肝X受体-α对炎症的调节与神经元衍生孤核受体-1的关联

发布时间：2018-03-06 20:37

  本文选题：肝X受体　切入点：枯否细胞　出处：《重庆医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:通过体外细胞试验,探讨肝X受体-α(LXRα)抑制脂多糖(LPS)诱导的小鼠枯否细胞(KCs),炎症反应途径与神经元衍生孤核受体-1(NOR-1)的关系。 方法:用密度梯度离心法分离雄性KM小鼠肝脏中的KCs,并将获得的细胞用含20% FCS的RPMI1640培养液培养24h后随机分为四组:(1)对照组:不含血清的RPMI1640培养30h。(2)LPS处理组:不含血清的RPMI1640培养24h,再以含10μg/ml LPS的无血清培养液培养6h。(3)T0901317组:不含血清的RPMI1640培养6h后,再以T0901317终浓度为1μM的无血清培养液培养24h。(4)LPS+T0901317组:以T0901317终浓度为1μM的无血清培养液培养24h后,再用含10μg/ml LPS的无血清培养液培养6h。实时荧光定量PCR(Real-Time PCR)检测各组细胞的LXRα和NOR-1的mRNA表达水平;Western blotting及免疫细胞荧光染色检测各组细胞的LXRα和NOR-1的蛋白表达水平;酶联免疫吸附法检测各组上清液的TNF-α和IL-10含量。 结果:(1)Real-Time PCR结果显示:LXRαmRNA在联合处理组中表达量高于LPS处理组,其2~(-△△CT)2,结果有显著性差异;LXRαmRNA在T0901317处理组中表达量高于LPS处理组,其2-△△CT2,结果有显著性差异;其在空白组中的表达量高于LPS处理组,其2~(-△△CT)2,结果有统计学意义。NOR-1 mRNA在联合处理组中表达量高于LPS处理组,其2~(-△△CT)2,结果有显著性差异;NOR-1 mRNA在T0901317处理组中表达量高于LPS处理组,其2~(-△△CT)2,结果有显著性差异;其在空白组中的表达量高于LPS处理组,其2~(-△△CT)2,结果有统计学意义。(2)Western blot检测结果显示:LXRα及NOR-1蛋白为单一条带,其相对分子质量分别为50kDa、60kDa,用内参照对待测物灰度值进行标准校正。LPS组LXRα和NOR-1表达最低(其值分别为0.569±0.046和0.335±0.042),明显低于其它三组(对照组分别为:1.014±0.050和0.541±0.017,联合处理组分别为:1.232±0.027和0.819±0.022,T0901317处理组分别为:1.736±0.039和1.031±0.040),差别具有统计学意义(P0.05);与其它三组相比,LXRα和NOR-1蛋白表达都在T0901317处理组最高(P0.05);LPS处理组中,LXRα及NOR-1蛋白表达都低于对照组(P0.05)。(3)免疫细胞荧光染色结果显示:LXRα阳性细胞为胞浆及胞核内绿色荧光颗粒,NOR-1阳性细胞为胞浆及胞核内红色荧光颗粒。LXRα荧光染色强度在对照组、T0901317处理组、LPS处理组及联合处理组分别为(97.52±10.39; 110.04±14.54; 56.98±7.95; 105.58±14.03);NOR-1染色强度在对照组、T0901317处理组、LPS处理组及联合处理组分别为(80.15±4.88; 115.65±8.47; 65.50±5.76; 94.14±4.48 ),表示其蛋白表达水平在 T0901317组最高(P0.05),在LPS处理组最低(P0.05)。此结果与western结果相符。(4)ELISA结果显示:肿瘤坏死因子-α(Tumor necrosis factor-α, TNF-α)在LPS处理组的含量(450.89±78.52)显著高于对照组(5.84±2.35)及T0901317处理组(6.73±1.90),差异具有统计学意义(P0.05);联合处理组TNF-α含量(61.21±17.45)比LPS处理组明显降低(P0.05)。白介素-10(Interleukin-10, IL-10)在联合处理组的含量(537.41±36.41)显著高于对照组(10.67±3.48)及T0901317处理组(10.17±1.82),差异具有统计学意义(P0.05);联合处理组IL-10含量比LPS处理组(61.85±12.41)明显提高(P0.05)。 结论:本实验结果表明,应用T0901317处理后,LXRα和NOR-1的表达都明显增加,LPS所诱生的炎症因子TNF-α大大降低,而抑炎因子IL-10含量大大增加。相对于对照组及T0901317处理组,在经过LPS处理后,LXRα和NOR-1的表达都有降低,TNF-α表达明显上升,IL-10表达明显下降。因此,LXRα与NOR-1存在着相互依赖的关系,LXRα能够通过调节NOR-1的表达而发挥抗炎效应,从而抑制LPS所诱导的Kupffer细胞活化。
[Abstract]:Objective: To investigate the inhibitory effect of liver X receptor alpha (LXR alpha) on lipopolysaccharide (LPS) induced Kupffer cells (KCs) and the relationship between inflammatory response pathway and neuronal derived nuclear receptor -1 (NOR-1) in vitro.
Methods: isolated from male KM mice by KCs density gradient centrifugation, and the cells obtained with 20% FCS RPMI1640 medium after 24h were randomly divided into four groups: (1) control group: Cultured in RPMI1640 30h. (2) LPS group: Cultured in RPMI1640 24h, then with 10 g/ml LPS serum-free medium for 6h. (3) T0901317 group: Cultured in RPMI1640 after 6h, then to T0901317 final concentration of serum-free medium for 24h. 1 M (4) LPS+T0901317 group: T0901317 to a final concentration of 1 M without serum cultured 24h, serum-free medium 6h. real-time fluorescence quantitative PCR with 10 g/ml LPS (Real-Time PCR) to detect the expression of LXR alpha and NOR-1 mRNA expression; the expression level of Western and blotting were assayed by immunofluorescence staining of LXR alpha and NOR-1 protein; ELISA The content of TNF- alpha and IL-10 in the supernatant of each group was detected by the method.
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