The Effects Of Lentivirus-mediated P16ink4a Silencing On Proliferation And Differentiation Of Aging Adipose-derived Mesenchymal Stem Cells

Background:Alzheimer’s disease(AD) is a kind of neurodegenerative disease threatening the life quality of elderly people, its pathogenesis is unclear and there is no effective treatment currently. There is a long history to treat Alzheimer’s disease by stem cells, demestic and overseas reseachers pay wide attention on ADSCs implantation to treat AD becuase of abundant source and easy auto-obtainment with little trauma. But stem cells implantation, including ADSCs, have to face the problem of recession and poor treatment effect cuased by physiological aging. P16ink4a, a tumor suppressor gene, shows overexpression not only in aging stem cells, but also in aging ADSCs in vitro. Therefore, using RNA interference(RNAi) mediated by lentivirus can promise to block the p16ink4a gene, and alleviate the function decline of stem cells, thus improve treatment effect of ADSCs to Alzheimer’s disease.Objective:Using RNAi mediated by lentivirus block the p16ink4a gene of aging ADSCs, try to figure out the difference of morphology, proliferation, neural differentiation of pl6ink4a silencing aging ADSCs.Methods:Inguinal and post peritoneal fat tissues were gotten from young/old rat, then to ADSCs were isolated and cultivated in media. For identifiying the stem cells common marker, the ADSCs were passed On confluence. The ADSCs were divided into four groups:p16ink4a silencing aging ADSCs、aging ADSCs+negative control virus、aging ADSCs+PBS and untreated aging ADSCs. P16ink4a expression was detected by real-time PCR and western blot in four groups. Then comparing with young ADSCs, explore the difference of proliferation through drawing growth curve by MTT in the five groups. The senescence of ADSCs was carried out by senescence β-galactosidase staning in the five groups. At last, ADSCs were induced into neurocytes with the two stages strategy, and expression of nestin, the neuron specific makers, was determined by immunofluorescence for comparing the function of differentiation in the five groups.Results:1. Indeed, ADSCs reaped from rat fat tissue which can designate a stem cell strongly expressed CD29/CD90but lowly express CD11b.2. The expression of pl6ink4a gene appears less in aging ADSCs+lentivirus9934-1than the other three groups. There were no significant difference between aging ADSCs+negative control virus、aging ADSCs+PBS and untreated aging ADSCs in the expression of pl6ink4a gene.3. Aging ADSCs+lentivirus9934-1appears weaker than young ADSCs and better than the other three groups in growing ability and exppression of Nestin than young ADSCs. There were no significant difference between aging ADSCs+negative control virus、aging ADSCs+PBS and untreated aging ADSCs in growing ability and exppression of Nestin.4. Aging ADSCs+lentivirus9934-1appears more than young ADSCs and much less than the other three groups in senile cells. There were no significant difference between aging ADSCs+negative control virus、 aging ADSCs+PBS and untreated aging ADSCs in senile cells.Conclusion:1. P16ink4a gene expression could be successfully suppressed in aging ADSCs by transfecting lentivirus.2. To a certain extent, p16ink4a silencing alleviated the cell senescence and improved the growing and neural differentiation ability in aging ADSCs.