氧化应激对大鼠真皮成纤维细胞生物学行为的影响

Influence of oxidative stress on the biological behaviors of rat dermal fibroblasts

目的:创面微环境中许多细胞都能不同程度的产生活性氧等自由基。观察氧化应激对大鼠真皮成纤维细胞生物学行为的影响,有利于认识创面愈合的机制。方法:实验于2005-09/2006-03在上海市烧伤研究所完成。①实验材料:体质量为200～220g的清洁级雄性SD大鼠,由中科院上海动物实验研究所提供。②实验方法:取SD大鼠背部皮肤组织,消化分离表皮真皮,剪成0.1cm×0.2cm真皮块,置于含体积分数为0.10 FBS的DMEM培养瓶中,在37℃、体积分数为0.05的CO2培养箱中培养,每周换液2次。约7～10d后,成纤维细胞逐渐从真皮块中迁移出来,并且大量增殖,待原代培养成纤维细胞生长融合成片,用0.05%胰酶-EDTA消化,用含体积分数为0.10FBS的DMEM终止消化,离心后,按1∶3分装传代。取对数生长期的成纤维细胞,培养24h后,分别以20,50,100,150μmol/LH2O2干预成纤维细胞,另设空白对照(不加H2O2)及调零组(只加Hank’s液不加细胞)。③实验评估:采用MTT法观察细胞活力,流式细胞仪观察细胞凋亡,荧光探针DCFH-DA进行活性氧检测,脂质过氧化物测试盒测试细胞丙二醛含量。结果:①MTT法观察细胞活力:随着H2O2浓度增加,成纤维细胞活力明显降低,各H2O2干预组吸光度值均较空白对照组低(P<0.01)。②流式细胞仪测定细胞凋亡:与空白对照组相比,20μmol/LH2O2干预组细胞凋亡无明显变化,100μmol/LH2O2干预组细胞凋亡明显升高(F=47.78,P<0.01)。③细胞内活性氧的测定:与空白对照组相比,20,100μmol/LH2O2干预组细胞活性氧产生明显升高(F=166.557,P<0.01,P<0.01)。④细胞内脂质过氧化物测定:与空白对照组相比,20μmol/LH2O2干预组细胞脂质过氧化产物丙二醛含量无明显变化,100μmol/LH2O2干预组细胞脂质过氧化产物丙二醛含量明显升高(F=5.756,P<0.01)。结论:氧化应激能导致大鼠真皮成纤维细胞细胞活力下降,凋亡增加,抗氧化治疗可以作为难愈性创面的治疗策略。

AIM： In wound microenvironment, many cells could generate active oxygen and other free radicles, in this paper, we investigate the mechanism and effect of oxidative stress on the biological behavior of dermal fibroblasts of normal rats. METHODS： The experiment was performed in Shanghai Bum Institute from September 2005 to March 2006. ①The back skin was harvested from the male SD rats of clean grade and 200-220 g （Shanghai Animal Experiment Institute, Chinese Academy of Sciences）, and digested to separate epidermis and dermis, then cut into pieces of 0.1 cm × 0.2 cm, and cultured in DMEM containing 0.10 volume fraction fetal bovine serum （FBS） in incubator with 0.05 volume fraction CO2 at 37 ℃. The medium was changed twice a week. About 7 to 10 days later, the flbroblasts gradually moved out of dermis, and proliferated massively. When the pnmary culture mixed together, the film was digested with 0.05% pancreatic enzyme-EDTA and terminated digestion with DMEM containing 0.01 volume fraction FBS, centrifuged, subpackage and passaged by 1：3. The fibroblast in log phase growth were. cultured for 24 hours and incubated with 20, 50, 100, and 150 μmol/L H2O2 （in Hanks＇ balanced salt solution）, at the same time, the blank control group （with no H2O2） and the zero alignment group （only Hank＇ s fluid with no cell） were set up. ②Cell survival was observed with MTT; call apoptosis with flow cytometry; reactive oxygen species （ROS） was detected by fluorescent probe of DCFH-DA, and malonaldehyde （MDA） by lipid peroxidate test box. RESULTS：①With the increasing of H2O2, cell survival was gradually decreased, and the absorbance value of each H2O2 group was lower than that of control group （P 〈 0.01 ）. ②Comparad with that in control group, cell apoptosis did not change obviously in 20 μmol/L H2O2 group, while abundant apoptotic dermal fibroblasts were observed in 100μmol/L H202 group （F =47.78, P 〈 0.01）. ③ROS was more produced in 20 and 100 μmol/L H2O2 groups compared with that in control group （F =166.557, P 〈 0.01, P 〈 0.01）. ④Compared with that in control group, the content of MDA in 20 μmol/L H2O2 group did not change, while the content of MDA in 100 μmol/L H2O2 group was increased （F=5.756, P 〈 0.01）. CONCLUSION： Oxidative stress results in decrease in survival, and increase in apoptosis of dermal fibroblasts of rats. Anti-oxidation therapy provides a treating approach for refractory wound.