ABCA1调节大鼠肺泡巨噬细胞炎症反应过程机制的研究
发布时间:2018-02-21 17:31
本文关键词: 肺泡巨噬细胞 ATP结合盒转运体A1 小干扰RNA 筛选 转染复合物 细胞毒性 脂多糖 巨噬细胞 肺泡 ATP结合盒转运体A1 出处:《苏州大学》2013年硕士论文 论文类型:学位论文
【摘要】:目的筛选有效抑制大鼠肺泡巨噬细胞上ABCA1(ATP-binding cassette transporterA1)表达的小干扰RNA(simall interference RNA,siRNA)序列,并检测转染复合物的细胞毒性。 方法设计并合成针对ABCA1的siRNA3条(siRNA1172, siRNA5948,siRNA6072)及1条与ABCA1基因无同源性的带绿色荧光标记的阴性对照siRNA,在HiPerFectTransfection Reagent介导下转染大鼠肺泡巨噬细胞。用RT-PCR方法测定干扰后大鼠肺泡巨噬细胞上ABCA1mRNA表达,流式细胞术方法测定干扰后ABCA1蛋白表达,比较其抑制率,筛选出有效抑制靶基因表达的siRNA。采用MTT法检测转染复合物的细胞毒性。 结果①siRNA(50nmol·L~(-1))和HiPerFect Transfection Reagent(6μL)有较高的转染效率;②与阴性对照组相比,ABCA1siRNA(50nmol·L~(-1))干扰大鼠肺泡巨噬细胞24h后ABCA1mRNA的相对表达量降低,分别降低:71.97%(siRNA1172),77.96%(siRNA5948),76.73%(siRNA6072)。③与阴性对照组相比,ABCA1siRNA(50nmol·L~(-1))干扰大鼠肺泡巨噬细胞48h后ABCA1蛋白表达降低,分别降低:65.88%(siRNA1172),63.88%(siRNA5948),62.17%(siRNA6072)。 结论①三条siRNA均可有效抑制大鼠肺泡巨噬细胞ABCA1的表达。②转染试剂HiPerFect Transfection Reagent6μl与终浓度为50nmol·L~(-1) siRNA,为合适的转染比例。 目的观察脂多糖(LPS)对大鼠肺泡巨噬细胞ATP结合盒转运体A1(ATP-bindingcassette transporter A1,ABCA1)表达的影响,以及ABCA1对于大鼠肺泡巨噬细胞表面Toll样受体4(Toll-like receptor4,TLR4)的影响,探索在LPS诱发的大鼠肺泡巨噬细胞炎症反应过程中,ABCA1的调节机制。 方法分别以不同浓度的LPS(0,0.2,2,20,200μg/L)作用于大鼠肺泡巨噬细胞,24h后采用RT-PCR方法测定大鼠肺泡巨噬细胞ABCA1mRNA表达,流式细胞术方法测定ABCA1蛋白表达。以相同浓度的LPS(20μg/L)作用于大鼠肺泡巨噬细胞,于不同的时间点(0,2,6,12,24h),采用上述方法分别测定ABCA1mRNA和蛋白表达。此外,联合使用20μg/L LPS以及特异性抑制ABCA1表达的siRNA干扰大鼠肺泡巨噬细胞,12h后,采用上述方法分别测定TLR4mRNA和蛋白表达。 结果①LPS呈浓度和时间依赖性抑制ABCA1mRNA及蛋白质的表达(P0.05)。②大鼠肺泡巨噬细胞ABCA1的表达被特异性siRNA抑制后LPS引起的TLR4上调较正常组更为明显(P0.05)。 结论在LPS诱发的大鼠肺泡巨噬细胞炎症反应过程中,,ABCA1可能通过影响TLR4的表达参与炎症的调节。
[Abstract]:Objective to screen the small interfering RNA(simall interference siRNAs that effectively inhibit the expression of ABCA1(ATP-binding cassette transporter A1 on rat alveolar macrophages, and to detect the cytotoxicity of the transfection complexes. Methods siRNA3 siRNA1172 and siRNA5948 siRNA6072 targeting ABCA1 were designed and synthesized, and a negative control siRNAwith no homology to ABCA1 gene was designed and synthesized. The siRNAs were transfected into rat alveolar macrophages mediated by HiPerFectTransfection Reagent. The lung was measured by RT-PCR method. ABCA1mRNA expression on alveolar macrophages, Flow cytometry was used to detect the expression of ABCA1 protein after interference, and the inhibition rate was compared. Sirna, which could effectively inhibit the expression of target gene, was screened out. The cytotoxicity of the transfected complex was detected by MTT assay. Results (1) siRNA-50 nmol 路L ~ (-1) and HiPerFect Transfection Reagent(6 渭 L) had a higher transfection efficiency. Compared with the negative control group, the relative expression of ABCA1mRNA in rat alveolar macrophages was decreased 24 hours after the treatment with 50 nmol 路L ~ (-1) siRNA-50 nmol 路L ~ (-1) siRNA-1). The ABCA1 protein expression of alveolar macrophages was decreased after 48 hours after treatment with 71.97% siRNA1172 and 77.96 siRNA5948 and 76.73 siRNA60723.Compared with the negative control group, the expression of ABCA1 protein in alveolar macrophages was decreased, and the expression of ABCA1 protein in alveolar macrophages was decreased after 48h, and the siRNA59484872siRNA592.172siRNA594.72siRNA 55.88siRNA was decreased respectively. Conclusion 1 three siRNA can effectively inhibit the expression of ABCA1 in rat alveolar macrophages. 2. The transfection reagent HiPerFect Transfection Reagent6 渭 l and the final concentration of 50 nmol 路L ~ (-1) siRNAs are suitable for transfection. Objective to observe the effect of lipopolysaccharide (LPS) on the expression of ATP binding cassette transporter A1A1ABCA1 in rat alveolar macrophages and the effect of ABCA1 on Toll like receptor 4Toll-like receptor 4TLR4 on the surface of alveolar macrophages in rats. To explore the regulatory mechanism of ABCA1 in the inflammatory response of alveolar macrophages induced by LPS in rats. Methods the expression of ABCA1mRNA in rat alveolar macrophages was measured by RT-PCR method after treated with 200 渭 g 路L ~ (-1) of different concentrations of LPS0 ~ (0.2) and 20 ~ (20 渭 g / L) on rat alveolar macrophages for 24 hours. The expression of ABCA1 protein was measured by flow cytometry. The same concentration of LPS(20 渭 g / L was used to treat rat alveolar macrophages. At different time points, the expression of ABCA1mRNA and protein were measured at different time points. After combined use of 20 渭 g / L LPS and siRNA which specifically inhibited the expression of ABCA1, the expression of TLR4mRNA and protein in alveolar macrophages was determined by the above method for 12 h. Results 1LPs inhibited the expression of ABCA1mRNA and protein in a concentration-and time-dependent manner. The expression of ABCA1 in rat alveolar macrophages was inhibited by specific siRNA. The up-regulation of TLR4 induced by LPS was more obvious than that in normal group. Conclusion during the inflammatory reaction induced by LPS in rat alveolar macrophages, ABCA1 may be involved in the regulation of inflammation by affecting the expression of TLR4.
【学位授予单位】:苏州大学
【学位级别】:硕士
【学位授予年份】:2013
【分类号】:R563.8;R3416
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