临床医学工程
臨床醫學工程
림상의학공정
Clinical Medical & Engineering
2015年
11期
1429-1431
,共3页
亚甲蓝%脂多糖%巨噬细胞%炎性反应
亞甲藍%脂多糖%巨噬細胞%炎性反應
아갑람%지다당%거서세포%염성반응
Methylene-blue%Lipopolysaccharide (LPS)%Macrophage%Inflammation reaction
目的 观察亚甲蓝对脂多糖 (LPS) 诱导小鼠RAW246.7巨噬细胞炎性反应因子表达和细胞NF-κB活性的影响, 以初步探讨亚甲蓝在LPS诱导巨噬细胞炎性反应中的作用. 方法 体外培养小鼠巨噬细胞系RAW264.7细胞, 利用脂多糖 (LPS)刺激RAW264.7细胞建立体外内毒素炎性反应模型. 进行实验分组: Ⅰ组 (对照组): 完全无血清培养基孵育; Ⅱ组 (模型组):在无血清培养基中加入浓度为1 g/mL的LPS进行孵育; Ⅲ组 (亚甲蓝组): 完全无血清培养基中加入亚甲蓝20μmo1/L, 2 h后加入1 g/mL的LPS孵育, 细胞培养24 h后: ①实时荧光定量聚合酶链反应 (rea1-time PCR) 观察IL-6、 IL-8、 MCP-1 mRNA的表达; ②酶联免疫吸附法 (ELISA) 检测细胞培养上清液中IL-6、 IL-8、 MCP-1蛋白水平; ③荧光素酶报告实验 (luciferase activity assay) 检测细胞中NF-κB的活性. 结果 LPS刺激巨噬细胞后, 细胞中炎性反应因子mRNA和蛋白表达增高, 而亚甲蓝预处理能够减弱LPS对炎性反应因子的诱导作用; LPS可以诱导巨噬细胞NF-κB活化, 亚甲蓝能够抑制NF-κB的激活. 结论 亚甲蓝可以通过抑制NF-κB的激活调控LPS诱导的炎性反应.
目的 觀察亞甲藍對脂多糖 (LPS) 誘導小鼠RAW246.7巨噬細胞炎性反應因子錶達和細胞NF-κB活性的影響, 以初步探討亞甲藍在LPS誘導巨噬細胞炎性反應中的作用. 方法 體外培養小鼠巨噬細胞繫RAW264.7細胞, 利用脂多糖 (LPS)刺激RAW264.7細胞建立體外內毒素炎性反應模型. 進行實驗分組: Ⅰ組 (對照組): 完全無血清培養基孵育; Ⅱ組 (模型組):在無血清培養基中加入濃度為1 g/mL的LPS進行孵育; Ⅲ組 (亞甲藍組): 完全無血清培養基中加入亞甲藍20μmo1/L, 2 h後加入1 g/mL的LPS孵育, 細胞培養24 h後: ①實時熒光定量聚閤酶鏈反應 (rea1-time PCR) 觀察IL-6、 IL-8、 MCP-1 mRNA的錶達; ②酶聯免疫吸附法 (ELISA) 檢測細胞培養上清液中IL-6、 IL-8、 MCP-1蛋白水平; ③熒光素酶報告實驗 (luciferase activity assay) 檢測細胞中NF-κB的活性. 結果 LPS刺激巨噬細胞後, 細胞中炎性反應因子mRNA和蛋白錶達增高, 而亞甲藍預處理能夠減弱LPS對炎性反應因子的誘導作用; LPS可以誘導巨噬細胞NF-κB活化, 亞甲藍能夠抑製NF-κB的激活. 結論 亞甲藍可以通過抑製NF-κB的激活調控LPS誘導的炎性反應.
목적 관찰아갑람대지다당 (LPS) 유도소서RAW246.7거서세포염성반응인자표체화세포NF-κB활성적영향, 이초보탐토아갑람재LPS유도거서세포염성반응중적작용. 방법 체외배양소서거서세포계RAW264.7세포, 이용지다당 (LPS)자격RAW264.7세포건입체외내독소염성반응모형. 진행실험분조: Ⅰ조 (대조조): 완전무혈청배양기부육; Ⅱ조 (모형조):재무혈청배양기중가입농도위1 g/mL적LPS진행부육; Ⅲ조 (아갑람조): 완전무혈청배양기중가입아갑람20μmo1/L, 2 h후가입1 g/mL적LPS부육, 세포배양24 h후: ①실시형광정량취합매련반응 (rea1-time PCR) 관찰IL-6、 IL-8、 MCP-1 mRNA적표체; ②매련면역흡부법 (ELISA) 검측세포배양상청액중IL-6、 IL-8、 MCP-1단백수평; ③형광소매보고실험 (luciferase activity assay) 검측세포중NF-κB적활성. 결과 LPS자격거서세포후, 세포중염성반응인자mRNA화단백표체증고, 이아갑람예처리능구감약LPS대염성반응인자적유도작용; LPS가이유도거서세포NF-κB활화, 아갑람능구억제NF-κB적격활. 결론 아갑람가이통과억제NF-κB적격활조공LPS유도적염성반응.
Objective To observe the effect of methylene-blue on lipopolysaccharide (LPS)-induced inflammatory cytokines production and NF-κB activition, and to explore the roles of methylene-blue in LPS-induced inflammatory reaction in macrophages. Methods Murine macrophage line RAW264.7 cells were stimulated with LPS (1 g/mL) to establish endotoxin inflammation reaction model in v itro. The experiment groups were divided: groupⅠ(control group): completely serum-free mediun incubation; groupⅡ(model group): the concentration of 1 g/mL LPS in serum-free medium for incubation;groupⅢ(methylene-blue group):completely serum-free media culture medium with methylene-blue 20μmol/L, and 1 g/mL LPS incubation after 2 hours, cell culture for 24 hours: ①The expressions of IL-6 mRNA, IL-8 mRNA, MCP-1 mRNA in each group were observed by real-time PCR method;②The protein levels of IL-6, IL-8 and MCP-1 in the culture supernatants were determined; ③The luciferase acivity of NF-κB was detected by luciferase reporter assay. Results The mRNA and protein levels of IL-6, IL-8 and MCP-1 in cytoplasm and supernantants after LPS stimulation significantly increased, and mehylene-blue pretreatment could inhibit the induction of these cytokines by LPS stimulation; LPS stimulation markedly increased the activity of NF-κB, whereas methylene-blue inhibited it. Conclusions Methylene-blue inhibites LPS-induced inflammation reaction through suppressing the activation of NF-κB.
