SIGIRR过表达抑制LPS诱导的肺泡上皮细胞NF-κB的活化

目的: 研究单免疫球蛋白白介素1受体相关蛋白(SIGIRR)对脂多糖(LPS)刺激的肺泡上皮细胞核因子-κB信号通路的调节作用。方法: 以LPS刺激人Ⅱ型肺泡上皮细胞A549。将含有SIGIRR cDNA 全长的真核表达载体瞬时转染A549细胞,使SIGIRR在A549细胞过表达。用双萤光素酶报告系统检测LPS刺激后A549细胞NF-κB的转录活性,用ELISA法检测LPS刺激后细胞因子白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)以及白细胞介素-6(IL-6)的水平,对比转染与否对上述因子在A549细胞中释放水平的影响。结果: 在A549细胞中,过表达SIGIRR可显著抑制NF-κB的转录活性,同时抑制LPS诱导的细胞因子IL-1β、TNF-α以及IL-6的表达。 结论: SIGIRR过表达可以抑制LPS触发的肺泡上皮细胞中TLR4信号的转导,从而发挥减轻炎症反应、保护肺泡上皮细胞的作用。     

腺病毒E1A蛋白对大鼠肺泡上皮细胞及人肺腺癌细胞凋亡的影响

目的:探讨腺病毒E1A蛋白对大鼠肺泡上皮细胞(CCL149)及人肺腺癌细胞(A549)在致凋亡因素TNF-α诱导下细胞凋亡影响。方法:将含腺病毒E1A基因完全编码区的Pneo-E1A质粒分别转染CCL149、A549细胞,用G418筛选抗性细胞克隆,用RT-PCR、免疫组化方法对单个细胞克隆进行筛选鉴定;将经鉴定确定的稳定转染E1A基因的阳性细胞克隆、对照质粒转染细胞克隆用致凋亡因素TNF-α刺激,用Hoechest荧光染色分析及流式细胞仪结合膜联蛋白V-FITC标记法检测细胞凋亡情况。结果:稳定转染E1A基因的CCL149细胞(C-E1A+)在30μg/LTNF-α作用前、后细胞的凋亡率分别为(2.63±0.8)%和(25.38±0.9)%,明显高于对照质粒转染细胞(C-E1A-)作用前的(0.62±0.3)%和作用后的(6.08±0.2)%,两组相比差别有统计学意义(P0.01);稳定转染E1A基因的A549细胞(A-E1A+)在30μg/LTNF-α作用前、后细胞的凋亡率分别为(5.12±0.5)%和(19.82±1.6)%,明显高于对照质粒转染细胞(A-E1A-)作用前的(2.02±0.7)%和作用后的(9.15±1.2)%,两组相比差别有统计学意义(P0.01)。结论:E1A蛋白能够增加细胞对致凋亡因素的敏感性,上调TNF-α诱导下的细胞凋亡。     

沉默NF-κB/p65对TNF-α诱导的肺泡Ⅱ型上皮NOX1基因表达的影响

目的构建急性肺损伤肺泡Ⅱ型上皮A549细胞炎症模型,通过沉默NF-κB基因,观察NOX1基因表达水平及氧化应激指标变化,探讨NOX1基因与NF-κB/p65的相关性。方法运用RNA干扰技术沉默NF-κB/p65基因,以TNF-α(10 ng/ml)刺激肺泡Ⅱ型上皮细胞(A549),采用RT-PCR及Western blot检测NOX1基因表达水平,DCFH-DA探针法检测细胞内活性氧水平,比色法检测细胞的总抗氧化能力、总谷胱甘肽、总超氧化物歧化酶活力和丙二醛的浓度。结果采用TNF-α刺激肺泡Ⅱ型上皮A549细胞可以分别在基因和蛋白水平上调NOX1基因表达;同时,增加细胞丙二醛和活性氧浓度,降低总抗氧化能力、总谷胱甘肽及超氧化物歧化酶浓度,氧化应激程度放大(P0.05)。预转染NF-κB/p65 siRNA,可下调NOX1基因的表达,减少细胞丙二醛、活性氧生成,提高总抗氧化能力、总谷胱甘肽及超氧化物歧化酶浓度,氧化应激程度降低(P0.05)。结论沉默NF-κB/p65基因,可有效下调TNF-α诱导A549细胞氧化应激程度及NOX1基因表达水平,提示NF-κB/p65可能参与调控TNF-α诱导的NOX1基因表达。     

包裹E1A基因的纳米粒子制备及转染人肺腺癌细胞A549实验研究

目的制备包裹E1A基因（腺病毒早期表达基因）的纳米粒子,并观察其介导E1A基因转染人肺腺癌细胞A549的可行性和效率。方法应用聚乳酸聚乙醇酸共聚物和聚乙烯醇包载E1A基因,制备纳米级粒子混合物,检测其包埋率、体外释放情况及粒径大小。用制备的包裹DNA纳米粒子转染人肺腺癌细胞A549,并以阳离子脂质体为对照,用PCR、RT-PCR方法分别检测转染细胞中E1A基因DNA整合和mRNA表达。结果制备的纳米粒子粒径为150～280nm,包埋率为0.78%,体外释放约为22d;在转染相等质量的DNA情况下,纳米组所得克隆数较脂质体组多（P〈0.05）;PCR、RT-PCR结果表明纳米粒子和脂质体转染细胞均有E1A基因整合和mRNA表达。结论成功制备了纳米粒子,纳米粒子可携带外源基因进行基因转染。     

腺病毒E1A蛋白抑制大鼠肺泡上皮细胞γ-谷氨酰半胱氨酸合成酶催化亚单位表达的机制研究

目的： 研究腺病毒E1A蛋白抑制大鼠肺泡上皮细胞γ-谷氨酰半胱氨酸合成酶催化亚单位（GCLC）表达的机制。方法: 构建稳定表达腺病毒E1A蛋白的大鼠肺泡上皮细胞，将GCLC基因5’-上游调控序列不同长度的缺失体-萤火虫荧光素酶报道系统转染后分析转录活性的变化；检测AP-1、NF-κB、USF与GCLC基因调控序列的结合活性。结果: GCLC基因5’-上游调控序列报道系统检测结果显示大部分（9/11）缺失体的转录活性抑制，AP-1、NF-κB、USF与GCLC基因的结合活性增强，而对应的功能元件的转录活性降低。结论: 腺病毒E1A蛋白通过抑制GCLC基因 5’-上游调控序列的转录活性，扩大大鼠肺泡上皮细胞氧化应激时的氧化/抗氧化失衡,其机制可能涉及E1A对辅助转录因子的抑制。     

RNA干扰对人肺泡上皮A549细胞巨噬细胞移动抑制因子基因表达抑制作用

目的探讨巨噬细胞移动抑制因子(MIF)小干扰RNA(siRNA)干扰对人肺泡上皮A549细胞株MIF基因表达的影响.方法将MIF siRNA用转染剂Interferin介导转染体外培养的A549细胞,通过RT-PCR方法观察不同浓度MIF siRNA转染后,对MIF mRNA表达的影响;采用细胞免疫荧光方法分析MIF蛋白表达的强弱;应用MTT方法测定MIF siRNA转染对细胞的毒性,及脂多糖(LPS)刺激对A549细胞生存率的影响.结果 (1)MIF siRNA转染人肺泡上皮A549细胞株后,可下调细胞中MIF mRNA的表达,只有当MIF siRNA转染浓度大于50 nmol/L时,才能明显下调MIF mRNA的表达;(2)MIF siRNA转染人肺泡上皮A549细胞株后,A549细胞MIF的荧光蛋白表达也明显降低,在此基础上再加入MIF刺激,MIF的蛋白表达又明显增加;(3)MIF siRNA转染对A549细胞几乎没有毒性,且对LPS刺激A549细胞导致细胞死亡及细胞的生存率下降有一定的保护作用.结论 MIF siRNA能在A549细胞特异沉默目的基因MIF和抑制MIF蛋白的表达,且对LPS刺激导致细胞死亡有一定的保护作用.     

miR-221靶向AdipoR1基因对LPS诱导的肺泡上皮细胞A549炎症分泌及凋亡影响

目的:探讨miR-221是否靶向脂联素受体1 AdipoR1基因影响脂多糖(LPS)诱导的肺泡上皮细胞A549炎症分泌和凋亡。方法:随机将人肺泡上皮细胞A549分为对照组、LPS组和LPS+转染组,RT-qPCR法检测miR-221和AdipoR1 mRNA表达,Western blot法检测AdipoR1蛋白及凋亡相关因子Bax、Bcl-2蛋白表达,流式细胞仪检测细胞凋亡率,ELISA法检测细胞培养上清液中炎性因子IL-1β、IL-6和肿瘤坏死因子-α(TNF-α)浓度,双荧光素酶报告基因检测miR-221是否靶向AdipoR1。结果:与对照组比较,LPS组细胞中miR-221及Bax蛋白表达升高(P<0.05),AdipoR1 miRNA和蛋白及Bcl-2蛋白表达降低(P<0.05),细胞培养上清液中IL-1β、IL-6、TNF-α浓度升高(P<0.05),细胞凋亡率升高(P<0.05);双荧光素酶报告基因实验证实miR-221靶向负调控AdipoR1的表达;抑制miR-221和过表达AdipoR1均能抑制LPS诱导肺泡上皮细胞分泌炎症因子,抑制细胞凋亡;抑...     

靶向髓样细胞表达的激发受体1基因短发夹RNA真核质粒表达载体的构建及筛选

目的 构建编码髓样细胞表达的激发受体1(TREM-1)基因短发夹RNA(shRNA)真核质粒表达载体,并筛选出基因沉默效果最明显的shRNA质粒表达载体.方法 针对TREM-1的靶位点设计含shRNA的靶序列,分别构建pGCsi-TREM-1 shRNA1和pGCsi-TREM-1 shRNA2的质粒表达载体和pGCsi-NegshRNA阴性质粒表达载体,通过酶切及测序进行鉴定.鉴定后以脂质体包裹转染肺泡上皮A549细胞,分为未转染对照组(A组)、转染空质粒载体pGCsi对照组(B组)、转染阴性shRNA质粒表达载体pGCsi-NegshRNA对照组(C组)、转染TREM-1 shRNA1质粒表达载体组(D组)和转染TREM-1shRNA2质粒表达载体组(E组),48 h后观察转染细胞绿色荧光蛋白表达效果.转染24、48、72 h后,以A组为对照采用MTT法测定B、C、D、E组细胞的存活率.转染48 h后,通过荧光定量PCR检测各组TREM-1 mRNA的表达水平并与转染前相比较.结果 经酶切和测序鉴定证实,目的 TREM-1基因shRNA1和shRNA2片段已被克隆到pGCsi载体中.荧光显微镜下,B、C、D、E组A549细胞有明显的绿色荧光蛋白表达.各时间点B、C、D、E组细胞存活率均达90%以上.A、B、C 3组转染前后TREM-1mRNA的表达水平差异无统计学意义,D、E组转染的重组质粒均能有效抑制A549细胞的TREM-1mRNA的表达([(1.945±0.252)×105比(1.010±0.194)×105,(1.933±0.216)×105比(1.202±0.171)×105,均P＜0.05],抑制率分别为48.07%和37.82%.结论 成功构建了靶向TREM-1基因的shRNA质粒表达载体,可有效抑制细胞中TREM-1目的 基因的表达,为后续脓毒症的基因治疗提供了依据.     

Polo-like激酶1反义RNA对肺癌细胞A549生长的实验研究

目的:探讨Polo-like激酶1(Plk1)基因表达下调对肺癌细胞周期分布及其生长的影响。方法:培养肺腺癌细胞株A549,构建表达Plk1反义RNA的质粒pcDNA3-Plk1,通过脂质体介导转染A549细胞,采用RT-PCR和Western blotting的方法检测Plk1基因的表达,细胞计数、BrdU脉冲标记检测细胞增殖,流式细胞仪分析细胞周期变化和凋亡,MTT法检测长春瑞宾(NVB)对各组细胞的生长抑制率。结果:A549细胞转染pcDNA3-Plk1后24h,Plk1mRNA及蛋白表达均下降;细胞变圆、漂浮、增殖减慢;S期细胞百分数(BrdU标记指数)显著低于对照组(P<0.05);转染后48hA549细胞出现G2/M期阻滞(P<0.05)并发生凋亡;等浓度化疗药物诺维本对转染pcDNA3-Plk1细胞的抑制率明显高于各对照组(P<0.05),转染pcDNA3与未转染的对照细胞差异无显著(P>0.05)。结论:pcDNA3-Plk1的转染能下调Plk1基因的表达,抑制A549细胞增殖,诱导凋亡,并能增加A549细胞对化疗药物的敏感性。     

miR-486调控PTEN影响LPS诱导的肺泡上皮细胞A549凋亡的实验研究

目的:探讨微小RNA-486(miR-486)对脂多糖(LPS)诱导的肺泡上皮细胞A549凋亡的影响和机制。方法:以LPS处理肺泡上皮细胞,RT-qPCR测定miR-486表达变化。在肺泡上皮细胞中转染miR-486模拟物(miR-486 mimics),RT-qPCR检测LPS条件下的转染效果。流式细胞术测定凋亡变化,Western blot检测细胞中cleaved caspase-3(C-caspase-3)和C-caspase-9蛋白表达变化。靶基因预测软件预测第10号染色体同源缺失性磷酸酶-张力蛋白(PTEN)可能是miR-486的靶基因,利用萤光素酶报告载体鉴定靶向关系。在肺泡上皮细胞中共转染pcDNA 3.1-PTEN和miR-486 mimics,检测上调PTEN对miR-486 mimics调控LPS诱导的A549细胞凋亡的作用。结果:LPS处理后的肺泡上皮细胞中miR-486的表达水平显著下降(P0.05)。miR-486 mimics可以显著上调LPS条件下肺泡上皮细胞中miR-486的表达水平(P0.05)。LPS处理后的肺泡上皮细胞凋亡率及C-caspase-3和C-caspase-9蛋白表达水平显著升高(P0.05);上调miR-486可以显著下调LPS诱导的肺泡上皮细胞凋亡(P0.05)。miR-486靶向负调控PTEN的表达。pcDNA 3.1-PTEN可以显著提高miR-486 mimics转染后LPS诱导的肺泡上皮细胞中PTEN的表达,促进细胞凋亡和细胞中C-caspase-3和C-caspase-9蛋白的表达(P0.05)。结论:miR-486靶向抑制PTEN的表达,减少LPS诱导的肺泡上皮细胞凋亡。     

谷胱甘肽硫转移酶M1和T1基因多态性与肺癌易感性关系的研究

目的和方法:采用病例-对照研究方法和多重PCR技术检测肺癌病例组161人和健康对照组165人的GSTM1(glutathioneS-transferaseM1)和GSTT1(glutathioneS-transferaseT1)基因缺陷型的频率,以多因素Logistic回归模型评价GSTM1和GSTT1基因型之间以及基因型与吸烟之间的交互作用。以探讨谷胱甘肽硫转移酶M1和T1的基因多态性与肺癌发病的关系。结果:GSTM1基因缺陷型和GSTT1基因缺陷型的频率在病例组和对照组之间均无显著的差异。在不吸烟(SI=0)的人群中,GSTM1基因缺陷型携带者患肺癌的危险性显著增加。此外,该基因型还可显著增加年龄≥60岁者患肺腺癌的危险性。多因素Logistic回归分析显示吸烟和GSTM1基因缺陷型是肺癌的危险因素,吸烟与GSTM1和GSTT1基因型不存在交互作用。分层分析表明GSTT1基因功能型与GSTM1基因缺陷型存在明显的交互作用,在年龄≥60岁的人群及不吸烟的人群中,GSTT1基因功能型可以使得GSTM1基因缺陷型携带者患肺腺癌的危险度分别降低48.5%和45.3%。结论:GSTM1基因缺陷型是非吸烟者和年龄≥60岁者患肺癌,尤其是患肺腺癌的危险因素,GSTT1基因功能型可以降低不吸烟或年龄≥60岁的GSTM1基因缺陷型携带者患肺腺癌的危险度。在肺癌的发生过程中GSTM1和GSTT1基因缺陷型与吸烟不存在交互作用。     

Soluble ICAM-1 and VCAM-1 as markers of endothelial activation

Activated endothelium releases the soluble adhesion molecules vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1). Measurement of fluid-phase adhesion molecules is therefore used to quantify endothelial activation, but it is unclear which is the better marker. The aims of the study were to compare the relationships between mRNA, surface and total expression and released VCAM-1 and ICAM-1 in endothelial cell cultures during activation, and to compare human umbilical vein endothelial cells (HUVEC) with the microvascular cell line HMEC-1. sVCAM-1 better represented mRNA and surface expression changes in HUVEC undergoing endotoxin stimulation than did sICAM-1. Very little VCAM-1 was released from endotoxin-stimulated HMEC-1, and sICAM-1 seemed a better activation marker for these cells. During incubation of HUVEC in media with glucose concentrations of 5.6, 10.6 or 20.6 m m , VCAM-1 was released to the media in a dose-dependent way without changes in surface expression. ICAM-1 was not influenced by the glucose concentration. There are situations when VCAM-1 concentrations in the media do not mirror the surface expression on HUVEC in culture, indicating that measurements of soluble adhesion molecules may not necessarily be representative of the conditions on the cell surface. Endothelium from different locations showed varying responses with respect to VCAM-1 and ICAM-1 liberation upon endotoxin stimulation. Thus, both sVCAM-1 and sICAM-1 should be quantified in clinical studies of endothelial activation until their characteristics are better clarified.     

Interleukin 1 as a cytokine inducer

Human interleukin 1 (IL 1) exerts an interferon-like antiviral activity in certain strains of human diploid fibroblasts. It is shown that this antiviral effect is indirect, in that it is mediated by production of interferon-beta. IL 1 also induces synthesis of the mRNA of another secreted protein, 26K, whose biological function is still controversial. Finally, IL 1 has bone marrow colony stimulating activity which is shown to be due to induction of colony stimulating factor in adherent phagocytic cells present in the bone marrow cultures. These findings emphasize the concept that several of the varied biological effects of IL 1 may be indirect.     

Virus-like particles as HIV-1 vaccines

Traditional successful antiviral vaccines have relied mostly on live-attenuated viruses. Live-attenuated HIV vaccine candidates are not ideal as they pose risks of reversion, recombination or mutations. Other current HIV vaccine candidates have difficulties generating broadly effective neutralising antibodies and cytotoxic T cell immune responses to primary HIV isolates. Virus-like-particles (VLPs) have been demonstrated to be safe to administer to animals and human patients as well as being potent and efficient stimulators of cellular and humoral immune responses. Therefore, VLPs are being considered as possible HIV vaccines. Chimeric HIV-1 VLPs constructed with either HIV or SIV capsid protein plus HIV immune epitopes and immuno-stimulatory molecules have further improved on early VLP designs, leading to enhanced immune stimulation. The administration of VLP vaccines via mucosal surfaces has also emerged as a promising strategy with which to elicit mucosal and systemic humoral and cellular immune responses. Additionally, new information on antigen processing and the presentation of particulate antigens by dendritic cells (DCs) has created new strategies for improved VLP vaccine candidates. This paper reviews the field of HIV-1 VLP vaccine development, focusing on recent studies that will likely uncover promising prospects for new HIV vaccines.     

Interleukin-1 receptor antagonist (IL-1ra) as a probe and as a treatment for IL-1 mediated disease.

The natural human IL-1 receptor antagonist (IL-1ra) has been produced in a recombinant organism and has been used to study IL-1 action in vivo. The receptor antagonist mitigates the pathophysiology associated with animal models of ulcerative colitis through reducing IL-1 mediated neutrophil recruitment into the affected tissue. It also reduces joint swelling and damage in an animal model of rheumatoid arthritis, possibly by reducing IL-1 mediated synthesis of proteases by the synovial fibroblasts and chondrocytes of the joint. The receptor antagonist is not immunosuppressive in rodents, indicating that it is working by blocking the inflammatory reaction rather than any underlying defect in specific immunity.