Dalkowski A, Fimmel S, Beutler C, Zouboulis Ch C
Department Dermatology, University Medical Center Benjamin Franklin, The Free University of Berlin, Fabeckstrasse 60-62, 14195 Berlin, Germany.
Exp Dermatol. 2003 Oct;12(5):673-81. doi: 10.1034/j.1600-0625.2003.00015.x.
In order to obtain a persuasive explanation for the beneficial clinical effect of cryotherapy on keloids, we developed a reproducible model to apply freezing temperatures on cell cultures, and investigated their influence on proliferation, viability, synthetic activity and differentiation of dermal fibroblasts in vitro. Cell cultures were established from 13 untreated keloids and 10 healthy skin specimens matched for age and skin localization to the donors. No significant influence of cell freezing on the proliferation rates of both keloidal and normal fibroblasts was documented, but mechanical cell destruction with a wide variation in lethality rates (29% average lethal effect on keloidal fibroblasts and 41% on normal ones) was observed. When comparing specimens of keloidal and normal tissue derived from the same four donors, the keloidal fibroblasts were similar regarding their synthetic activity but presented enhanced tenascin-C expression compared with the normal fibroblasts. After cryotherapy, delayed collagen III increase was detected in both cell types (P = 0.03). The collagen II/collagen I ratio increased from 1.6 to 2.8 in the keloidal and only from 1.9 to 2.2 in the normal fibroblasts after subcultivation. Normal fibroblasts exhibited a significantly lasting increase in fibronectin synthesis after freezing (P = 0.03). The intensity of staining against tenascin-C was decreased in five of nine keloidal fibroblast cultures after cryotherapy (P < 0.05) but increased in four of five normal fibroblast cultures (P = 0.016), so that the intensity of tenascin-C staining after freezing became identical in both cell types. Immunoblot studies in four patients and two controls confirmed a temporary decrease of tenascin-C in keloidal but not in normal fibroblasts immediately after freezing. Significantly decreased staining with two markers of myogenic differentiation, myosin in keloidal fibroblasts (P = 0.002) and desmin (P = 0.007) in normal fibroblasts, could also be detected after treatment. In summary, with the help of a model for controlled cell freezing in vitro, cryotherapy was found to modify collagen synthesis and differentiation of keloidal fibroblasts.
为了获得关于冷冻疗法对瘢痕疙瘩临床疗效的有说服力的解释，我们建立了一个可重复的模型，将冷冻温度应用于细胞培养，并研究其对体外真皮成纤维细胞增殖、活力、合成活性和分化的影响。从13个未经治疗的瘢痕疙瘩以及10个与供体年龄和皮肤部位匹配的健康皮肤标本中建立细胞培养。未发现细胞冷冻对瘢痕疙瘩和成纤维细胞增殖率有显著影响，但观察到机械性细胞破坏，致死率差异很大（对瘢痕疙瘩成纤维细胞的平均致死效应为29%，对正常成纤维细胞为41%）。当比较来自相同四个供体的瘢痕疙瘩和正常组织标本时，瘢痕疙瘩成纤维细胞的合成活性相似，但与正常成纤维细胞相比，其肌腱蛋白-C表达增强。冷冻疗法后，两种细胞类型均检测到Ⅲ型胶原蛋白延迟增加（P = 0.03）。传代培养后，瘢痕疙瘩成纤维细胞中Ⅱ型胶原蛋白/Ⅰ型胶原蛋白的比例从1.6增加到2.8，而正常成纤维细胞中仅从1.9增加到2.2。正常成纤维细胞冷冻后纤连蛋白合成显著持续增加（P = 0.03）。冷冻疗法后，9个瘢痕疙瘩成纤维细胞培养物中有5个的肌腱蛋白-C染色强度降低（P < 0.05），而5个正常成纤维细胞培养物中有4个增加（P = 0.016），因此冷冻后两种细胞类型的肌腱蛋白-C染色强度变得相同。对4名患者和2名对照的免疫印迹研究证实，冷冻后瘢痕疙瘩成纤维细胞中的肌腱蛋白-C暂时减少，而正常成纤维细胞中未减少。治疗后还可检测到两种成肌分化标志物的染色显著降低，瘢痕疙瘩成纤维细胞中的肌球蛋白（P = 0.002）和正常成纤维细胞中的结蛋白（P = 0.007）。总之，借助体外可控细胞冷冻模型，发现冷冻疗法可改变瘢痕疙瘩成纤维细胞的胶原蛋白合成和分化。
