幾丁聚醣對以生長因子誘導產生老化之人類纖維母細胞之影響

Abstract

人類纖維母細胞的體外培養造成複製性老化的概念已提出超過50年。從那時起，從各種動物體取出的許多類型的细胞，已被證明有一個有限的壽命。此外，在許多文獻中顯示生長因子能增進细胞生長、增殖和细胞分化。但在我們的研究中，我們對生長因子抱持著一個不同的態度。雖然表皮生長因子(EGF)可以刺激增殖和提高複製的潛力，但長期培養的結果與没有加EGF的组別之間没有顯著差異。透過檢視老化的標準，經表皮生長因子處理的细胞顯示了衰老的特徵。因此，我們用表皮生長因子誘導人類包皮纖維母细胞產生老化，製造不同程度的可逆老化模型。我們進一步將老化的細胞培養於幾丁聚醣上，使他們形成球型，造成類似低氧的環境，使老化的細胞回春。於此，我們指出於幾丁聚醣表面培養3天足以使老化的細胞呈現年輕的狀態，且不走向癌轉移。我們採用偵測SA-s-gal的活性、p53基因和p21基因表現及BrdU增殖試驗，來鑑定此老化的細胞是否回春。

Replicative senescence was first described formally more than 50 years ago, for human fibroblasts in culture. Since then, many cell types from a variety of animal species have been shown to have a finite life span. In addition, growth factors were considered to enhance cellular growth, proliferation and cellular differentiation in many studies. In this study, we approached a different angle about growth factor. Although EGF could stimulate proliferation and increase replicative potential, the fate of long-term culture had no difference between the groups without EGF. EGF treated cells displayed senescent characteristics, which was provided by examining senescence marker. Thus, we used EGF to induced human foreskin fibroblast to express a senescence phenotype, and founded a model to make different degrees of reversible senescence. We further transferred these senescent cells on chitosan, and made them form spheroids, which could be a hypoxia circumstance, to rejuvenate. Herein, we reported that three days culture on chitosan was sufficient to rejuvenate senescent cells to an apparently youthful state without transformation. The identity of the rejuvenated cells as having been derived from senescent cells has been confirmed by a variety of methods, including SA-s-gal activity, p53-p21 gene expression and BrdU proliferation assay.