The Expression Of Nampt In Replicative Senescence Of Rat Bone Marrow Mesenchymal Stem Cells And Its Significance

The deficiency of seeding cells in basic and industrial research of tissueengineering caused by replicative senescence of mesenchymal stem cells (MSCs) invitro affects the application in tissue regeneration and repair, which is one of the majorscientific issues in the field of stem cell research. Nicotinamidephosphoribosyltransferase (Nampt) has recently become the study focus because of theNAD+theory for the control of aging and longevity in mammals. Nampt, as arate-limiting enzyme in NAD+biosynthesis, determines NAD+levels in the metabolism.While activity of Sirt1, an anti-aging protein is absolutedly dependent on NAD+concerntration. Previous studies on relationship between Nampt and cellularsenescence mostly concentrated on the somatic cells. Nampt may be an effective factorto postpone somatic cellular senescence. Our previous study found that the decreasedgrowth of rat BMSCs occurred during the cell cultivation in vitro. Nampt expression inMSCs from aged individuals was reduced. Therefore, we speculate that Nampt couldplay a regulatory role in the senescence of stem cells.In this study, rat bone marrow mesenchymal stem cells (rBMSCs) replicativesenescence model was established by the use of serially passaged cultivation in vitro.Then model was systematically evaluated by investigation of cell morphology, cell cyle,SA-β-gal activity and expression of senescence-related factors. Furthermore, theexpression and significance of Nampt on the basis of rBMSCs replicative senescencemodels in vitro were explored by using quantitative RT-PCR, Western blot andimmunofluorescent staining.The results are as follows:1. rBMSCs were successfully isolated and obtained, which expressed the specificmarkers of mesenchymal stem cells (CD44, CD90and CD105), but CD31and CD45 are negative. Meanwhile, rBMSCs had potentiatls of osteogenic and adipogenicdifferentiation.2. rBMSCs replicative senescence model in vitro was set up. With the increasingpassages, rBMSCs displayed the senescent morphology, decreased cell aspect ratio andenlarged cell area. The growth of rBMSCs slowed down, while population doublingtime extended. The cell cycle arrest was in G0/G1phase at the expense of S phase.Both osteogenic and adipogenic differentiation were declined. In addition, SA-β-galactivity and the expression of p16INK4awere elevated.3. There was Nampt expression in rBMSCs. With the extended subculture inrBMSCs replicative senescence model, Nampt expression at both mRNA and proteinlevels were reduced time-dependently.In conclusions, Nampt expression is downregulated in rBMSCs replicativesenescence, which implies that Nampt could have regulatory effects on rBMSCssenescence. It will provide important theoretic and experimental basis for elucidatingthe molecular mechanisms of stem cell senescence, maintaining the quantity andfunctions of stem cells, meeting the needs of seeding cells and delaying stem cellssenescence.