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学科主题外科学
白藜芦醇对软骨细胞炎症因子及SREBP-2、MMP-13表达的影响
袁昊
2016-05-16
导师曾晖
专业外科学
授予单位北京大学
授予地点北京大学深圳医院
学位硕士
关键词骨关节炎 软骨细胞 白藜芦醇 核因子-κB 炎症因子
其他题名Effect of resveratrol on the expression of inflammatory cytokines, MMP-13 and SREBP-2 in chondrocytes
分类号R681.3
摘要

目的: 1. 通过体外培养小鼠膝关节软骨细胞并建立骨关节炎软骨细胞模型,探讨白藜芦醇对小鼠膝关节软骨细胞炎症因子及SREBP-2、MMP-13表达的影响; 2. 为白藜芦醇应用于临床治疗骨关节炎提供新的理论依据。方法: 1. 体外分离培养新生1d的C57BJ/6L品系小鼠膝关节软骨细胞,实验选取传代至P3的细胞,按一定细胞密度接种于6孔板中,根据加入培养物的不同将细胞分为3组: LPS组(1 μg/ml LPS处理6 h),白藜芦醇组(100 μM 白藜芦醇预处理2 h后加1 μg/ml LPS处理6 h),空白对照组(不做任何处理); 2. 采用Ⅱ型胶原免疫荧光染色法进行软骨细胞的鉴定; 3. 采用酶联免疫吸附试验法(ELISA)检测各组软骨细胞中SREBP-2、I-κBa 蛋白的表达量; 4. 采用软骨细胞免疫荧光染色的方法检测NF-κB p65蛋白核转位的情况; 5. 实时定量聚合酶链反应(RT-PCR)测 IL-1β、TNF-α mRNA 表达量; 6. Western Blot检测MMP-13蛋白表达情况。结果: 1. 软骨细胞胞核在荧光显微镜下呈现蓝色荧光,而胞质镜下则呈红色荧光; 2. ELISA检测I-κBa表达量,与空白对照组相比,LPS组表达下降,白藜芦醇组的表达量高于LPS组,差异均具有统计学意义; 3. 软骨细胞经免疫荧光染色检测发现,在LPS组中NF-κB p65发生明显的核转位,而在白藜芦醇组中软骨细胞NF-κB p65核转位被抑制; 4. 荧光定量PCR检测结果显示,LPS组IL-1β和TNF-α mRNA的表达量与空白组相比表达增多。与LPS组比,白藜芦醇组IL-1β、TNF-α mRNA的表达被抑制,差异均有统计学意义(P<0.05)。 5. 脂多糖组SREBP-2、MMP-13表达量较空白组明显升高,白藜芦醇组SREBP-2、MMP-13表达量较脂多糖组下降,但仍高于空白组。结论: 1. 白藜芦醇可通过NF-κB信号通路影响软骨细胞IL-1β、TNF-α mRNA的表达,进而降低软骨细胞炎症反应,延缓关节软骨细胞退变。 2. 白藜芦醇可抑制小鼠软骨细胞SREBP-2和MMP-13的表达,保护软骨细胞,延缓关节软骨退变

英文摘要

Objective 1. To study the effect of resveratrol on the expression of inflammatory factors, SREBP-2 `s\vl VE`s\vl Vicular chondrocytes of mice by culture articular chondrocytes and establish a model of osteoarthritis chondrocytes; 2. Provide a new theoretical basis for the application of resveratrol in the clinical treatment of osteoarthritis. Methods 1. Isolated and cultured the Knee chondrocytes of C57BJ/6L strain 1d mice in vitro, P3 cells were ed for the experiment, The cells were seeded on 6 well plates at a certain density. The experiment was divided into three groups according to the different addition materials: lipopolysacide intervention group (1 μg/ml) and resveratrol intervention group (100 M resveratrol +1 μg/ml lipopolysacide), the blank group without any processing; 2. Mouse chondrocytes were identified by immunofluorescence staining with collagen type II; 3. Enzyme linked immunosorbent assay (ELISA) method was used to detect the expression of SREBP-2 and I-κBa protein in chondrocytes; 4. NF-κB nuclear translocation was observed by immunofluorescence staining; 5. The expression of IL-1β、TNF-α mRNA were measured by real-time quantitative polymerase chain reaction (RT-PCR); 6. Western blot was used to detect the expression level of MMP-13 protein in chondrocytes. Results 1. The cell nucleus of chondrocytes was blue fluorescence and cytoplasmic red fluorescence under the fluorescent microscope; 2. ELISA detection: the expression of I-κBa in LPS group was significantly higher than that in control group (P < 0.05), and the resveratrol group was significantly higher than that in LPS group (P < 0.01); 3. Immunofluorescence staining cells:the nuclear translocation of NF- κB in LPS group was significantly changed compared with the control group, while the nuclear translocation of NF-κB in resveratrol group was inhibited; 4. RT-PCR test results:as compared with the control group, the expressions of IL-1β and TNF-α mRNA in LPS group were upregulated, while resveratrol group were downregulated compared to LPS group, differences were statistically significant (P <0.05). 5. Compared with the blank control group, the expression of SREBP-2 and MMP-13 in LPS group was significantly higher, and the expression of SREBP-2 and MMP-13 in resveratrol group was lower than that in LPS group, but it was still higher than that in the blank control group. Conclusion 1. Resveratrol can influence the expression of IL-1β and TNF-α mRNA in the cartilage cells through NF- κB pathway, decrease the inflammatory reaction of the chondrocytes, and then delay the degeneration of articular cartilage cells; 2. Resveratrol can inhibit the expression of SREBP-2 and MMP-13 in mouse chondrocytes. protect cartilage cells and delay the degeneration of articular cartilage

语种中文
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文献类型学位论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/125368
专题北京大学深圳医院
作者单位北京大学深圳医院
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袁昊. 白藜芦醇对软骨细胞炎症因子及SREBP-2、MMP-13表达的影响[D]. 北京大学深圳医院. 北京大学,2016.
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