通过 microRNA/SIRT1 通路介导的人真皮成纤维细胞的转基因永生化。
Transgenic Immortalization of Human Dermal Fibroblasts Mediated Through the MicroRNA/SIRT1 Pathway.
机构信息
Laboratory of Cell-Based Assays and Innovations, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
Laboratory of Cell-Based Assays and Innovations, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand
出版信息
In Vivo. 2022 Jan-Feb;36(1):140-152. doi: 10.21873/invivo.12685.
BACKGROUND/AIM: Human dermal fibroblasts (HDFs) are widely used as a skin model in cosmetic and pharmaceutical industry due their advantages for the cosmetic industry and medical aspects. Telomeres are key players in controlling cellular aging, in which telomeres and the telomerase enzyme (hTERT) can maintain proliferative capacity and prolong cellular senescence. The primary aim of the study was to elucidate the underlying mechanisms of hTERT/SV40 immortalization of human dermal fibroblasts.
MATERIALS AND METHODS
Transgenic expression of hTERT and SV40 large antigen, as well as co-transfection of both factors was performed and their significance evaluated in terms of HDF immortalization efficiency.
RESULTS
The results showed that the immortalized fibroblasts of all conditions can be cultured in over 60 passages and maintain their telomere length. Further, key markers of skin cells, such as COL1A1, KRT18 and ELASTIN, were up-regulated in immortalized cells. In addition, p53 expression was enhanced in all immortalized cells, in accordance with activation of the SIRT1 gene upon transgenic immortalization. The significant role of SIRT1 in fibroblast proliferation was assessed by shRNA-knockdown, and it was found that SIRT1 silencing led to loss of Ki67, a proliferation marker. Moreover, miR-93, a SIRT1-targeted miRNA, also had a significantly reduced expression in the co-transfected immortalized cells, highlighting the linkage of the miRNA and SIRT1 pathway in the immortalization of human dermal fibroblasts.
CONCLUSION
This evidence from this study could benefit the efficient development of human skin cell lines for use in the cosmetic industry in the future.
背景/目的：由于人真皮成纤维细胞（HDF）在化妆品和制药行业具有优势，因此被广泛用作皮肤模型。端粒是控制细胞衰老的关键因素，其中端粒和端粒酶（hTERT）可以维持增殖能力并延长细胞衰老。本研究的主要目的是阐明 hTERT/SV40 永生化人真皮成纤维细胞的潜在机制。
材料和方法
转染 hTERT 和 SV40 大抗原的表达，以及这两种因子的共转染，并根据 HDF 永生化效率评估其意义。
结果
结果表明，所有条件下的永生化成纤维细胞均可在 60 多代中培养，并保持其端粒长度。此外，在永生化细胞中上调了关键的皮肤细胞标志物，如 COL1A1、KRT18 和 ELASTIN。此外，所有永生化细胞中的 p53 表达均增强，符合 SIRT1 基因在转基因永生化过程中的激活。通过 shRNA 敲低评估了 SIRT1 在成纤维细胞增殖中的重要作用，发现 SIRT1 沉默导致增殖标志物 Ki67 的丢失。此外，miR-93 是 SIRT1 的靶向 miRNA，在共转染的永生化细胞中也有明显降低的表达，突出了 miRNA 和 SIRT1 途径在人真皮成纤维细胞永生化中的联系。
结论
本研究的证据可能有助于未来在化妆品行业中高效开发人皮肤细胞系。
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