De Mattei Monica, Ongaro Alessia, Magaldi Simona, Gemmati Donato, Legnaro Andrea, Palazzo Annunziata, Masieri Federica, Pellati Agnese, Catozzi Linda, Caruso Angelo, Zamboni Paolo
Department of Morphology and Embryology, Ferrara, Italy.
Dermatol Surg. 2008 Mar;34(3):347-56. doi: 10.1111/j.1524-4725.2007.34068.x. Epub 2007 Dec 20.
Wound healing is a biologic process that is altered in patients affected by chronic venous ulcers. The wound microenvironment is reflected in the chronic wound fluid (CWF), an exudate containing serum components and tissue-derived proteins.
We investigated the effects of increasing doses of CWF collected from patients suffering from chronic venous ulcers on human adult dermal fibroblasts cultured in vitro and the relationship among CWF effects and treatment length.
Fibroblasts were treated with 60, 240, and 720 microg/mL CWF for 3 and 7 days. We evaluated cell proliferation and viability by MTT and Trypan blue assay, cell morphology by light microscopy, F-actin microfilaments organization by tetramethylrhodamine B isothiocyanate-conjugated phalloidin, alpha-smooth muscle actin expression by immunofluorescence, and senescence-associated beta-galactosidase activity.
CWF induced an increase in cell proliferation in the first 3 days of treatment. In contrast, at 7 days, a strong decrease in cell viability was observed. These changes were related to a cytoskeletal F-actin reorganization and not to fibroblast-myofibroblast differentiation nor to changes in cellular senescence.
This study shows a dose-dependent and biphasic effect of CWF on dermal fibroblasts, suggesting that a continuous exposure to chronic wounds microenvironment may induce late cellular dysfunctions possibly involved in the delayed wound healing.
伤口愈合是一个生物学过程，在慢性静脉溃疡患者中会发生改变。伤口微环境反映在慢性伤口液（CWF）中，这是一种含有血清成分和组织衍生蛋白的渗出液。
我们研究了从慢性静脉溃疡患者收集的不同剂量CWF对体外培养的成人皮肤成纤维细胞的影响，以及CWF效应与治疗时长之间的关系。
用60、240和720微克/毫升的CWF处理成纤维细胞3天和7天。我们通过MTT和台盼蓝试验评估细胞增殖和活力，通过光学显微镜观察细胞形态，用异硫氰酸四甲基罗丹明标记的鬼笔环肽评估F-肌动蛋白微丝组织，通过免疫荧光评估α-平滑肌肌动蛋白表达，以及衰老相关β-半乳糖苷酶活性。
在治疗的前3天，CWF诱导细胞增殖增加。相反，在7天时，观察到细胞活力大幅下降。这些变化与细胞骨架F-肌动蛋白的重组有关，与成纤维细胞-肌成纤维细胞分化无关，也与细胞衰老的变化无关。
本研究表明CWF对真皮成纤维细胞具有剂量依赖性和双相效应，提示持续暴露于慢性伤口微环境可能会诱导晚期细胞功能障碍，这可能与伤口愈合延迟有关。
