摘要
目的 探讨机械力诱导氧化应激对人子宫旁韧带成纤维细胞Ⅰ、Ⅲ型胶原表达的影响.方法 选取2014年1月至10月于本院行阴式全子宫切除术患者10例,术中取切除子宫的子宫旁韧带行成纤维细胞原代培养及鉴定.采用四点弯曲细胞力学加载系统对成纤维细胞进行力学加载构建机械力加载模型.使培养板产生形变位移1 mm的加力大小定义为1 333 μstrain.加力大小分别为0 μstrain（0 mm）、1 333 μtstrain（1 mm）、5 333 μstrain（4 mm）,加载频率为0.5 Hz,加载时间为4h.同时采用不同浓度H2O2孵育成纤维细胞构建氧化应激模型.H2O2浓度分别为0、0.2、0.4、0.8和1.6 mmol/L,孵育时间均为4h.采用实时荧光定量PCR和免疫印迹检测两种模型中成纤维细胞Ⅰ、Ⅲ型胶原mRNA和蛋白表达,采用细胞免疫荧光检测机械力模型中细胞氧化损伤产物8-羟基脱氧鸟苷（8-OHdG）水平变化.结果 机械力加载模型中,实时荧光定量PCR和免疫印迹显示,与对照组（0μstrain）比较,加载1 333 μstrain机械力后成纤维细胞Ⅰ、Ⅲ型胶原mRNA和蛋白表达均增加,而加载5 333 μstrain机械力后成纤维细胞Ⅰ、Ⅲ型胶原mRNA和蛋白表达均减少（均P＜0.05）.细胞免疫荧光检测显示,与对照组（0 μstrain）比较,加载1 333、5 333 μstrain机械力4h后,成纤维细胞内8-OHdG荧光强度明显增强,且后者更为显著.氧化应激模型中,实时荧光定量PCR显示,与对照组（0 mmol/LH2O2）比较,0.2 mmol/L H2O2孵育4h后成纤维细胞Ⅰ、Ⅲ型胶原mRNA表达均显著增加（均P＜0.05）;0.4 mmol/L H2O2孵育4h后成纤维细胞Ⅰ型胶原mRNA表达增加（P＜0.05）,而Ⅲ型胶原mRNA表达差异无统计学意义（P＞0.05）;0.8、1.6 mmol/L H2O2孵育4h后成纤维细胞Ⅰ、Ⅲ型胶原mRNA的表达均显著下降（均P＜0.05）.氧化应激模型中,免疫印迹检测显示,与对照组（0mmol/L H2O2）比较,0.2 mmol/LH2O2孵育4h后成纤维细胞Ⅰ型胶原�
Objective To investigate the effect of mechanical strain-induced oxidative stress on expression of types Ⅰ and Ⅲ collagen from fibroblasts in human parametrial ligament.Methods Ten cases of vaginal hysterectomy conducted at our hospital between January and October 2014 were included in this study.At surgery,parametrial ligaments were harvested and subjected to primary culture and identification of fibroblasts.A four-point bending system was used to establish the mechanical strain model in which fibroblasts were stretched as reflected by displacement of culture plate due to strain-induced deformation.A plate displacement of 1 mm corresponded to a mechanical force of 1 333 μstrain.The strains were 0 μstrain （0 mm）,1 333 μ strain （1 mm） and 5 333 μstrain （4 mm）,loaded with a frequency of 0.5 Hz over a loading time of 4 h.Meanwhile,fibroblasts were incubated in different concentrations of H2O2 to establish the model of oxidative stress.The H2O2 concentrations were 0,0.2,0.4,0.8 and 1.6 mmol/L,respectively,during an incubation time of 4 h.The types Ⅰ and Ⅲ collagen mRNA and protein expression of fibroblasts in both mnodels were determined by real-time fluorescent quantitative PCR and Western blotting.Immunofluorescence was used to detect the levels of 8-hydroxy-deoxyguanosine （8-OHdG）,a product of cellular oxidative damage in the mechanical strain model.Results In the mechanical strain model,real-time PCR and Western blotting showed that compared with the control group （0 μstrain）,1 333 μmstrain mechanical strain resulted in significantly higher expression levels,whereas 5 333 μstrain mechanical strain resulted in significantly lowered expression levels,of both types Ⅰ and Ⅲ collagen mRNA and protein in fibroblasts （all P〈0.05）.Immunofluorescence showed that compared with the control group （0 μstrain）,loading 1 333 or 5 333 μstrain mechanical strain for 4 h resulted in higher fluorescence intensity of 8-OHdG in fibroblasts,which was more obvious with 5 333 μstrain.In
出处
《中华生物医学工程杂志》
CAS
2016年第5期357-364,共8页
Chinese Journal of Biomedical Engineering
基金
国家自然科学基金面上项目（81270684）
