We previously developed the technique of conditional reprogramming (CR), which allows primary epithelial cells from fresh or cryopreserved specimens to be propagated long-term in vitro, while maintaining their genetic stability and differentiation potential. This method requires a combination of irradiated fibroblast feeder cells and a Rho-associated kinase (ROCK) inhibitor. In the present study, we demonstrate increased levels of full-length p53 and its natural isoform, Δ133p53α, in conditionally reprogrammed epithelial cells from primary prostate, foreskin, ectocervical, and mammary tissues. Increased Δ133p53α expression is critical for CR since cell proliferation is rapidly inhibited following siRNA knockdown of endogenous Δ133p53α. Importantly, overexpression of Δ133p53α consistently delays the onset of cellular senescence of primary cells when cultured under non-CR conditions in normal keratinocyte growth medium (KGM). More significantly, the combination of Δ133p53α overexpression and ROCK inhibitor, without feeder cells, enables primary epithelial cells to be propagated long-term in vitro. We also show that Δ133p53α overexpression induces hTERT expression and telomerase activity and that siRNA knockdown of hTERT causes rapid inhibition of cell proliferation, indicating a critical role of hTERT for mediating the effects of Δ133p53α. Altogether, these data demonstrate a functional and regulatory link between p53 pathways and hTERT expression during the conditional reprogramming of primary epithelial cells.

译文

我们先前开发了条件重编程 (CR) 技术,该技术允许新鲜或冷冻保存的标本中的原代上皮细胞在体外长期繁殖,同时保持其遗传稳定性和分化潜力。此方法需要辐照的成纤维细胞饲养细胞和Rho相关激酶 (ROCK) 抑制剂的组合。在本研究中,我们证明了来自原发性前列腺,包皮,宫颈外和乳腺组织的有条件重编程的上皮细胞中全长p53及其天然同工型 Δ133p53α 的水平增加。增加的 Δ133p53α 表达对于CR至关重要,因为在siRNA敲低内源性 Δ133p53α 后,细胞增殖被迅速抑制。重要的是,在正常角质形成细胞生长培养基 (KGM) 中,在非CR条件下培养时,Δ133p53α 的过表达始终会延迟原代细胞的细胞衰老。更重要的是,Δ133p53α 过表达和ROCK抑制剂的组合 (没有饲养细胞) 使原代上皮细胞能够在体外长期繁殖。我们还表明,Δ133p53α 过表达诱导hTERT表达和端粒酶活性,hTERT的siRNA敲低导致细胞增殖的快速抑制,表明hTERT在介导 Δ133p53α 作用中的关键作用。总之,这些数据证明了在原代上皮细胞的条件重编程过程中,p53途径与hTERT表达之间的功能和调节联系。

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