烧伤患者深Ⅱ度创面成纤维细胞的体外培养

Long-term in vitro culture of the fibroblasts from the deep partial thickness burn wound in burn patients

目的 建立烧伤患者深Ⅱ度创面成纤维细胞体外培养方法。 方法 切取正常人皮肤及烧伤患者伤后 5、10、14、2 1、2 8、35d的深Ⅱ度创面组织 (各 3例 )。去除非真皮组织 ,用 2 .5g/L的洗必泰溶液浸泡后 ,剪碎并用胰蛋白酶 EDTA(终浓度分别为 1.2 5 g/L、0 .1g/L)消化。离心收集细胞、接种培养。细胞生长近 10 0 %融合时 ,按常规方法进行多次传代培养、冻存与复苏。培养期间随时进行细胞形态学观察 ,计算细胞贴壁存活率和倍增时间。 结果 原代培养的创面成纤维细胞纯度较高 ,活力较好 ;原代、继代培养的及经冻存复苏后的细胞在生长特性、大体形态等方面能够保持稳定 ;经多次复苏的创面成纤维细胞贴壁存活率保持在 80 %以上。 结论 该细胞体外培养方法的建立 ,可为深入探讨瘢痕的形成机制及防治瘢痕提供良好的研究手段。

Objective To establish a long-term in vitro culture of the fibroblasts obtained from burn wounds. Methods Skin samples were harvested from normal volunteers and the deep partial thickness burn wound in burn patients on the 5th, 10th, 21st, 28th and 35th postburn days (PBDs). The non-dermal tissue was removed from the samples and primed by chlorhexidine solution in concentration of 2.5 g/L. The skin sample was then digested by trypsin-EDTA in concentration of 1.25 g/L and was centrifuged before the cells were harvested and cultured. When the cells grew nearly to form sheet, multiple passage culture, freezing storage and revivification were carried out with routine methods. The cell morphology was continuously observed during the culture. And the cell doubling time was calculated. Results The wound-origin fibroblasts exhibited higher purity and better activity. The cellular growth features and gross morphology kept stable during primary and secondary culture, and during freezing storage and after revivification. The cells kept their activity above 80% of their original after many times of revivification. Conclusion The establishment of the in vitro culture of fibroblasts from burn wounds might be useful in the exploration of the pathogenesis and therapeutic measures of scars. [