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发布日期: Jun 1, 2020
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Chronic expression of p16INK4a in the epidermis induces Wnt-mediated hyperplasia and promotes tumor initiation

Results .Epidermal p16 induction causes partial senescence features . To study the effects of p16-expressing cells on the adult skin we crossed mice carrying a doxycycline-activated human p16 gene (tet-p16)21with K5-rtTA mice31, allowing its inducible activation in the basal epidermis. Transgenic p16 protein was detected in ~40% of basal keratinocytes in the interfollicular epidermis (IFE) after 2 days of doxycycline (dox) treatment at 3 weeks of age (Fig.? 1a–c ). Tissues showed reduced phosphorylated Rb levels, consistent with expected Rb activation (Fig.? 1d ). Staining for Ki67 revealed a dramatic reduction in the number of proliferating IFE basal cells after 2 days of p16 activation (Fig.? 1e, f ). However, after 2 weeks of p16 activation, overall proliferation rates returned to those of control animals.Fig. 1: p16 induces cell-cycle arrest and cell hypertrophy, and disrupts hair-follicle growth.a Immunostaining of human p16 (brown) in back skin sections of K5-rtTA/tet-p16 and control tet-p16 mice treated with doxycycline (dox) for 2 days (2d) starting at 3 weeks of age. b Skin sections from the same mice stained for p16 (red) and K14 (green), which labels the basal epidermis. c Percentage of p16 + keratinocytes in the interfollicular epidermis (IFE) in mice treated with dox for 2 days (2d), 2 weeks (2w), or 6 months (6?m). Dots indicate individual mice, n ?=?5, 5, 4 in respective groups. d Skin sections from mice treated with dox for 2 days, stained for Rb phosphorylated on Thr-821/826 (p-Rb) (red) and K14 (green). e Immunostaining of the proliferation marker Ki67 (brown) in sections from mice treated with dox for 2 days (2d) or 2 weeks (2w). f Percentage of IFE keratinocytes expressing Ki67 in the same mice (dots). n ?=?4, 6, 4, 4. g SA-βGal staining (blue) of skin sections from?indicated mice, treated with dox for 2 weeks. h Percentage of SA-βGal positive IFE cells from indicated mice (dots) treated with dox for 2 weeks. n ?=?9, 10. i Skin sections from indicated mice stained for E-Cadherin (brown) to indicate cell circumference. j Area of epidermal keratinocytes from indicated mice (dots). Shown are values for control mice (tet-p16), and for p16 ? and p16 + keratinocytes in K5-rtTA/tet-p16 mice. n ?=?3, 6, 6 mice, >60 cells were scored per mouse. k H&E-stained skin sections from the indicated mice treated with dox for 2 weeks, showing hair-follicle morphology. l Number of hair follicles per field in indicated mice (dots), following dox treatment for 6 months. n ?=?9 per group. m FACS analysis of epidermal cells from indicated mice treated with dox for 6 months, stained for CD34, CD49f, and Sca1. Charts show Sca1 ? (follicular) cells only. Gate indicates percentage of CD34 + /CD49f high hair-follicle stem cells. Cells were pooled from three mice per group. All graphs indicate mean across mice?±?S.E.M, all scores were conducted visually from images. Blue labels DNA in all fluorescence images. P ?