探讨胎球蛋白-A对磷酸钙晶体诱导巨噬细胞表达及释放HMGB1的抑制作用

发布时间：2018-03-12 18:35

  本文选题：胎球蛋白-A　切入点：肾结石　出处：《广西医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：探讨胎球蛋白-A(Fetuin-A)干预对结石晶体磷酸钙（CaP）诱导人巨噬细胞表达及释放晚期炎症因子高迁移率蛋白（HMGB1）的抑制作用。 方法：分别采用100ug/ml CaP、100ug/ml CaP+1000ng/ml Fetuin-A、100ug/ml CaP+2500ng/ml Fetuin-A、100ug/ml CaP+5000ng/ml Fetuin-A刺激已成功诱导为巨噬细胞的U937，于刺激后24h分别提取各组培养细胞总RNA，，RT-PCR检测细胞HMGB1mRNA表达水平；提取各组细胞总蛋白、胞浆蛋白、胞核蛋白及细胞培养上清蛋白，Western Blot检测细胞HMGB1总蛋白、胞浆蛋白、胞核蛋白及细胞培养上清蛋白含量;干预4h后分别收集每组细胞培养液，ELISA法检测早期炎症因子IL-1β、IL-6、TNF-α及单核细胞趋化蛋白MCP-1含量； 结果：CaP+1000ng/ml Fetuin-A组、CaP+2500ng/ml Fetuin-A、CaP+5000ng/ml Fetuin-A组巨噬细胞HMGB1mRNA的表达均低于CaP晶体组，和CaP晶体组比较，差异有统计学意义（P0.05）；CaP+1000ng/mlFetuin-A组、CaP+2500ng/ml Fetuin-A、CaP+5000ng/ml Fetuin-A组细胞内总HMGB1蛋白、胞浆HMGB1蛋白以及细胞上清HMGB1蛋白含量均少于CaP晶体组，而胞核HMGB1蛋白含量均高于CaP晶体组，和CaP晶体组比较，差异有统计学意义(P 0.05)；CaP+1000ng/ml Fetuin-A组、CaP+2500ng/ml Fetuin-A、CaP+5000ng/ml Fetuin-A组细胞培养上清内早期炎症因子IL-1β、IL-6、TNF-α及单核/巨噬细胞趋化蛋白MCP-1含量均少于CaP晶体组，和CaP晶体组比较，差异有统计学意义（P 0.05）； 结论：1. Fetuin-A可以降低CaP晶体诱导巨噬细胞HMGB1mRNA转录水平，抑制HMGB1蛋白的合成；2. Fetuin-A可以抑制CaP晶体诱导巨噬细胞HMGB1胞核向胞浆转位及释放，降低CaP晶体干预后细胞培养液中HMGB1的含量；3. Fetuin-A可以抑制CaP晶体诱导巨噬细胞释放早期炎症因子IL-1β、IL-6、TNF-α及单核/巨噬细胞趋化蛋白MCP-1，降低CaP晶体刺激后细胞培养上清中早期炎症因子IL-1β、IL-6、TNF-α及单核/巨噬细胞趋化蛋白MCP-1含量。
[Abstract]:Aim: to investigate the inhibitory effect of fetuin-A on the expression of human macrophages and the release of advanced inflammatory factor high mobility protein (HMGB1) induced by calcium phosphate (CaP). Methods: the U937 cells were successfully induced to macrophage by 100ugr / ml CaPU 100ug / ml CaP 1000ng / ml Fetuin-Ang-1 / ml CaP 2500ng / ml Fetuin-Ang-1 ml CaP 5000ng / ml Fetuin-A. After 24 hours of stimulation, the total RNA-RNA RT-PCR was extracted from the cultured cells of each group to detect the expression of HMGB1mRNA, and the total protein, cytosolic protein, cytosolic protein of the cells were extracted. The contents of total protein, cytoplasmic protein, nuclear protein and supernatant protein in cell culture were determined by Western Blot. Four hours after intervention, the levels of IL-1 尾 -IL-6 TNF- 伪 and monocyte chemoattractant protein (MCP-1) were detected by Elisa in each group. Results the HMGB1mRNA expression of macrophages in the cap 2500ng / ml Fetuin-Ang-1 Fetuin-A group was lower than that in the CaP crystal group. Compared with the CaP crystal group, there was a significant difference in the total HMGB1 protein in the CaP2500ng-1 / ml Fetuin-A group and in the CAP2500ng-1 / ml Fetuin-Atuin-CaP5000ng-1 Fetuin-A group. The content of cytoplasmic HMGB1 protein and supernatant HMGB1 protein was lower than that of CaP crystal group, but the content of HMGB1 protein in nucleus was higher than that in CaP crystal group, which was higher than that in CaP crystal group. There was significant difference in the content of IL-1 尾 -IL-6TNF- 伪 and monocyte / macrophage chemoattractant protein (MCP-1) in the supernatant of the cell culture supernatant in the Fetuin-A group (P < 0.05). The difference was statistically significant compared with that in the CaP crystal group (P 0.05). Conclusion Fetuin-A can decrease the level of HMGB1mRNA transcription in macrophages induced by CaP crystal and inhibit the synthesis of HMGB1 protein. Fetuin-A can inhibit the translocation and release of HMGB1 nucleus into cytoplasm induced by CaP crystal. Fetuin-A could inhibit the release of early inflammatory factor IL-1 尾 -IL-6TNF- 伪 and monocyte / macrophage chemoattractant protein MCP-1 by CaP crystal, and decrease the supernatant of cell culture stimulated by CaP crystal, and decrease the content of HMGB1 in the cell culture medium after the intervention of CaP crystal. Fetuin-A can inhibit the release of the early inflammatory factor IL-1 尾 -tNF- 伪 and monocyte / macrophage chemotactic protein MCP-1 by CaP crystal. The content of IL-1 尾 -IL-6 TNF- 伪 and monocyte / macrophage chemoattractant protein (MCP-1).
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.4

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