Mouse Anti E. coli Beta-galactosidase Hybridoma [51-2b]

Over the past 20 years, various identifiers of cellular senescence have been used to quantify the abundance of these cells in different tissues. These include classic markers such as p16, senescence-associated beta-gal, and gamma H2AX, in addition to more recent markers (Sudan Black B and HMGB1). In vivo data on the usefulness of these markers in skeletal muscle are very limited and inconsistent. In the present study, we attempted to identify senescent cells in frozen human skeletal muscle biopsies using these markers to determine the effects of age and obesity on senescent cell burden; however, we were only able to assess the abundance of DNA-damaged nuclei using gamma H2AX immunohistochemistry. The abundance of gamma H2AX+ cells, including satellite cells, was not higher in muscle from old compared to young individuals; however, gamma H2AX+ cells were higher with obesity. Additionally, terminally differentiated, postmitotic myofiber nuclei from obese individuals had elevated gamma H2AX abundance compared to muscle from lean individuals. Analyses of gene expression support the conclusion that the elevated DNA damage and the senescence-associated secretory phenotype are preferentially associated with obesity in skeletal muscle. These data implicate obesity as a larger contributor to DNA damage in skeletal muscle than aging; however, more sensitive senescence markers for human skeletal muscle are needed to determine if these cells are in fact senescent.

Background: Allogeneic disc cell is the main cellular resource in tissue engineering (TE)-based strategy to retard disc degeneration. However, the accessible disc cells often exhibit senescent phenotype when they are subcultured in vitro. Hence, alleviating senescence of human disc cells during cell subculture is important for TE-based strategy to regenerate degenerative disc tissue. Objective: The present study was aimed to investigate whether bone morphogenetic protein-7 (BMP-7) can alleviate subculture-induced senescence of human nucleus pulposus (NP) cells in vitro. Methods: NP cells from human disc tissue were subcultured in vitro for six passages. Exogenous BMP-7 was added along with the culture medium to investigate its effects on senescence of NP cells. The inhibitor LY294002 was used to investigate the role of the PI3K/Akt pathway. Results: Compared with the human disc NP cells cultured in the baseline culture medium, addition of BMP-7 increased cell proliferation potency and telomerase activity, decreased senescence-associated beta-galactosidase (SA-beta-Gal) activity and G(0)/G(1) phase fraction, and down-regulated the expression of p16 and p53. Moreover, these positive effects of BMP-7 against senescence of human disc NP cells coincided with activation of the PI3K/Akt pathway. Further analysis showed that inhibitor LY294002 partly inhibited these protective effects of BMP-7 against senescence of human disc NP cells. Conclusion: BMP-7 alleviates subculture-induced senescence of human disc NP cells through activating the PI3K/Akt pathway. The present study provides new knowledge on allogeneic disc NP cell-based TE strategy to regenerate degenerative human disc tissue.