靶向高迁移率族蛋白A1基因RNA干扰真核表达载体的构建

目的构建针对人高迁移率族蛋白A1(HMGA1)基因的RNA干扰真核表达载体,为研究HMGA1基因在肿瘤细胞中的作用奠定实验基础。方法设计合成特异性针对人HMGA1基因的寡核苷酸序列,梯度退火后,与pU6mRFP载体连接,构建重组载体,转染人肝癌细胞SMMC-7721。通过激光共聚焦显微镜观察红色荧光,估测转染效率,RT-PCR法检测转染细胞HMGA1mRNA水平,流式细胞仪检测细胞凋亡和细胞周期。结果DNA测序证实,成功构建了特异性HMGA1siRNA真核表达载体,转染后72h,转染效率为40%左右。所构建的载体能够特异性沉默转染细胞HMGA1基因的表达。转染细胞HMGA1基因沉默后,细胞凋亡率(27·86%±2·44%)明显高于空载体对照组和SMMC-7721对照组(分别为2·82%±2·39%和2·04%±0·70%),G0-G1期细胞百分数(77·73%±1·78%)明显高于空载体对照组和SMMC-7721对照组(分别为42·19%±3·28%和39·23%±3·63%),G2-S期细胞百分数(22·27%±1·78%)明显低于空载体对照组和SMMC-7721对照组(分别为57·81%±3·28%和60·77%±3·63%)。结论成功构建了HMGA1的RNA干扰真核表达载体。     

脂多糖诱导小鼠巨噬细胞系RAW264.7细胞的活化凋亡作用

目的探讨脂多糖(LPS)诱导小鼠巨噬细胞系RAW264.7细胞活化凋亡的作用。方法体外培养小鼠巨噬细胞系RAW264.7细胞,分别用0.5、1.0、2.5μg/mlLPS刺激RAW264.7细胞24h,一氧化氮(NO)试剂盒检测细胞培养上清中NO水平;用1.0μg/mlLPS分别刺激细胞3d和6d,台盼蓝拒染法检测细胞增殖情况;用1.0μg/mlLPS刺激细胞6d,流式细胞术分析细胞周期及细胞凋亡情况。结果经LPS刺激后24h,RAW264.7细胞培养上清中NO含量明显增加,且具有剂量依赖性;LPS刺激3d和6d后,细胞的增殖均受到抑制,且呈时间依赖性;LPS刺激6d时,细胞周期被阻滞在S期,并出现明显的凋亡。结论LPS具有诱导小鼠巨噬细胞系RAW264.7细胞活化凋亡的作用。     

氨基葡萄糖对小鼠腹腔巨噬细胞免疫功能的影响

目的探讨氨基葡萄糖对小鼠腹腔巨噬细胞(PMФ)免疫功能的影响。方法用壳聚糖制备氨基葡萄糖纯品,通过体外实验测定其对小鼠PMФ增殖、吞噬中性红、产生NO和IL-1能力的影响。结果氨基葡萄糖能够显著增强PMФ对中性红的吞噬能力,提高NO和IL-1的生成量,而对PMФ的增殖能力无明显影响。结论氨基葡萄糖能够活化PMФ,增强小鼠的免疫功能。     

激活素A抑制脂多糖活化巨噬细胞的作用机制

目的探讨激活素A(Activin A)抑制脂多糖(Lipopolysaccharides,LPS)活化巨噬细胞的作用机制。方法取小鼠巨噬细胞系RAW264.7细胞,分别添加Activin A(5 ng/ml)、LPS(1μg/ml)和Activin A(5 ng/ml)+LPS(1μg/ml),同时设以含单纯2.5%胎牛血清的DMEM培养液培养的细胞作为对照孔,培养24 h后,采用还原酶法检测细胞分泌一氧化氮(Nitric oxide,NO)的水平,流式细胞术分析TLR2和TLR4蛋白的表达水平,RT-PCR分析细胞ActRⅡA和ActRⅡB基因mRNA的转录水平。结果 Activin A和LPS单独作用均促进RAW264.7细胞分泌NO,但二者联合使用时,Activin A可抑制LPS刺激RAW264.7细胞的NO分泌;Activin A能抑制LPS上调RAW264.7细胞TLR4蛋白的表达,但对TLR2蛋白的表达无影响;LPS可促进RAW264.7细胞ActRⅡA基因mRNA的转录水平,但对ActRⅡB基因mRNA的转录水平无影响。结论 Activin A通过调控TLR4途径抑制LPS的作用,LPS可能通过促进ActRⅡA的表达进一步增强Activin A的负反馈调节作用。     

激活素A对小鼠腹腔巨噬细胞分泌细胞因子的影响

目的探讨激活素A对小鼠腹腔巨噬细胞分泌细胞因子的影响。方法分离小鼠腹腔巨噬细胞,将其分为激活素A处理组和对照组,瑞氏-吉姆萨染色观察小鼠腹腔巨噬细胞形态学变化;流式细胞术分析小鼠腹腔巨噬细胞表面分子CD68的表达;ELISA法检测小鼠腹腔巨噬细胞分泌IL-10及TNFα的水平;Griess法检测小鼠腹腔巨噬细胞分泌NO的水平。结果经激活素A刺激后,显微镜下可见呈不规则、多边型的活化巨噬细胞增多,细胞表达巨噬细胞成熟标志CD68增加,Ⅱ型巨噬细胞(M2)产生的细胞因子IL-10及NO分泌水平升高,而Ⅰ型巨噬细胞(M1)产生的细胞因子TNFα水平无变化。结论激活素A可能主要促进小鼠Ⅱ型巨噬细胞分泌细胞因子。     

免疫原性细胞死亡及其在肿瘤治疗应用中的研究进展

免疫原性细胞死亡(immunogenic cell death,ICD)是细胞死亡的一种形式,通过释放肿瘤相关抗原(tumor asso-ciated antigen,TAA)和肿瘤特异性抗原(tumor specific antigen,TSA),暴露"危险信号"以刺激机体免疫系统产生免疫应答,其特点是释放和/或增加...     

穿心莲内酯对脂多糖诱导的小鼠急性肺损伤中NF-κB信号通路的影响

目的探讨穿心莲内酯(Andrographolide,AP)对脂多糖(Lipopolysaccharide,LPS)诱导的小鼠急性肺损伤(Acute lung injury,ALI)中NF-κB信号通路的影响。方法将雄性C57BL/6小鼠随机分为3组:空白对照组、LPS组和AP+LPS组。AP+LPS组腹腔内注射10 mg/kg的AP,空白对照组腹腔内注射等体积的NS,2次/d,连续3 d,第3天注射后2 h,LPS组和AP+LPS组气管内雾化吸入LPS(1 mg/kg),空白对照组气管内雾化吸入等剂量的NS,12 h后处死小鼠,开胸取肺,10%甲醛溶液固定,随后进行石蜡切片及常规HE染色,光镜下观察各组小鼠肺组织的病理学变化;免疫组化法观察肺组织中血管细胞黏附分子-1(Vascular cell adhesion molecule-1,VCAM-1)蛋白的表达;Western blot法检测肺组织中磷酸化抑制蛋白IκB(Phospho-inhibitor ofκB,p-IκBα)和p-NF-κB(P65)蛋白的表达。结果 LPS组小鼠肺损伤明显,有明显的炎症反应;AP+LPS组小鼠肺损伤明显减轻,炎症细胞浸润明显减少。与LPS组相比,AP+LPS组小鼠肺组织中VCAM-1蛋白的表达明显抑制;p-IκBα和p-NF-κB(P65)蛋白的表达明显降低(P<0.05)。结论 AP可通过抑制磷酸化的IκBα的降解及P65单体的磷酸化来调节NF-κB通路,从而减轻小鼠肺损伤的炎症变化。     

Ⅱ型B族链球菌表面免疫相关蛋白基因的克隆和原核表达

目的重组表达Ⅱ型B族链球菌表面免疫相关蛋白(Surfaceimmunogenicprotein,SIP)基因,为进一步免疫学研究提供目标蛋白。方法用PCR的方法从GBSⅡ型标准株的基因组DNA中扩增出SIP基因,用T/A克隆法将其插入pMD18T载体,构建原核表达载体pET32aSIP,用BL21(DE3)/pET系统表达TrixSIP融合蛋白,SDSPAGE和质谱分析鉴定表达产物,并对表达蛋白进行初步纯化。结果PCR扩增产物经测序,证实与GenBank中Ⅰa/c型GBS的SIP的基因序列同源性为99%。SDSPAGE显示,经IPTG诱导后BL21(DE3)/pET32aSIP总蛋白中出现一条相对分子质量为66000的新蛋白带。质谱分析和蛋白质库的比较证实其为B族链球菌表面免疫相关蛋白(SIP)的可能性分数为74。结论已成功表达并初步纯化SIP,为SIP在细菌致病中的作用研究以及相关疫苗的制备奠定了基础。     

High Mobility Group Box 1 Protein in Cerebral Thromboemboli

High-mobility group box 1 protein (HMGB1) is a damage-associated molecular pattern (DAMP) involved in neutrophil extracellular trap (NET) formation and thrombosis. NETs are regularly found in cerebral thromboemboli. We here analyzed associated HMGB1 expression in human thromboemboli retrieved via mechanical thrombectomy from 37 stroke patients with large vessel occlusion. HMGB1 was detected in all thromboemboli, accounting for 1.7% (IQR 0.6–6.2%) of the total thromboemboli area and was found to be colocalized with neutrophils and NETs and in spatial proximity to platelets. Correlation analysis revealed that the detection of HMGB1 was strongly related to the number of neutrophils (r = 0.58, p = 0.0002) and platelets (r = 0.51, p = 0.001). Our results demonstrate that HMGB1 is a substantial constituent of thromboemboli causing large vessel occlusion stroke.     

High Mobility Group Box 1 Promotes Lung Cancer Cell Migration and Motility via Regulation of Dynamin-Related Protein 1

High mobility group box 1 (HMGB1) has been demonstrated to promote the migration and invasion of non-small cell lung cancer (NSCLC). However, the mechanism of action of HMGB1 in regulating tumor mobility remains unclear. Therefore, we aimed to investigate whether HMGB1 affects mitochondria distribution and regulates dynamin-related protein 1 (DRP1)-mediated lamellipodia/filopodia formation to promote NSCLC migration. The regulation of mitochondrial membrane tension, dynamics, polarization, fission process, and cytoskeletal rearrangements in lung cancer cells by HMGB1 was analyzed using confocal microscopy. The HMGB1-mediated regulation of DRP1 phosphorylation and colocalization was determined using immunostaining and co-immunoprecipitation assays. The tumorigenic potential of HMGB1 was assessed in vivo and further confirmed using NSCLC patient samples. Our results showed that HMGB1 increased the polarity and mobility of cells (mainly by regulating the cytoskeletal system actin and microtubule dynamics and distribution), promoted the formation of lamellipodia/filopodia, and enhanced the expression and phosphorylation of DRP1 in both the nucleus and cytoplasm. In addition, HMGB1 and DRP1 expressions were positively correlated and exhibited poor prognosis and survival in patients with lung cancer. Collectively, HMGB1 plays a key role in the formation of lamellipodia and filopodia by regulating cytoskeleton dynamics and DRP1 expression to promote lung cancer migration.     

Stimulation of THP-1 Macrophages with LPS Increased the Production of Osteopontin-Encapsulating Exosome

Osteopontin (OPN) mediates bone remodeling and tissue debridement. The OPN protein is cleaved, but it is unclear how full-length (FL)-OPN or its cleaved form perform their biological activities in target cells. We, therefore, performed the molecular characterization of OPN in exosomes (Exo). The Exo were isolated from lipopolysaccharide (LPS)-stimulated phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages. The Exo were also isolated from PMA-differentiated THP-1 macrophages. The Exo were identified using the qNano multiple analyzer (diameter 59–315 nm) and western blotting with a CD9 antibody. LPS-stimulated cells produced more particles than non-stimulated cells. The presence of the FL or the cleaved form of OPN was confirmed using western blot analysis. A mixture of FL and cleaved OPN was also measured using an ELISA system (Ud-OPN) and their presence in the Exo was confirmed. Ud/FL ratios became low after LPS stimulation, indicating the enhanced encapsulation of FL-OPN in the Exo by LPS. These findings suggest that LPS stimulation of human macrophages facilitates the synthesis of FL-OPN, which is cleaved in cells or the Exo after release. These findings indicate that Exo is a suitable vehicle to transfer OPN to the target cells.     

Role of Glycated High Mobility Group Box-1 in Gastric Cancer

Advanced glycation end products (AGEs) are produced in response to a high-glucose environment and oxidative stress and exacerbate various diseases. N^ε-(Carboxymethyl)lysine (CML) is an AGE that is produced by the glycation of lysine residues of proteins. There are a few reports on alterations in protein function due to CML modification; however, its association with cancer is not clear. We investigated the significance of CML modification in high mobility group box protein-1 (HMGB1), a cytokine that is significantly associated with cancer progression. Treatment of the gastric cancer cell lines TMK1 and MKN74 with glyoxal or glucose resulted in increased CML modification compared to untreated cells. CML-HMGB1 was modified via oxidation and more pronouncedly activated the receptor for AGE and downstream AKT and NF-κB compared to naïve HMGB1 and oxidized HMGB1. CML-HMGB1 bound with reduced affinity to DNA and histone H3, resulting in enhanced extranuclear translocation and extracellular secretion. Treatment of gastric cancer cells with CML-HMGB1 enhanced cell proliferation and invasion, sphere formation, and protection from thapsigargin-induced apoptosis, and decreased 5-FU sensitivity in comparison to HMGB1. Further, CML-HMGB1 was detected at various levels in all the 10 gastric cancer tumor specimens. HMGB1 levels correlated with primary tumor progression and distant metastasis, whereas CML-HMGB1 levels were associated with primary tumor progression, lymph node metastasis, distant metastasis, and stage. In addition, CML-HMGB1 levels correlated with oxidative stress in cancer tissues and resistance to neoadjuvant therapy. Therefore, CML modification of HMGB1 enhanced the cancer-promoting effect of HMGB1. In this study, CML-HMGB1 has been highlighted as a new therapeutic target, and analysis of the molecular structure of CML-HMGB1 is desired in the future.     

Role of HMGB1 in Chemotherapy-Induced Peripheral Neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN), one of major dose-limiting side effects of first-line chemotherapeutic agents such as paclitaxel, oxaliplatin, vincristine, and bortezomib is resistant to most of existing medicines. The molecular mechanisms of CIPN have not been fully understood. High mobility group box 1 (HMGB1), a nuclear protein, is a damage-associated molecular pattern protein now considered to function as a pro-nociceptive mediator once released to the extracellular space. Most interestingly, HMGB1 plays a key role in the development of CIPN. Soluble thrombomodulin (TMα), known to degrade HMGB1 in a thrombin-dependent manner, prevents CIPN in rodents treated with paclitaxel, oxaliplatin, or vincristine and in patients with colorectal cancer undergoing oxaliplatin-based chemotherapy. In this review, we describe the role of HMGB1 and its upstream/downstream mechanisms in the development of CIPN and show drug candidates that inhibit the HMGB1 pathway, possibly useful for prevention of CIPN.     

Activation of Peripheral Blood Mononuclear Cells and Leptin Secretion: New Potential Role of Interleukin-2 and High Mobility Group Box (HMGB)1

Activation of innate immunity and low-grade inflammation contributes to hyperglycemia and an onset of Type 2 Diabetes Mellitus (T2DM). Interleukin-2 (IL-2), leptin, High Mobility Group Box-1 (HMGB-1), and increased glucose concentrations are mediators of these processes also by modulating peripheral blood mononuclear cells (PBMCs) response. The aim of this study was to investigate if HMGB-1 and IL-2 turn on PBMCs and their leptin secretion. In isolated human PBMCs and their subpopulations from healthy individuals and naïve T2DM patients, leptin release, pro-inflammatory response and Toll-like Receptors (TLRs) activation was measured. After treatment with IL-2 and HMGB1, NK (Natural Killer) have the highest amount of leptin secretion, whilst NK-T have the maximal release in basal conditions. TLR4 (TAK242) and/or TLR2 (TLR2-IgA) inhibitors decreased leptin secretion after IL-2 and HMGB1 treatment. A further non-significant increase in leptin secretion was reported in PBMCs of naive T2DM patients in response to IL-2 and HMGB-1 stimulation. Finally, hyperglycemia or hyperinsulinemia might stimulate leptin secretion from PBMCs. The amount of leptin released from PBMCs after the different treatments was enough to stimulate the secretion of IL-1β from monocytes. Targeting leptin sera levels and secretion from PBMCs could represent a new therapeutic strategy to counteract metabolic diseases such as T2DM.     

激活素A对巨噬细胞系RAW264.7细胞吞饮活性及TLR4表达的双向调节作用

目的探讨激活素A(ActivinA)对巨噬细胞系RAW264.7细胞活性的调节作用及其可能的机制。方法取对数生长期的小鼠巨噬细胞系RAW264.7细胞,加入1μg/ml脂多糖(LPS),继续培养8h,采用ELISA法检测细胞分泌ActivinA水平;分别加入ActivinA、LPS和ActivinA+LPS,中性红染料法检测细胞吞饮活性;流式细胞术分析细胞表面分子MHCⅠ、MHCⅡ及Toll样受体4(TLR4)的表达水平。结果LPS呈时间依赖性刺激RAW264.7细胞分泌ActivinA;ActivinA可明显促进静息RAW264.7细胞的吞饮活性,而对MHCⅠ、MHCⅡ及TLR4的表达水平无明显影响;ActivinA和LPS共同作用,ActivinA明显抑制了LPS活化的RAW264.7细胞的吞饮活性,并下调TLR4的表达。结论ActivinA可能以自分泌/旁分泌形式参与巨噬细胞活性调节,其抑制LPS作用与TLR4表达有关。