共培养的角膜上皮细胞诱导人羊膜上皮细胞分化为角膜上皮样细胞

Differentiation of human amniotic epithelial cells (HAECs) into corneal epithelial cells induced by co-culture of HAECs and corneal epithelial cells in vitro

目的探讨体外培养人羊膜上皮细胞(HAEC)并定向诱导分化为角膜上皮样细胞的可行性。方法采用Ⅱ型胶原酶和胰蛋白酶消化分离培养HAEC,通过Transwell^(TM)非接触共培养系统与人角膜上皮细胞(CEC)进行共培养2周,诱导HAEC分化;通过免疫荧光细胞化学染色法检测诱导后HAEC中细胞角蛋白3+12(CK3+12)、CK14、CK19、增殖细胞核抗原(PCNA)蛋白的表达,实时荧光定量PCR检测分化细胞CK12、CK14、CK19、P63 m RNA水平,Western blot法检测HAEC诱导前后CK3+12、CK14、CK19、P63的蛋白水平。结果体外培养的HAEC和CEC形态相似,免疫荧光细胞化学染色检测到HAEC诱导分化细胞CK3+12、CK14、CK19、PCNA呈阳性表达,诱导后HAEC中P63、CK12、CK19、CK14 mRNA相对表达量分别为共培养前的3.35、6.76、2.42、1.06倍。结论在与CEC共培养2周后,HAEC可以转分化为角膜上皮样细胞,HAEC可用作组织工程角膜重建的种子细胞。

Objective To investigate the feasibility of inducing human amniotic epithelial cells （HAECs） differentiation into corneal epithelial cells （CECs） in vitro. Methods HAECs were isolated by type II collagenase and typsin. The cells were induced by human CECs in a Transwell TM co-culture system for 2 weeks. The expressions of cytokeratin 3 ＋ 12 （CK3 ＋ 12）, CK14, CK19, proliferating cell nuclear antigen （PCNA） proteins in the induced HAECs were detected by immunofluorescence staining; CK12, CK14, CK19, P63 mRNAs were examined by fluorescence quantitative real-time PCR （qRT-PCR）; CK3 ＋ 12, CKI4, CK19, P63 proteins were tested by Western blotting. Results HAECs were similar to human CECs in morphology in vitro. After co-culture with CECs, the induced HAECs were positive for CK3 ＋ 12, CK14, CK19 and P63. The relative expression levels of P63, CK12, CK19 and CK14 mRNAs in the induced HAECs increased 3.35, 6.76, 2.42 and 1.06 times, respectively. Conclusion HAECs can differentiate into corneal epithelial-like cells under the condition of co-culture with CECs. The differentiation potential of HAECs may make it as seed cells for tissue engineering corneal reconstruction.