慢病毒介导的nhe1基因干扰对raw264.7细胞内胆固醇含量的影响-influence of nhe 1 gene interference mediated by lentivirus on cholesterol content in raw 264.7 cells.docxVIP

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慢病毒介导的nhe1基因干扰对raw264.7细胞内胆固醇含量的影响-influence of nhe 1 gene interference mediated by lentivirus on cholesterol content in raw 264.7 cells.docx

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慢病毒介导的nhe1基因干扰对raw264.7细胞内胆固醇含量的影响-influence of nhe 1 gene interference mediated by lentivirus on cholesterol content in raw 264.7 cells

万方数据万方数据色谱法结果显示，有效抑制 NHE1 可以明显降低细胞内 TC 及 CE 含量，CE/TC 值降低。提示 NHE1 可能参与 Ox-LDL 干预下巨噬细胞内脂代谢的调节。【关键词】钠氢交换体1；脂代谢；ATP结合盒转运蛋白 A1；RNA干扰；高效液相色谱法 III Effect of NHE1 shRNA Mediated by lentiviral on Intracellular Cholesterol Content in RAW264.7 Cell Major: Geriatric Medicine Postgraduate: Zhang Luochao Supervisor:Professor Zhang Li [Abstract] Objective: The aim of this study is to explore the effect of NHE1 shRNA mediated by lentiviral on intracellular cholesterol content in RAW264.7 cell.Methods: NHE1-shRNA lentiviral vector and scramble small RNA lentiviral vector as negative control were obtained commercially,sequence analysis and sequence alignment were used to test sequence.Transfected RAW264.7 cells with the Packed lentiviral, RAW264.7 were infected by the lentiviral vector by virus dilution MOI 100, 50, 25, 10, 5, 1, in order to determine the appropriate MOI. Experimental groups include: the lentiviral-specific transfection group, the scramble virus negative control group and the uninfected control group,transfection efficiency was detected by immunofluorescence technique.Combined enzymatic and HPLC was carried out to assay intracellular free cholesterol (FC) and total cholesterol (TC) content ,thereby worked out cholesteryl ester(CE) content and CE/TC.Results: Packed lentiviral sequence analysis and sequence alignment were accord with Genbank.RAW264.7 cells were optimally infected at MOI 50.The result of qPCR had shown that NHE1 mRNA in NHE1-shRNA lentiviral interfering group was reduced (0.237±0.061) (P＜ 0.05)，compared with negative control group, whereas there was no statistically different between negative control group (1.145±0.475) and blank control group (0.977±0.118).The result of western blotting show that ， NHE1 protein in NHE1-shRNA lentiviral interfering group was reduced (4.697±1.233)(P＜0.05)， compared with negative control group，whereas there was no statistically different between negative control group (31.196±2.419) and blank co