, we evaluated the effects of consecutive passages on cellular senescence and the immunomodulatory abilities of UC-MSCs. Long term-cultured UC-MSCs showed decreased proliferation, senescence phenotypes and impaired immunosuppressive effects on PHA induced peripheral blood mononuclear cell (PBMC) proliferation. We found that Nrf2, a transcription factor that responds to oxidative stress, that showed decreased expression in long term-cultured UC-MSCs, and the further knock-down of Nrf2 in UC-MSCs induced premature senescence, decreased proliferation ability and immunosuppressive abilities. Furthermore, the protein expression of IDO-1 were decreased in response to the downregulation of Nrf2 in UC-MSCs, suggesting that Nrf2 regulates the immunosuppressive properties of UC-MSCs via Nrf2-mediated IDO-1 expression. In conclusion, our results demonstrate that Nrf2 plays a key role in the regulation of the immunosuppressive properties of UC-MSCs, and we suggest that these findings might provide a strategy to enhance the functionality of UC-MSCs for use in therapeutic applications.
Subject(s)
Cellular Senescence , Immunosuppression Therapy , Mesenchymal Stem Cells/cytology , NF-E2-Related Factor 2/metabolism , Umbilical Cord/cytology , Cell Culture Techniques , Cell Proliferation , Cells, Cultured , Down-Regulation , Gene Knockdown Techniques , Humans , PhenotypeABSTRACT
Objective To construct the p300 specific siRNA vector in mice and observe its effect on cardiac transcription factor,GATA4,for further study of the effect of p300-mediated histone acetylation on heart growth. Methods Three recombinant plasmid vectors ( p300 RNAi1,p300 RNAi2,and p300 RNAi3s) ,designed and constructed according to the conservative sequence of mice p300,were respectively tansfected into in vitro cultured cardiac muscle cells of suckling mice. Expression of p300 and GATA4 at mRNA and protein levels was detected by RT-PCR and immunofluorescence,respectively. Results The expression level of p300 mRNA was not significantly decreased in pSOS-HUS and p300 RNAi2 groups,but significantly decreased in p300 RNAi1 and p300 RNAi3 groups ( 0. 220 8 ? 0. 020 0 and 0. 170 8 ? 0. 040 0 vs 0. 509 5 ? 0. 030 0,P
ABSTRACT
Objective To detect the expression of nesprin-1 gene mRNA and protein and to localize the sub-cellular protein during the development of mouse heart.Methods Samples of embryonic heart tissue were collected from postnatal and adult mice on embryo 12.5 days(E12.5),E14.5,E16.5,and E18.5,respectively.Expression levels of nesprin-1 mRNA and protein and the protein sub-cellular location in samples of embryonic heart tissue from mice were detected by reverse transcriptase PCR,Western blot analysis and immunofluorescence at different time points.Results The expression level of nesprin-1 mRNA and protein in samples of embryonic heart tissue from postnatal and adult mice was measured on E12.5,E16.5 and E18.5,respectively,which increased with the development of mouse heart,reached it peak on E16.5 and E18.5.The lowest expression level of nesprin-1 mRNA and protein was found in adult mice.Immunofluorescence showed that nesprin-1 was distributed in nuclear envelope and in spaces around the nuclei.Conclusion nesprin-1 is dynamically expressed during the development of mouse heart,and highly expressed in the mature period of cardiac conduction system and cardiac muscle cells,indicating that it plays an important role in the development of cardiac conduction system and cardiac muscle cells.
