靶向精氨酸转运体siRNA阻断诱生型一氧化氮合酶下游途径的实验研究

发布时间：2018-05-17 23:06

本文选题：L-arginine + CAT-2　；参考：《南京大学》2011年硕士论文

【摘要】：目的利用RNA干扰(RNA interference, RNAi)技术,设计并构建可以表达CAT-2 siRNA的重组质粒载体,将其转染RAW264.7细胞后,观察RNAi技术抑制CAT-2表达后iNOS下游调控途径的变化。方法1.根据基因库收录的小鼠CAT-2基因全序列及RNAi原理,设计并构建可以表达CAT-2 siRNA的四组重组质粒载体及阴性对照质粒,pSilencer-CAT-2-1、pSilencer-CAT-2-2、pSilencer-CAT-2-3、pSilencer-CAT-2-4、pSilencer-control。2.将5种质粒分别转染RAW264.7细胞后,筛选出稳定转染且CAT-2抑制效率高的细胞系。3.检测CAT-2B抑制率高的细胞系经LPS活化后NO合成、L-arginine的跨膜转运、iNOS及CAT-2表达的变化。结果1.成功构建表达CAT-2 siRNA质粒载体,pSilencer-CAT-2-1、pSilencer-CAT-2、pSilencer-CAT-2-3、pSilencer-CAT-2-4、pSilencer-control; 2筛选出CAT-2抑制率高的稳定转染细胞系pSilencer-CAT2-4。3.与对照组比较,pSilencer-CAT2-4细胞系经LPS活化后,NO合成、L-arginine跨膜转运、CAT-2表达明显下降,而iNOS表达无明显变化。结论pSilencer-CAT2-4重组质粒表达的siRNA可以沉默CAT-2基因表达,进而阻断L-arginine的跨膜转运,从而在底物水平上抑制NO合成。为进一步实验研究提供理论及技术基础。
[Abstract]:Objective to design and construct a recombinant plasmid vector expressing CAT-2 siRNA by RNA interference RNA interference (RNAi) technique, and to observe the change of downstream regulation pathway of iNOS after RNAi technique inhibited CAT-2 expression in RAW264.7 cells. Method 1. According to the whole sequence of mouse CAT-2 gene and the principle of RNAi, four groups of recombinant plasmids expressing CAT-2 siRNA were designed and constructed, and the negative control plasmids, pSilencer-CAT-2-2, pSilencer-CAT-2-3, pSilencer-CAT-2-4, pSilencer-control.2were designed and constructed. After transfection of five plasmids into RAW264.7 cells, a stable transfection cell line. 3. 3 was selected with high inhibition efficiency of CAT-2. The transmembrane transport of L-arginine and the expression of CAT-2 in the cell lines with high inhibition rate of CAT-2B were detected after the activation of LPS. Result 1. The expression of CAT-2 siRNA plasmid pSilencer-CAT-2-1 pSilencer-CAT-2 + pSilencer-CAT-2-3 pSilencer-CAT-2-4 pSilencer-control. 2 stable transfection cell line pSilencer-CAT2-4.3 with high inhibition rate of CAT-2 was successfully constructed. Compared with the control group, the expression of L-arginine transporter CAT-2 was significantly decreased in pSilencer-CAT2-4 cell line activated by LPS, but the expression of iNOS was not changed. Conclusion siRNA expressed by pSilencer-CAT2-4 recombinant plasmid can silence the expression of CAT-2 gene and block the transmembrane transport of L-arginine, thus inhibiting the synthesis of no at substrate level. To provide theoretical and technical basis for further experimental research.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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