压力下角质形成细胞与成纤维细胞共培养对细胞增殖及胶原合成的影响

目的 研究压力下人角质形成细胞（human keratinocytes, HKC）和人成纤维细胞（human fibroblasts, HFB）的三维共培养对细胞增殖及胶原蛋白合成的影响。方法 将HKC与HFB分别接种于壳聚糖-明胶支架2 d后，将气 液界面诱导分化1 d后的HKC-壳聚糖-明胶复合物与HFB-壳聚糖-明胶复合物共培养12 h，3.4 kPa气体压力加载24 h，并以压力单独培养、无压力单独培养或无压力共培养作为对照。HE染色观察细胞在支架中的分布及生长情况，MTT法测定细胞增殖情况，羟脯氨酸试剂盒测定上清液中的胶原含量。结果 HE染色发现，HKC与HFB均可以在壳聚糖 明胶支架上正常增殖成片；3.4 kPa压力或共培养均可以促进HKC增殖和胶原合成，抑制HFB增殖及胶原合成。结论 压力及共培养是影响HKC与HFB增殖及胶原蛋白合成的重要因素，研究结果为手术切除瘢痕后移植组织工程表皮结合压力法治疗增生性瘢痕的可能机制提供参考。
Objective To investigate effects of 3D co-culture of human keratinocytes (HKC) and human fibroblasts (HFB) under pressure on cell proliferation and collagen synthesis. Methods The HKC and HFB were planted on chitosan-gelatin scaffolds, respectively, for 2 d. The HKC-chitosan-gelatin complex (3D HKC) was cultured at air-liquid interface for 1 d to induce differentiation, and then co-cultured with the HFB-chitosan-gelatin complex (3D HFB) for 12 h. 3.4 kPa pressure was applied on the co-culture group for 24 h. The group of single culture with pressure, the group of single culture without pressure and the group of co-culture without pressure were used as control. HE staining was used to observe distribution and growth of HKC and HFB on chitosan-gelatin scaffolds. MTT method was used to test proliferation of HKC and HFB. Hydroxyproline kit was used to observe collagen concentration of the supernatant fluids. Results HE staining showed that HKC and HFB could grow confluently on chitosan-gelatin scaffolds；3.4 kPa pressure or co-culture both could promote the HKC proliferation and collagen synthesis, while restrain the HFB proliferation and collagen synthesis. Conclusions Pressure and co-culture play an important role in HKC and HFB proliferation and collagen synthesis. This research finding provides some reference for exploring the therapeutic mechanism of hyperplastic scar from clinical operation of resecting scar by transplanting tissue-engineered skin to the wound and then combined with pressure treatment.