摘要
背景:肿瘤坏死因子α是诱导骨关节炎软骨细胞凋亡的主要细胞因子,对骨关节炎的病理进程具有重要调节作用。目的:比较不同质量浓度肿瘤坏死因子α诱导大鼠软骨细胞凋亡模型,为进一步探讨药物对软骨细胞凋亡的调控作用提供理论支持。方法:清洁级4周龄SD大鼠,采用机械-Ⅱ型胶原酶消化法获取膝关节软骨细胞,并用不同质量浓度的肿瘤坏死因子α(0,10,20,30,40μg/L)诱导建立软骨细胞的凋亡模型,倒置相差显微镜观察软骨细胞的形态结构,Ⅱ型胶原免疫组化鉴定软骨细胞,MTT检测软骨细胞的活性,DAPI检测软骨细胞的凋亡情况。结果与结论:(1)体外培养软骨细胞,Ⅱ型胶原免疫组化染色可见胞浆呈棕黄色,为阳性细胞;(2)软骨细胞凋亡率,干预48 h肿瘤坏死因子α10,20,30μg/L明显高于0μg/L(P<0.01),肿瘤坏死因子α40μg/L明显高于10μg/L(P<0.01);(3)软骨细胞活性,干预48 h肿瘤坏死因子α10,20,40μg/L明显低于0μg/L(P<0.01),肿瘤坏死因子α20μg/L明显低于10μg/L(P<0.05),肿瘤坏死因子α40μg/L明显低于10μg/L(P<0.01)、20μg/L(P<0.05)。(4)结果说明,20μg/L肿瘤坏死因子α能成功建立炎症介导软骨细胞凋亡模型。
BACKGROUND： Tumor necrosis factor-α（TNF-α）, a main cytokine inducing chondrocyte apoptosis of osteoarthritis, plays a regulatory role in the process of osteoarthritis. OBJECTIVE： To compare the rat models of chondrocyte apoptosis induced by different mass concentrations of TNF-α, thus providing theoretical basis for further study on the regulation of drugs on chondrocyte apoptosis. METHODS： Chondrocytes were isolated from the knee cartilage of 4-week-old Sprague-Dawley rats of clean grade by mechanical ll collagenase digestion and were then incubated with different mass concentrations of TNF-α to induce apoptosis. The morphology of chondrocytes was observed under inverted phase contrast microscope, cells were identified using immunohistochemical staining of type II collagen, as well as the cell viability and apoptosis were detected by MTT and DAPI, respectively. RESULTS AND CONCLUSION：（1） In vitro, the cytoplasm of chondrocytes was stained brown-yellow by using immunohistochemical staining of type II collagen.（2） At 48 hours, the apoptosis rate of chondrocytes induced by 10, 20 and 30 μg/L TNF-α was significantly higher than that of the 0 μg/L TNF-α（P〈0.01）, and the apoptosis rate of chondrocytes induced by 40 μg/L TNF-α was significantly higher than that of the 10 μg/L TNF-α（P〈0.01）.（3） The viability of chondrocytes induced by 10, 20 and 40 μg/L TNF-α was significantly lower than that of the 0 μg/L TNF-α（P〈0.01）. In detail, the viability of chondrocytes induced by 20 μg/L TNF-α was lower than that of the 10 μg/L TNF-α（P〈0.05）; the viability of chondrocytes induced by 40 μg/L TNF-α was significantly lower than that of the 10 and 20 μg/L TNF-α（P〈0.01, P〈0.05）.（4） These results suggest that 20 μg/L TNF-α can successfully induce the chondrocyte apoptosis model.
作者
陈后煌
邵翔
李俐
吴明霞
李西海
Chen Hou-huang Shao Xiang Li Li Wu Ming-xia Li Xi-hai(Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China the Second Affiliated People＇s Hospital of Fujian University of Traditional Chinese Medicine, Fuzhou 350003, Fujian Province, China)
出处
《中国组织工程研究》
CAS
北大核心
2017年第4期527-531,共5页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(81373719)
陈可冀中西医结合发展基金(CKJ2015009)
福建省科技厅科技计划项目(2015J01479)~~
作者简介
陈后煌，男，1990年生，江西省丰城市人，汉族，福建中医药大学在读硕士，主要从事中西医结合骨伤基础与临床研究。
通讯作者：吴明霞，博士，主任医师，教授，福建中医药大学附属第二人民医院，福建省福州市350003
并列通讯作者：李西海，博士，副教授，福建中医药大学中西医结合研究院，福建省福州市350122
