巨噬细胞与肝实质细胞在肝脏代谢性炎症发生中的作用的实验研究

发布时间：2018-05-19 08:17

本文选题：内科学 + 肝脏代谢性炎症　；参考：《重庆医科大学》2014年硕士论文

【摘要】：目的：研究巨噬细胞与肝实质细胞在肝脏代谢性炎症发生中的作用及其可能机制。方法：选用不同浓度的软脂酸（Palmitic Acid，PA）（0mmol/L、0.08mmol/L、0.16mmol/L、0.32mmol/L）分别单独处理HepG2细胞以及THP-1来源巨噬细胞（以下简称：巨噬细胞），24h后运用MTT法检测不同浓度PA对细胞增殖的影响，同时运用实时荧光定量聚合酶链反应（real-time PCR）以及免疫印迹法(western blot)分别检测两种细胞中各炎症因子（IL-1β、IL-6、TNF-a）的基因表达水平以及蛋白表达水平；然后选用0.4μm孔径大小的Transwell小室建立HepG2细胞与巨噬细胞的共培养体系，选用PA(0.16mmol/L)处理细胞24h后分别检测HepG2细胞及巨噬细胞中（IL-1β、IL-6、TNF-a）及巨噬细胞（MCP-1、Mannose-R、CD163）的基因表达水平；同时，采用5μm孔径大小的Transwell小室建立HepG2细胞与巨噬细胞共培养体系，选用0.1%结晶紫染色法（crystal violet staining）观察巨噬细胞的迁移情况。结果：在不同浓度PA（0.08mmol/L、0.16mmol/L、0.32mmol/L）处理下，PA对HepG2细胞及巨噬细胞的增殖的影响无统计学意义，在两种细胞中各炎症因子（IL-1β、IL-6、TNF-a）基因表达水平及蛋白表达水平均较对照组增加；将HepG2细胞与巨噬细胞共培养后，HepG2细胞及巨噬细胞中各炎症因子基因表达水平均较对照组（HepG2细胞或巨噬细胞单独培养组）升高，，其中，在有PA（0.16mmol/L）处理下时，这种升高趋势更为明显；结晶紫染色显示，共培养后巨噬细胞迁移的数量明显增加，巨噬细胞中MCP-1的基因表达水平均较对照组明显升高；而CD163、Mannose-R基因表达较对照组明显下降。结论：软脂酸能分别诱导HepG2细胞和巨噬细胞的炎症产生，当两种细胞共培养后，细胞各自的炎症反应进一步加剧，其机制可能与巨噬细胞迁移能力增加，及细胞表型的转换有关。
[Abstract]:Aim: to study the role of macrophages and hepatic parenchyma cells in the development of hepatic metabolic inflammation and its possible mechanism. Methods: HepG2 cells and macrophages derived from THP-1 were treated separately with different concentrations of Palmitic acid (Palmitic acid) 0 mmol / L 0.08 mmol / L ~ (0.08) mmol / L ~ (0.16) mmol / L ~ (0.32) mmol 路L ~ (-1) and macrophages derived from THP-1 for 24 h. The effects of different concentrations of PA on cell proliferation were detected by MTT assay. At the same time, real-time PCR and Western blot were used to detect the gene expression and protein expression of IL-1 尾 -IL-6 TNF-a in the two kinds of cells. Then the co-culture system of HepG2 cells and macrophages was established with 0.4 渭 m pore size Transwell chamber. After 24 hours of treatment with PAA 0.16 mmol / L, the expression levels of IL-1 尾 -IL-6IL-6 TNF-a and MCP-1 / Mannose-RCD163 were detected in HepG2 cells and macrophages, respectively. The co-culture system of HepG2 cells and macrophages was established by Transwell chamber with 5 渭 m pore size. The migration of macrophages was observed by 0.1% crystal violet staining. Results: there was no significant difference in the proliferation of HepG2 cells and macrophages treated with different concentrations of PA-0.08 mmol / L 0.16 mmol / L (0.32 mmol 路L ~ (-1). The expression level of IL-1 尾 IL-6 / TNF-a gene and protein in the two kinds of cells were higher than those in the control group. After co-culture of HepG2 cells and macrophages, the expression level of inflammatory factor genes in HepG2 cells and macrophages was higher than that in the control group (HepG2 cells or macrophages alone). Crystal violet staining showed that the number of macrophage migration was significantly increased, the expression of MCP-1 gene in macrophages was significantly higher than that in control group, and the expression of CD163 Mannose-R gene was significantly lower than that in control group. Conclusion: palmitate can induce inflammation of HepG2 cells and macrophages, and the inflammatory response of HepG2 cells and macrophages increases after co-culture, and the mechanism may be related to the migration ability of macrophages. And the transformation of cell phenotype.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575

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