本文關(guān)鍵詞： 肺泡巨噬細(xì)胞 ATP結(jié)合盒轉(zhuǎn)運(yùn)體A1 小干擾RNA 篩選 轉(zhuǎn)染復(fù)合物 細(xì)胞毒性 脂多糖 巨噬細(xì)胞 肺泡 ATP結(jié)合盒轉(zhuǎn)運(yùn)體A1　出處：《蘇州大學(xué)》2013年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的篩選有效抑制大鼠肺泡巨噬細(xì)胞上ABCA1(ATP-binding cassette transporterA1)表達(dá)的小干擾RNA(simall interference RNA，siRNA)序列，并檢測(cè)轉(zhuǎn)染復(fù)合物的細(xì)胞毒性。 方法設(shè)計(jì)并合成針對(duì)ABCA1的siRNA3條(siRNA1172, siRNA5948,siRNA6072)及1條與ABCA1基因無(wú)同源性的帶綠色熒光標(biāo)記的陰性對(duì)照siRNA，在HiPerFectTransfection Reagent介導(dǎo)下轉(zhuǎn)染大鼠肺泡巨噬細(xì)胞。用RT-PCR方法測(cè)定干擾后大鼠肺泡巨噬細(xì)胞上ABCA1mRNA表達(dá)，流式細(xì)胞術(shù)方法測(cè)定干擾后ABCA1蛋白表達(dá)，比較其抑制率，篩選出有效抑制靶基因表達(dá)的siRNA。采用MTT法檢測(cè)轉(zhuǎn)染復(fù)合物的細(xì)胞毒性。 結(jié)果①siRNA(50nmol·L~(-1))和HiPerFect Transfection Reagent(6μL)有較高的轉(zhuǎn)染效率；②與陰性對(duì)照組相比，ABCA1siRNA(50nmol·L~(-1))干擾大鼠肺泡巨噬細(xì)胞24h后ABCA1mRNA的相對(duì)表達(dá)量降低，分別降低：71.97%(siRNA1172）,77.96%(siRNA5948）,76.73%(siRNA6072）。③與陰性對(duì)照組相比，ABCA1siRNA(50nmol·L~(-1))干擾大鼠肺泡巨噬細(xì)胞48h后ABCA1蛋白表達(dá)降低，分別降低：65.88%(siRNA1172）,63.88%(siRNA5948）,62.17%(siRNA6072）。 結(jié)論①三條siRNA均可有效抑制大鼠肺泡巨噬細(xì)胞ABCA1的表達(dá)。②轉(zhuǎn)染試劑HiPerFect Transfection Reagent6μl與終濃度為50nmol·L~(-1) siRNA，為合適的轉(zhuǎn)染比例。 目的觀察脂多糖（LPS)對(duì)大鼠肺泡巨噬細(xì)胞ATP結(jié)合盒轉(zhuǎn)運(yùn)體A1(ATP-bindingcassette transporter A1，ABCA1)表達(dá)的影響,以及ABCA1對(duì)于大鼠肺泡巨噬細(xì)胞表面Toll樣受體4（Toll-like receptor4,TLR4)的影響，探索在LPS誘發(fā)的大鼠肺泡巨噬細(xì)胞炎癥反應(yīng)過(guò)程中，ABCA1的調(diào)節(jié)機(jī)制。 方法分別以不同濃度的LPS（0，0.2，2，20，200μg/L)作用于大鼠肺泡巨噬細(xì)胞，24h后采用RT-PCR方法測(cè)定大鼠肺泡巨噬細(xì)胞ABCA1mRNA表達(dá)，流式細(xì)胞術(shù)方法測(cè)定ABCA1蛋白表達(dá)。以相同濃度的LPS（20μg/L）作用于大鼠肺泡巨噬細(xì)胞，于不同的時(shí)間點(diǎn)（0，2，6，12，24h),采用上述方法分別測(cè)定ABCA1mRNA和蛋白表達(dá)。此外，聯(lián)合使用20μg/L LPS以及特異性抑制ABCA1表達(dá)的siRNA干擾大鼠肺泡巨噬細(xì)胞，12h后，采用上述方法分別測(cè)定TLR4mRNA和蛋白表達(dá)。 結(jié)果①LPS呈濃度和時(shí)間依賴性抑制ABCA1mRNA及蛋白質(zhì)的表達(dá)（P0.05)。②大鼠肺泡巨噬細(xì)胞ABCA1的表達(dá)被特異性siRNA抑制后LPS引起的TLR4上調(diào)較正常組更為明顯（P0.05)。 結(jié)論在LPS誘發(fā)的大鼠肺泡巨噬細(xì)胞炎癥反應(yīng)過(guò)程中，，ABCA1可能通過(guò)影響TLR4的表達(dá)參與炎癥的調(diào)節(jié)。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R563.8;R3416

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