亲磷脂酸磷脂酶A1基因沉默对胰岛素分泌影响的研究

发布时间：2018-03-09 06:37

本文选题：亲磷脂酸磷脂酶A1　切入点：基因表达　出处：《桂林医学院》2012年硕士论文　论文类型：学位论文

【摘要】：目的：研究亲磷脂酸磷脂酶A1(phosphatidic acid-preferring phospholipase Al, PA-PLA1)基因沉默对小鼠胰岛p细胞株MIN6胰岛素分泌的影响。方法：采用invitrogen网站提供的在线RNAi设计软件设计、合成小鼠PA-PLA1靶向shRNA,以Lipofectamin2000为转染试剂转染小鼠胰岛素分泌细胞MIN6,用荧光显微镜检测干扰载体的转染效率,实时荧光定量PCR检测MIN6细胞中PA-PLA1mRNA的表达水平,Western Blotting检测PA-PLA1蛋白的表达水平,同时对MIN6细胞行葡萄糖刺激胰岛素分泌实验,放免法测定细胞上清液中胰岛素分泌量。结果：本实验设计、合成了小鼠PA-PLA1靶向shRNA1、2,测序结果证实shRNA编码序列与设计的片段完全一致。转染MIN6细胞48h后荧光显微镜检测其干扰载体的转染效率约为50%。实时荧光定量PCR和Western Blotting检测,干扰载体1、2PA-PLA1mRNA表达水平分别下降11%和53%,蛋白表达水平分别下降53%和59%。对经RNAi处理过的MIN6细胞行葡萄糖刺激胰岛素分泌实验,测得细胞胰岛素分泌水平下降30%。结论：本文设计、合成了小鼠PA-PLA1靶向shRNA。所构建的小鼠PA-PLA1靶向shRNA干扰载体能有效转染小鼠胰岛分泌细胞MIN6。PA-PL A1基因沉默后的MIN6细胞胰岛素的分泌水平明显下降,表明PA-PLA1基因表达与MIN6细胞胰岛素分泌存在相关性,提示该酶对胰岛β细胞胰岛素分泌可能发挥正调节作用。本研究结果为进一步研究胰岛β细胞信号转导和胰岛素分泌的分子机制提供了新的线索。
[Abstract]:Objective: To study the phosphatidic acid phospholipase A1 (phosphatidic acid-preferring phospholipase Al, PA-PLA1) of MIN6 gene silencing on insulin secretion in mouse islet cell line P. Methods: the design of online RNAi design software available on the Invitrogen website, the synthesis of mouse PA-PLA1 targeting shRNA to Lipofectamin2000, and then was transfected into mouse insulin secreting cells by fluorescence MIN6. Microscope interference vector transfection efficiency and expression level of PA-PLA1mRNA was detected by real-time fluorescent quantitative PCR in MIN6 cells, the expression level of PA-PLA1 protein was detected by Western Blotting, while the MIN6 cells for glucose stimulated insulin secretion test, radioimmunoassay in insulin secretion in the supernatant. Results: the experimental design, synthesis of mouse PA-PLA1 targeting shRNA1,2 sequencing results confirmed that the design of shRNA encoding sequence and fragment identical. Transfection of MI N6 cells were detected by fluorescence microscopy after 48h interference vector transfection efficiency was about 50%. PCR and Western Blotting real-time fluorescence quantitative detection, interference vector 1,2PA-PLA1mRNA expression levels were decreased to 11% and 53%, protein expression levels were decreased by 53% and 59%. RNAi treated MIN6 cells for glucose stimulated insulin secretion test measured insulin conclusion: the decreased 30%. secretion of mouse PA-PLA1 mouse PA-PLA1 target design, target construction to synthesize shRNA. to shRNA interference vector can effectively transfected MIN6 cells insulin secretion of mouse islet secreting cells MIN6.PA-PL A1 gene silence was obviously decreased, suggesting that the PA-PLA1 gene expression correlated with MIN6 cell insulin secretion, suggesting that the enzyme on the islet beta cell insulin secretion may play a positive regulatory role. The results of this study for further study of islet beta cell signal The molecular mechanisms of signal transduction and insulin secretion provide new clues.

【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

【参考文献】