摘要
目的构建体外压疮深部组织细胞氧化应激模型,为进一步在细胞层面探讨压疮深部组织损伤机制建立基础。方法将对数生长期大鼠骨骼肌细胞分为对照组及5个实验组,对照组行常规培养,5个实验组分别采用50、100、150、200、250μmol/L过氧化氢处理1～4h后分别检测四氮甲基唑蓝（MTT）、乳酸脱氧酶（LDH）指标并观察骨骼肌细胞Hoechst染色及形态学变化。结果对照组1～4h细胞存活100%,MTT、LDH指标及组织形态无明显变化。5个实验组2h时除50μmol/L组外,均呈现明显损伤变化;至4h,大鼠骨骼肌细胞存活率为20%～75%;显微镜下观察大鼠骨骼肌细胞随氧化应激程度加重而缩收,细胞间隙增大;Hoechst细胞荧光染色显示100μmol/L过氧化氢作用4h已有较多肌细胞趋向凋亡;细胞LDH释放率约为对照组的1.5倍。结论大鼠骨骼肌细胞随氧化应激程度加重,趋向凋亡;经100μmol/L过氧化氢作用4h的骨骼肌细胞可作为研究压疮深部组织细胞氧化应激的体外模型。
Objective To develop in vitro deep tissue injury（DTI）under oxidative stress,and to lay foundation for further study of DTI mechanism at cellular level.Methods The rat skeletal muscle cells which were at the logarithmic growth phase were divided into a control group and five experimental groups.The control group was cultured routinely,while the five experimental groups were treated with 50,100,150,200,and 250μmol/L hydrogen peroxide for 1to 4hours.Then methyl thiazolyl tetrazolium（MTT）and lactate dehydrogenase（LDH）were detected,and morphological changes by Hoechst staining were observed.Results The myoblast survival rate in the control group from 1to 4hours was 100%,and no obvious changes were found in MTT,LDH and cell morphology.The experimental groups except the 50μmol/L group had obvious injury at 2nd h.The cell survival rates at 4th h in the experimental groups varied from 20%to 75%.The myoblasts were gradually shrinked under the microscope.Hoechst staining showed that myobalsts treated with 100μmol/L hydrogen peroxide for 4hours started to develop cell apoptosis and the LDH release rate was 1.5times faster than the control group.Conclusion Myoblasts of rats tended to develop apoptosis along with the increased oxidative stress.Skeletal muscle cells treated with 100μmol/L hydrogen peroxide for 4hours could be used as in vitro model for further study of DTI.
基金
国家自然科学基金资助项目(81372064)
