Delanian S, Martin M, Bravard A, Luccioni C, Lefaix J L
Département d'Oncologie-Radiothérapie, Hôpital Saint-Louis, APHP, 75010 Paris, France.
Radiother Oncol. 2001 Mar;58(3):325-31. doi: 10.1016/s0167-8140(00)00332-7.
As we previously observed that bovine liposomal Cu/Zn SOD (LipSOD) reduces cutaneous radiation-induced fibrosis (RIF) in human therapeutic assays the mechanisms involved were investigated here by an in vitro study of the LipSOD effects on cellular antioxidant metabolism and regulation of matrix degradation.
Primary cultures of human fibroblasts harvested from normal or RIF skin were treated with various doses of LipSOD. Catalase, Cu/Zn and Mn SOD endogenous cell enzyme activities and protein amounts were assayed by polyacrylamide gel electrophoresis and western blotting. Gene expressions of tissue inhibitor of metalloproteinases (TIMP) and TGF-beta1 was investigated by northern blot analysis.
A deficiency of endogenous Mn SOD, considered to favour cell proliferation, was observed in cultured RIF cell. The present study showed that bovine Cu/Zn SOD entered the cells. Exposure to LipSOD (a) enhanced endogenous Mn SOD activity and protein level, without changes of endogenous Cu/Zn SOD and catalase, and (b) significantly reduced TIMP and TGF-beta1 gene expression, in RIF cells. No changes in these parameters were noted in treated control skin fibroblasts.
Modulation of RIF skin fibroblasts by LipSOD seems effective via indirect endogenous Mn SOD activation, which might explain the cell phenotype reversion observed. TIMP reduction accounts for the elimination of collagenase activity inhibition and the subsequent digestion of excess extracellular matrix deposition, as well as RIF reversibility in vivo. The reduction of TGF-beta1 expression might explain the breaking of maintaining fibrotic cell activation connected with this growth factor.
正如我们之前所观察到的，在人体治疗试验中牛脂质体铜/锌超氧化物歧化酶（LipSOD）可减轻皮肤辐射诱导的纤维化（RIF），在此通过对LipSOD对细胞抗氧化代谢和基质降解调节作用的体外研究来探究其中涉及的机制。
用不同剂量的LipSOD处理从正常或RIF皮肤中获取的人成纤维细胞原代培养物。通过聚丙烯酰胺凝胶电泳和蛋白质印迹法检测过氧化氢酶、铜/锌和锰超氧化物歧化酶的内源性细胞酶活性及蛋白量。通过Northern印迹分析研究金属蛋白酶组织抑制剂（TIMP）和转化生长因子-β1（TGF-β1）的基因表达。
在培养的RIF细胞中观察到内源性锰超氧化物歧化酶缺乏，这被认为有利于细胞增殖。本研究表明牛铜/锌超氧化物歧化酶可进入细胞。在RIF细胞中，暴露于LipSOD（a）可增强内源性锰超氧化物歧化酶活性和蛋白水平，而内源性铜/锌超氧化物歧化酶和过氧化氢酶无变化，并且（b）显著降低TIMP和TGF-β1基因表达。在处理过的对照皮肤成纤维细胞中未观察到这些参数的变化。
LipSOD对RIF皮肤成纤维细胞的调节似乎通过间接激活内源性锰超氧化物歧化酶而有效，这可能解释了所观察到的细胞表型逆转。TIMP减少可解释胶原酶活性抑制的消除以及随后过量细胞外基质沉积的消解，以及体内RIF的可逆性。TGF-β1表达的降低可能解释了与该生长因子相关的维持纤维化细胞激活的打破。
