Department of Pathology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Cheras, Kuala Lumpur, Malaysia.
J Biosci Bioeng. 2012 Aug;114(2):220-7. doi: 10.1016/j.jbiosc.2012.03.021. Epub 2012 May 10.
Human amnion epithelial cells (HAECs) hold great promise in tissue engineering for regenerative medicine. Large numbers of HAECs are required for this purpose. Hence, exogenous growth factor is added to the culture medium to improve epithelial cells proliferation. The aim of the present study was to determine the effects of epidermal growth factor (EGF) on the proliferation and cell cycle regulation of cultured HAECs. HAECs at P1 were cultured for 7 days in medium containing an equal volume mix of HAM's F12: Dulbecco's Modified Eagles Medium (1:1) supplemented with different concentrations of EGF (0, 5, 10, 20, 30 and 50 ng/ml EGF) in reduced serum. Morphology, growth kinetics and cell cycle analysis using flow cytometry were assessed. Quantitative gene expression for cell cycle control genes, pluripotent transcription factors, epithelial genes and neuronal genes were also determined. EGF enhanced HAECs proliferation with optimal concentration at 10 ng/ml EGF. EGF significantly increased the proportion of HAECs at S- and G2/M-phase of the cell cycle compared to the control. At the end of culture, HAECs remained as diploid cells under cell cycle analysis. EGF significantly decreased the mRNA expression of p21, pRb, p53 and GADD45 in cultured HAECs. EGF also significantly decreased the pluripotent genes expression: Oct-3/4, Sox2 and Nanog; epithelial genes expression: CK14, p63, CK1 and Involucrin; and neuronal gene expression: NSE, NF-M and MAP 2. The results suggested that EGF is a strong mitogen that promotes the proliferation of HAECs through cell cycle regulation. EGF did not promote HAECs differentiation or pluripotent genes expression.
人羊膜上皮细胞（HAECs）在组织工程再生医学中具有广阔的应用前景。为此需要大量的 HAECs。因此，在培养基中添加外源性生长因子以提高上皮细胞的增殖。本研究旨在探讨表皮生长因子（EGF）对培养的 HAECs 增殖和细胞周期调控的影响。将 P1 代 HAECs 在含有 HAM's F12：Dulbecco's Modified Eagles Medium（1：1）等体积混合物的培养基中培养 7 天，培养基中添加不同浓度的 EGF（0、5、10、20、30 和 50ng/ml EGF）和低血清。评估形态、生长动力学和流式细胞术进行的细胞周期分析。还确定了细胞周期控制基因、多能转录因子、上皮基因和神经元基因的定量基因表达。EGF 增强 HAECs 的增殖，最佳浓度为 10ng/ml EGF。与对照组相比，EGF 显著增加了 HAECs 在 S 和 G2/M 期的细胞周期比例。在培养结束时，HAECs 在细胞周期分析中仍保持二倍体。EGF 显著降低了培养的 HAECs 中 p21、pRb、p53 和 GADD45 的 mRNA 表达。EGF 还显著降低了多能基因表达：Oct-3/4、Sox2 和 Nanog；上皮基因表达：CK14、p63、CK1 和 Involucrin；以及神经元基因表达：NSE、NF-M 和 MAP 2。结果表明，EGF 是一种强大的有丝分裂原，通过细胞周期调控促进 HAECs 的增殖。EGF 没有促进 HAECs 的分化或多能基因的表达。
