氧化低密度脂蛋白对小鼠DC2.4细胞CD47及microRNA-155表达的影响

发布时间：2018-05-24 02:51

本文选题：氧化低密度脂蛋白 + DC2.4　；参考：《广州医学院》2012年硕士论文

【摘要】：目的观察氧化低密度脂蛋白（oxidized low-density lipoprotein，ox-LDL）对小鼠树突状细胞株DC2.4细胞形态、表型成熟、膜蛋白CD47及microRNA-155表达的影响，初步探讨CD47和microRNA-155在动脉粥样硬化（atherosclerosis，AS）条件下在DC成熟和迁移中的潜在作用。方法 1. DC2.4接种于6孔板中，用含10%胎牛血清的RPMI1640完全培养液培养至第3天，改用无血清培养基培养并分为2组，实验组加入ox-LDL（100ug/mL），对照组加入磷酸缓冲液（PBS）。使用光学显微镜观察细胞形态学变化；分别于干预后12小时、24小时收集DC2.4细胞，采用流式细胞仪检测细胞表型（MHC-II，CD80和CD86）的表达。 2. DC2.4接种于6孔板中，用含10%胎牛血清的RPMI1640完全培养液培养至第3天，用无血清培养基培养并分为2组，实验组加入ox-LDL（100ug/mL），对照组加入磷酸缓冲液（PBS）。分别于干预后12小时、24小时收集DC2.4细胞，分别提取蛋白后采用Western blot方法测定细胞表面CD47分子的表达；提取RNA后采用荧光定量PCR方法测定DC2.4细胞中microRNA-155的表达。结果 1.1DC2.4形态学观察：对照组细胞形态呈圆形或类圆形、较小、透亮，周边少量毛刺状或棘状突起；ox-LDL刺激12h及24h后观察细胞，发现细胞体积增大，胞浆逐渐丰富，呈不规则多型状态，具有典型的树突状突起。 1.2DC2.4成熟表型变化：收集实验组和对照组细胞进行流式细胞检测，经过ox-LDL处理后的DC2.4表型（MHC-II，CD80和CD86）的表达明显增加，与PBS对照组相比，存在统计学差异（P＜0.05）。 2.1经过ox-LDL处理后的DC2.4CD47的表达下降，与PBS对照组相比，存在统计学差异（P＜0.05）。 2.2经过ox-LDL处理后的DC2.4microRNA-155的表达增高，，与PBS对照组相比，存在统计学差异（P＜0.05）。结论 1.ox-LDL可使DC2.4表面类分子MHC-II，CD80和CD86高表达，诱导DC2.4成熟。 2. ox-LDL可诱导DC2.4表面CD47分子下调表达、microRNA-155上调表达。
[Abstract]:Purpose To observe the effects of oxidized low density lipoprotein (low-density) on the morphology, phenotypic maturation, membrane protein CD47 and microRNA-155 expression of mouse dendritic cell line DC2.4, and to explore the potential role of CD47 and microRNA-155 in DC maturation and migration under the condition of atherosclerotic atherosclerosis. Method 1. DC2.4 was inoculated in 6-well plate and cultured in 10% fetal bovine serum RPMI1640 for 3 days, then cultured in serum-free medium and divided into two groups. The experimental group was added ox-LDLN 100ugmLX, and the control group was added phosphoric acid buffer solution (PBS). DC2.4 cells were collected at 12 hours and 24 hours after intervention, and the expression of MHC-IIP CD80 and CD86 were detected by flow cytometry. 2. DC2.4 was inoculated in a 6-well plate and cultured in 10% fetal bovine serum RPMI1640 for 3 days. The RPMI1640 was cultured in serum-free medium and divided into two groups. The experimental group was added ox-LDLN 100ugmLX, and the control group was added phosphoric acid buffer solution (PBS). DC2.4 cells were collected at 12 hours and 24 hours after intervention. The expression of CD47 on the cell surface was measured by Western blot method after protein extraction, and the expression of microRNA-155 in DC2.4 cells was detected by fluorescence quantitative PCR after RNA extraction. Result Morphological observation of 1.1DC2.4: the cells in the control group were round or round, small and bright, and a small amount of burrs or spikes around the cells were observed after 12 h and 24 hours of stimulation. It was found that the size of the cells increased, the cytoplasm was gradually rich, and the cells were irregular and polytype. Having typical dendritic protuberances. Changes of mature phenotype of 1.2DC2.4: the expression of MHC-IIP CD80 and CD86 in DC2.4 treated with ox-LDL was significantly increased, compared with that in control group (P < 0.05), and the expression of MHC-IIP CD80 and CD86 was significantly increased after ox-LDL treatment (P < 0.05). 2.1 the expression of DC2.4CD47 decreased after ox-LDL treatment, and there was significant difference between the two groups (P < 0.05). 2.2 the expression of DC2.4microRNA-155 was increased after ox-LDL treatment, and there was significant difference between the two groups (P < 0.05). Conclusion 1.ox-LDL could induce the expression of MHC-IIP CD80 and CD86 on the surface of DC2.4 and induce DC2.4 maturation. 2. Ox-LDL could induce down-regulation of CD47 expression on DC2.4 surface and upregulate the expression of microRNA-155.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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