摘要
目的建立肿瘤坏死因子-α(TNF-α)诱导的大鼠软骨细胞焦亡模型并评价模型的特点。方法两步酶消化法获取大鼠膝关节软骨细胞,倒置相差显微镜观察大鼠软骨细胞的形态结构,甲苯胺蓝染色、Ⅱ型胶原(ColⅡ)免疫组化染色鉴定大鼠软骨细胞,用不同质量浓度TNF-α(5、10、20、40 ng/ml)诱导建立大鼠软骨细胞的焦亡模型,以TNF-α(0 ng/ml)为对照组。CCK-8法检测大鼠软骨细胞活力,Western blot检测大鼠软骨细胞焦亡信号相关蛋白,确定最佳刺激浓度,ELISA法检测细胞培养上清液中IL-1β、IL-18的水平,免疫荧光检测大鼠软骨细胞gasdermin D(GSDMD)表达,扫描电镜观测大鼠软骨细胞焦亡的形态学改变。结果与对照组相比,大鼠软骨细胞的活力随着TNF-α浓度升高而逐渐下降,并且核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、半胱天冬酶1(caspase-1)、GSDMD、磷酸化核转录因子-κB p65(P-p65)蛋白的表达增加。TNF-α(20 ng/ml)可诱导大鼠软骨细胞基质金属蛋白酶-13(MMP-13)表达上调,ColⅡ表达减少,GSDMD荧光表达增强,培养上清液中IL-1β、IL-18水平增加,关节软骨细胞肿胀,显微结构破坏。结论成功建立大鼠软骨细胞焦亡模型。TNF-α(20 ng/ml)体外可致大鼠软骨细胞肿胀死亡,显微结构破坏,软骨基质降解,焦亡信号活化。
Objective To establish and evaluate the pyroptosis model of rat chondrocytes induced by tumor necrosis factor-α(TNF-α).Methods A two-step enzymatic digestion method was used to obtain rat articular chondrocytes,inverted phase contrast microscope was used to observe the morphological structure of chondrocytes.Toluidine blue staining and typeⅡcollagen(ColⅡ)staining were used to identify chondrocytes.Different mass concentrations of TNF-α(5,10,20,40 ng/ml)were used to establish the pyroptosis model with TNF-α(0 ng/ml)as the control group.Cell viability was detected by CCK-8 method and proteins of pyroptosis signal were detected by Western blot.The levels of IL-1βand IL-18 in cultured supernatants were examined by ELISA kits.The expression of gasdermin D(GSDMD)in chondrocytes was detected by immunofluorescence.Scanning electron microscope was used to observe the morphological changes of chondrocyte.Results Compared with the control group,the cellviability of rat chondrocytes gradually decreased with the increase of TNF-αconcentration and the expression of nucleotide-binding and oligomerization domain-like receptor containing protein 3(NLRP3),caspase-1,GSDMD and Phospho-NF-κB p65(P-p65)proteins increased.Furthermore,TNF-α(20 ng/ml)could up-regulate the expression of matrix metalloproteinase-13(MMP-13),the fluorescence expression of GSDMD and the levels of IL-1βand IL-18 while the expression of ColⅡwas distinctly reduced.What′s more,the articular chondrocytes were swollen,and the microstructure was destroyed.Conclusion TNF-α(20 ng/ml)can cause the swelling and death of rat chondrocytes,degradation of cartilage matrix and activation of pyroptosis signaling pathway.The pyroptosis model of rat chondrocytes was successfully established.
作者
徐靓
吴玉娇
袁晓阳
张峰
程刚
张运芳
魏伟
严尚学
Xu Liang;Wu Yujiao;Yuan Xiaoyang;Zhang Feng;Cheng Gang;Zhang Yunfang;Wei Wei;Yan Shangxue(Institute of Clinical Pharmacology, Anhui Medical University, Key Laboratory of Anti-inflammatory and Immune Medicine, Ministry of Education, Anhui Engineering Research Center of Anti-inflammatory and Immune Drugs, Hefei 230032)
出处
《安徽医科大学学报》
CAS
北大核心
2022年第5期781-786,共6页
Acta Universitatis Medicinalis Anhui
基金
安徽高校自然科学研究重大项目(编号:KJ2020ZD15)。
