Research Paper Volume 10, Issue 12 pp 3713—3735

Figure 2. The altered microenvironment in aged mice affects the activation of OCs. Young (2m) and aged (24m) mice were fed with DDC diet for 3 weeks. (A) Freshly isolated OCs from young (Fresh-2m-OC) and aged (Fresh-24m-OC) DDC-fed mice were cultured on type I collagen coated 96-well plates, 3 days later, CCK-8 test was performed (left panel, n=6, ** p < 0.01). Quantitative Real-time PCR analysis of Ccnd1 in 2m-OC and 24m-OC (right panel, n=6, * p < 0.05). (B) Freshly isolated OCs were passaged for 6 times, then CCK-8 test (left panel, n=6) and quantitative Real-time PCR analysis of Ccnd1 (right panel, n=6) were performed. (C) Freshly isolated OCs were passaged for 6 times, then were cultured in normal medium (Ctrl), with liver extract from young mice (2m-extract) and with liver extract from aged mice (24m-extract). CCK-8 test (left panel, n=6, * p < 0.05) and quantitative Real-time PCR analysis of Ccnd1 (right panel, n=6, * p < 0.05) were performed. (D) Young and aged mice were joined in parabiotic pairs for 3 weeks with DDC diet. Immunofluorescence staining for EpCAM⁺ cells (green) was performed. Quantification of EpCAM⁺ cells was shown (n=6, * p < 0.05, ** p < 0.01). (E) CCK-8 test was performed in the OCs freshly isolated from the parabiotic pair (Parabiosis) or individuals as controls (Control) (n=6, * p < 0.05, ** p < 0.01). (F) Young and aged mice were joined in parabiotic pairs and kept for 3 weeks under DDC diet. The transcript levels of Cdkn2a, and Cdkn1a in the liver tissues were measured by quantitative real-time PCR (n=5, * p < 0.05, ** p < 0.01).