永生化表皮细胞体外快速扩增的实验研究

In vitro amplification of immortalized keratinocytes with micro-carriers in rotary cell culture system

目的 探讨获取大量皮肤组织工程种子细胞的方法和技术。方法 通过真核表达载体,把人端粒酶逆转录酶基因(hTERT)转入兔表皮细胞,筛选、挑选阳性克隆在微载体 RCCS内快速扩增永生化表皮细胞。观测RCCS中微载体培养永生化表皮细胞的生长情况和检测细胞代谢率,进行抗人角细胞广谱角蛋白(AE1/AE3 )免疫组化染色,并与常规培养方法进行比较。结果 永生化表皮细胞在微载体 RCCS中生长迅速、细胞代谢率高、倍增时间缩短(P <0 0 1)。在培养第10天,细胞数量可达最初接种的64倍(P <0 0 1)。结论 联合应用微载体和RCCS培养技术,能够在体外快速、大量扩增永生化表皮细胞。

Objective To explore the way and technique to gain adequate seed cells for skin tissue engineering sufficiently. Methods Human telomerase reverse transcriptase (hTERT) gene was introduced into rabbit keratinocytes by eukaryotic vector. The positive clones were selected and cultured in microcarrier-RCCS. The growth of immortalized keratinocytes was observed, and the metabolic rate and pan-cytokeratins (AE1/AE3) expression of immortalized keratinocytes in experimental groups were detected and compared with those in the control group. Results The immortalized keratinocytes in the experimental groups grew rapidly and had a high metabolic rate and shorter population doubling time (PD) (P<0.01). The amount of immortalized keratinocytes reached 64 times of the initial amount after culture for 10 d (P<0.01). Immunohistochemical staining showed that AE1/AE3 was positive in both experimental and control groups. Conclusion Large number of immortalized keratinocytes could be obtained rapidly with microcarrier-RCCS culture system.