脂多糖刺激RAW264.7和Ana-1形态改变及细胞因子表达的差异

Objective To compare the morphologic change and cytokines expression in RAW264.7 and Ana-1 stimulated by lipopolysaccharide(LPS). Methods RAW264.7 and Ana-1 were cultivated with various concentrations of LPS(0.1 mg/L, 1 mg/L, 10 mg/L, 100 mg/L). MTT was performed to evaluate the proliferation ability of cells. Two kinds of cells were cultivated with 1 mg/L. Then,the concentrations of TNF-α,IL-1β,IL-6,IL-10 in culture medium were detected by ELISA in different times(0 h,4 h,8 h,12 h,24 h). Results The survival rates of RAW264.7 and Ana-1 (162.05±28.14)% and (159.92±20.43)% were significantly higher in 1 mg/L group than those of other groups. When stimulated with 1 mg/L LPS, both RAW264.7 and Ana-1 expressed cytokines in time-dependent manner (increased first and decreased finally). The concentrations of TNF-α and IL-10 in RAW264.7 group were higher than those Ana-1 group in 4 h only. IL-1β and IL-6,however,were in higher concentrations in RAW264.7 group than Ana-1 group all the time. Conclusions When stimulated with 1 mg/L LPS, both RAW264.7 and Ana-1 have higher survival rate. RAW264.7 and Ana-1 have different ability in expressing cytokines after being stimulated by LPS. Therefore,RAW264.7 and Ana-1 have variant response to LPS slightly.     

脂多糖刺激RAW264.7和Ana-1形态改变及细胞因子表达的差异

Objective To compare the morphologic change and cytokines expression in RAW264.7 and Ana-1 stimulated by lipopolysaccharide(LPS). Methods RAW264.7 and Ana-1 were cultivated with various concentrations of LPS(0.1 mg/L, 1 mg/L, 10 mg/L, 100 mg/L). MTT was performed to evaluate the proliferation ability of cells. Two kinds of cells were cultivated with 1 mg/L. Then,the concentrations of TNF-α,IL-1β,IL-6,IL-10 in culture medium were detected by ELISA in different times(0 h,4 h,8 h,12 h,24 h). Results The survival rates of RAW264.7 and Ana-1 (162.05±28.14)% and (159.92±20.43)% were significantly higher in 1 mg/L group than those of other groups. When stimulated with 1 mg/L LPS, both RAW264.7 and Ana-1 expressed cytokines in time-dependent manner (increased first and decreased finally). The concentrations of TNF-α and IL-10 in RAW264.7 group were higher than those Ana-1 group in 4 h only. IL-1β and IL-6,however,were in higher concentrations in RAW264.7 group than Ana-1 group all the time. Conclusions When stimulated with 1 mg/L LPS, both RAW264.7 and Ana-1 have higher survival rate. RAW264.7 and Ana-1 have different ability in expressing cytokines after being stimulated by LPS. Therefore,RAW264.7 and Ana-1 have variant response to LPS slightly.     

NFκB1基因启动序列-94ins／delATTG多态性与社区获得性肺炎易感性及严重程度相关性研究

目的探讨NFκBl基因启动序列-94ins／del ATTG基因多态性与中国广东省汉族人群社区获得性肺炎（CAP）易感性及严重程度的相关性。方法采用聚合酶链反应-高分辨率熔解曲线法（PCR—HRM）检测66例CAP患者和66例健康体检者的NFκB1基因启动序列-94ins／de1 ATTG基因型的分布,分析该基因多态性与CAP易感性及严重性的相关性。结果NFxB1基因启动序列-94ins／del ATTG基因型和等位基因分布频率在CAP组和健康对照组比较差异无统计学意义（P〉0．05）;在重症肺炎组与非重症肺炎组比较差异无统计学意义（P〉O．05）;NFκB1-94ins／delATTG的不同基因型与CAP患者的性别、年龄、住院时间、白细胞数、中性粒细胞数之间的相关性均无统计学意义（P〉0．05）。结论NFκB1基因启动序列-94ins／delATTG多态性与cAP易感性及严重程度无相关性。     

NF-κB p65基因在TNF-α诱导的肺泡上皮细胞氧化损伤中的作用

目的建立急性肺损伤肺泡上皮细胞炎症模型,探讨NF-κB p65基因在炎症诱导的氧化应激损伤中的作用。方法以肿瘤坏死因子α（TNF-α,10 ng/mL）刺激A549细胞,运用RNA干扰技术沉默核因子κB（NF-κB）p65基因,采用RT-PCR及Western blot法检测沉默效率,ELISA法检测细胞培养上清中白细胞介素1β（IL-1β）、IL-4、IL-6等炎症因子浓度,比色法检测细胞内丙二醛（MDA）及超氧化物歧化酶（SOD）浓度,MTT法检测细胞存活率。结果 TNF-α刺激A549细胞可在基因及蛋白水平上调NF-κB p65的表达,并增加NF-κB蛋白的核转位;同时细胞培养上清中IL-1β、IL-4、IL-6的浓度升高,细胞内MDA增多,SOD减少,细胞存活率降低。预转染NF-κB p65 siRNA可在基因水平及蛋白水平有效沉默NF-κB p65表达,降低TNF-α诱导的上述各炎症因子及MDA浓度的增高,减少SOD的耗损,A549细胞存活率升高（P〈0.05）。结论 NF-κB能介导TNF-α触发的过度炎症反应和氧化应激,导致细胞存活率明显降低;沉默NF-κB p65基因可有效下调炎症反应水平及其诱发的氧化应激,减轻肺结构细胞的损伤。     

IL-10对脂多糖诱导Ana-1细胞的MyD88/核因子κB活性影响的研究

目的探讨IL-10对小鼠巨噬细胞髓样分化因子88(MyD88)/核因子κB(NF-κB)炎症信号活化的影响。方法将小鼠巨噬细胞Ana-1分为脂多糖(LPS)组和LPS+IL-10组,分别于0.5、1及2h收集巨噬细胞和细胞培养上清液,Western blot检测细胞MyD88与胞浆、胞核NF-κBp65亚基表达,ELISA法检测培养上清中肿瘤坏死因子α(TNF-α)含量。结果在0～2h,LPS组细胞MyD88表达显著持续上升,LPS+IL-10组于LPS刺激后上升,0.5h达峰值,2h恢复至正常水平,1h和2h相对含量均低于LPS组(11.6±1.3比17.5±0.7,8.8±0.3比21.4±1.8,P0.05);总NF-κB表达量在两组间无明显差异。NF-κB核浆比变化趋势与MyD88类似,LPS+IL-10组1h及2h相对含量亦均低于LPS组(1.1±0.1比2.4±0.4,0.6±0.7比3.1±0.6,P0.05);相应的,LPS+IL-10组1h和2hTNF-α含量亦低于LPS组[(222.5±33.5)pg/mL比(365.2±22.7)pg/mL,(212.7±15.9)pg/mL比(566.2±31.5)pg/mL,P0.05]。结论 IL-10通过抑制减少MyD88/NF-κB信号通路活化,降低TNF-α表达,从而下调炎症反应强度。     

脂多糖刺激RAW264.7和Ana-1形态改变及细胞因子表达的差异

Objective To compare the morphologic change and cytokines expression in RAW264.7 and Ana-1 stimulated by lipopolysaccharide(LPS). Methods RAW264.7 and Ana-1 were cultivated with various concentrations of LPS(0.1 mg/L, 1 mg/L, 10 mg/L, 100 mg/L). MTT was performed to evaluate the proliferation ability of cells. Two kinds of cells were cultivated with 1 mg/L. Then,the concentrations of TNF-α,IL-1β,IL-6,IL-10 in culture medium were detected by ELISA in different times(0 h,4 h,8 h,12 h,24 h). Results The survival rates of RAW264.7 and Ana-1 (162.05±28.14)% and (159.92±20.43)% were significantly higher in 1 mg/L group than those of other groups. When stimulated with 1 mg/L LPS, both RAW264.7 and Ana-1 expressed cytokines in time-dependent manner (increased first and decreased finally). The concentrations of TNF-α and IL-10 in RAW264.7 group were higher than those Ana-1 group in 4 h only. IL-1β and IL-6,however,were in higher concentrations in RAW264.7 group than Ana-1 group all the time. Conclusions When stimulated with 1 mg/L LPS, both RAW264.7 and Ana-1 have higher survival rate. RAW264.7 and Ana-1 have different ability in expressing cytokines after being stimulated by LPS. Therefore,RAW264.7 and Ana-1 have variant response to LPS slightly.     

吉非替尼对博莱霉素致肺纤维化小鼠α平滑肌肌动蛋白的抑制作用

目的 观察吉非替尼对博莱霉素诱导肺纤维化小鼠a平滑肌肌动蛋白(a-SMA)表达的影响,探讨EGFR在肺纤维化上皮间质转分化中的作用.方法 将30只SPF级雌性BALB/c小鼠分为三组:对照组(气管滴人生理盐水)、纤维化组(气管滴人博莱霉素3 mg/kg )、纤维化吉非替尼干预组(气管滴人博莱霉素+吉非替尼灌胃20 mg/Kg ).实验第14天杀鼠取肺,肺组织石蜡切片行HE染色与Masson染色;RT-PCR法检测a-SMA的mRNA表达水平;免疫组化检测总EGFR,磷酸化EGFR及a-SMA表达.结果 纤维化吉非替尼干预组肺病理损伤较纤维化组减轻,气道上皮下胶原沉积减少,气道上皮及肺间质细胞磷酸化EGFR表达评分下降(P<0.05);同时,肺a-SMA的mRNA表达减少,免疫组化评分也明显降低(P<0.05).结论 吉非替尼抑制博莱霉素诱导小鼠肺纤维化可能与其抑制EGFR活性,下调a-SMA表达有关.     

脂多糖刺激RAW264.7和Ana-1形态改变及细胞因子表达的差异

Objective To compare the morphologic change and cytokines expression in RAW264.7 and Ana-1 stimulated by lipopolysaccharide(LPS). Methods RAW264.7 and Ana-1 were cultivated with various concentrations of LPS(0.1 mg/L, 1 mg/L, 10 mg/L, 100 mg/L). MTT was performed to evaluate the proliferation ability of cells. Two kinds of cells were cultivated with 1 mg/L. Then,the concentrations of TNF-α,IL-1β,IL-6,IL-10 in culture medium were detected by ELISA in different times(0 h,4 h,8 h,12 h,24 h). Results The survival rates of RAW264.7 and Ana-1 (162.05±28.14)% and (159.92±20.43)% were significantly higher in 1 mg/L group than those of other groups. When stimulated with 1 mg/L LPS, both RAW264.7 and Ana-1 expressed cytokines in time-dependent manner (increased first and decreased finally). The concentrations of TNF-α and IL-10 in RAW264.7 group were higher than those Ana-1 group in 4 h only. IL-1β and IL-6,however,were in higher concentrations in RAW264.7 group than Ana-1 group all the time. Conclusions When stimulated with 1 mg/L LPS, both RAW264.7 and Ana-1 have higher survival rate. RAW264.7 and Ana-1 have different ability in expressing cytokines after being stimulated by LPS. Therefore,RAW264.7 and Ana-1 have variant response to LPS slightly.     

RNAi沉默NF-κB p65对小鼠巨噬细胞细胞因子表达的影响

目的: 探讨RNA干扰(RNAi)技术在抑制NF-κB p65表达、调控LPS活化后巨噬细胞细胞因子表达中的应用。方法: 利用阳离子脂质体将NF-κB p65小干扰RNA(siRNA)瞬时转染入Ana-1细胞,RT-PCR及Western blotting法检测其沉默效率,ELISA法检测LPS(1 mg/L)刺激下0 h、4 h、12 h和24 h Ana-1细胞培养上清中TNF-α、IL-1β、IL-6和IL-10浓度。结果: NF-κB p65 siRNA转染24 h后,NF-κB p65在基因水平及蛋白水平表达均被明显抑制(P<0.05)。RNA干扰组Ana-1细胞培养上清中细胞因子TNF-α、IL-1β和IL-6表达在相应时点内均较对照组明显降低(P<0.05),IL-10表达显著升高,在12 h和24 h差异显著(P<0.05)。结论: 体外实验初步证实RNAi技术能有效沉默小鼠巨噬细胞NF-κB p65 基因的表达,下调其下游调控的促炎症细胞因子TNF-α、IL-1β、IL-6及上调抑炎症细胞因子IL-10的表达,从而抑制过度的炎症反应。     

不同浓度肿瘤坏死因子-α对肺腺癌A549细胞的影响

目的 观察不同浓度肿瘤坏死因子-α(TNF-α)对肺腺癌A549细胞产生的保护、促炎、促细胞凋亡效应.方法 分别以0、1、10、25、50 ng/mL的TNF-α诱导肺腺癌A549细胞6h.采用MTT比色法观察细胞存活率;应用活性氧(ROS)荧光探针DCFH-DA检测ROS强度;RT-PCR法检测Nrf-2、NF-κB基因的表达;DAPI细胞核荧光染色法处理细胞,荧光显微镜下观察细胞凋亡.结果 0、1、10、25、50 ng/mL TNF-α对肺腺癌A549细胞存活率无影响(P均＞0.05);100 ng/mL TNF-α诱导后,与对照组比较,细胞存活率降低(P＜0.05).随着TNF-α浓度增加,肺腺癌A549细胞产生ROS的量随之增加(P均＜0.05),Nrf-2 mRNA表达减少(P均＜0.05),NF-κB mRNA表达增加(P均＜0.05),细胞凋亡率有上升趋势.结论 不同浓度TNF-α诱导肺腺癌A549细胞可产生保护、促炎、促细胞凋亡等不同细胞效应,且随TNF-α浓度增加,细胞损伤加重;其机制可能与ROS产生的量有关.