CD14的表达及其在库普佛细胞激活与所致组织损伤中的意义

背景：如何减轻甚至消除脂多糖(LPS)引起的全身炎症反应是目前研究的热点；库普佛细胞(KC)的激活与LPS的这种作用密切相关，但关于其具体的机制尚有待于进一步研究。目的：探讨LPS对KccDl4表达的影响及CD14在LPS激活KC中的意义。设计：完全随机前后对照研究。地点和对象：在第三军医大学西南医院病理学研究所完成。大鼠KC来源于Wistar大鼠，购自第三军医大学实验动物中心。干预：在分离培养大鼠KC的基础上，作者应用LPS直接或LPS刺激KC后产生的介质刺激新培养的KC，或在血清存在的情况下加入抗CD14单抗或在无血清的情况下单独加入LPS等措施刺激KC细胞。主要观察指标：各种条件下CD14 mRNA的表达及其蛋白合成的变化、培养KC中上清中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)和一氧化氮浓度。结果：①CD14 mRNA表达及其蛋白合成在正常KC中极弱，分别为0．035和19．91&;#177;2．89，浓度为10mg／L的LPS分别为0．73和676．79&;#177;24．95，其量与LPS浓度呈剂量依赖性相关。浓度为10μg／L的LPS刺激后KC中CD14 mRNA表达及其蛋白合成在30min即明显增强，分别为O．56和548．86&;#177;40．43，至6h达高峰(0．54和673．91&;#177;35．08)。②在血清存在时加入抗CD14单抗或在无血清时单独加入LPS，可明显降低KC TNF-α，IL-6和一氧化氮的释放。而后者如果同时加入工LBP，则可明显上调培养KC中的TNF-α，IL-6和一氧化氮浓度。结论：①LPS及其刺激KC后产生的活性介质与CD14 mRNA的表达及其蛋白合成密切相关，并推测在实验1～3h的CD14表达的增强可能主要由LPS引起，而此后CD14表达的进一步增强可能与KC释放的细胞因子密切相关。②低浓度LPS对KC的激活是CD14依赖的。     

磷肌酰脂醇特异性磷脂酶C对内毒素介导枯否细胞激活的抑制作用

目的 研究磷肌酰脂醇特异性磷脂酶C（PI-PLC）对内毒素介导的肝Kupffer细胞（枯否细胞，KCs）激活及其CDl4表达的抑制作用。方法 Wistar大鼠20只，分为PI-PLC组和LPS组，测定细胞培养液中TNF-α和IL-6含量变化，用免疫组化观察KCs膜CD14和核NF-κB的相对活性，测定KCs中CD14、TNF-α和IL-6的mRNA表达和KCs膜CD14蛋白含量变化。结果 不同浓度的LPS刺激60min后，PI-PLC组培养液中TNF-α与IL-6的含量随LPS浓度的增高而增加，但明显低于LPS组（P〈0．01）；CD14抗体染色显示，PI-PLC组部分KCs为弱阳性，而LPS组KCs为阳性细胞；NF-κB P65抗体染色显示，PI-PLC组部分KCs为弱阳性细胞，而LPS组KCs为强阳性细胞；PI-PLC组NF-κB活性在10μg／mL以上LPS刺激下才有升高，其相对光密度值显著低于LPS组（P〈0．01）；在相同浓度LPS刺激后，PI-PLC组CD14、TNF-α和IL-6的mRNA表达显著低于LPS组（P〈0．01）；PI-PLC组在相同浓度LPS刺激120min后CD14蛋白表达才明显，LPS组在100μg／mL LPS刺激后30min CD14蛋白开始升高，两者有显著性差异（P〈0．01）。结论 PI-PLC对LPS介导的KCs激活有明显的抑制作用，其机制可能与抑制KCs中CD14蛋白的表达有关。     

脂多糖对间充质干细胞Toll样受体4基因的表达水平及活性的影响

目的 探讨脂多糖(LPS)对骨髓间充质干细胞(MSC)Toll样受体4(TLR-4)基因的表达及其功能的影响.方法 采用贴壁培养和密度梯度离心法从大鼠骨髓分离MSC,通过细胞形态、成骨分化潜能及流式细胞术检测表型以鉴定其纯度;半定量RT-PCR和流式细胞术分别检测MSC在不同浓度(1、10、100 μg/ml)LPS存在条件下培养24 h的TLR-4 mRNA相对表达量和共刺激分子(CD80、CD86、MHC-Ⅱ)的表达水平;ELISA法定量检测TNF-α的分泌水平.结果 骨髓MSC低表达TLR-4mRNA(相对表达量0.61±0.10),同时表达CD80[(9.56±0.69)%]、CD86[(22.03±2.03)%]、MHC-Ⅱ[(2.51±0.97)%],少量分泌TNF-α[(4.97±2.98)pg/ml].MSC经LPS处理后,其TLR-4mRNA、共刺激分子表达和TNF-oα分泌水平均升高,其中10 μg/ml LPS培养组升高显著,TLR-4 mRNA相对表达水平为1.55±0.02;CD80、CD86、MHC-Ⅱ阳性细胞率分别为(41.70±2.92)%、(59.72±2.00)%、(24.56±2.19)%;TNF-α分泌水平为(213.12±69.08)pg/ml,与对照组比较,P值均＜0.01.100μg/ml LPS处理组与10 μg/mlLPS处理组相比,各项检测指标均降低,但MHC-Ⅱ和TNF-α的降低水平无统计学意义(P＞0.05).结论 骨髓MSC低表达TLR-4,体外LPS可促进骨髓MSCTLR-4的表达,且与浓度相关.伴随TLR-4表达水平的升高,CD80、CD86、MHC-Ⅱ表达水平及TNF-α水平同时升高.     

中药当归主要活性成分阿魏酸钠抑制淀粉样β蛋白激活炎症因子的量剂反应

目的:观察中药当归提取的主要活性成分阿魏酸钠对淀粉样β蛋白激活的小鼠腹腔巨噬细胞α肿瘤坏死因子、诱导型一氧化氮合酶和一氧化氮水平的影响。方法:实验于2004-03/10在锦州医学院药理实验室和解剖实验室完成。选择8～10周龄的小鼠36只,随机分为6组,每组6只。正常对照组:巨噬细胞接种24h后用培养基继续培养。淀粉样β蛋白组:巨噬细胞接种24h后在培养基中加入经老化处理的终浓度为10μmol/L的淀粉样β蛋白。淀粉样β蛋白+10,100,500μmol/L,1mmol/L阿魏酸钠组:在加入淀粉样β蛋白的同时加入10,100,500μmol/L,1mmol/L的阿魏酸钠共同孵育。培养48h后采用酶联免疫吸附法、免疫组织化学方法和Griess反应分别检测肿瘤坏死因子α分泌量、诱导型一氧化氮合酶表达和一氧化氮的生成量。结果:36只小鼠均进入结果分析。①巨噬细胞的肿瘤坏死因子α分泌量:淀粉样β蛋白组明显高于正常对照组[(281.54±14.85),(17.55±4.84)ng/L,(P<0.01)]。淀粉样β蛋白+10,100,500μmol/L,1mmol/L阿魏酸钠组可以不同程度地减少由淀粉样β蛋白诱导产生的肿瘤坏死因子α的分泌量[(238.05±6.21),(186.90±7.44),(117.55±8.08),(71.77±10.50)ng/L,P<0.01],且有明显剂量依赖关系(P<0.05)。②巨噬细胞的一氧化氮生成量:淀粉样β蛋白组明显高于正常对照组[(85.04±6.16),(26.10±3.25)μmol/L,(P<0.01)],淀粉样β蛋白+10,100,500μmol/L,1mmol/L阿魏酸钠组可以不同程度地减少由淀粉样β蛋白诱导产生的一氧化氮的量[(69.80±4.96),(54.52±3.45),(43.88±4.04),(33.83±3.13)μmol/L,P<0.01],且有明显剂量依赖关系(P<0.05)。③诱导型一氧化氮合酶的表达:正常对照组只有零星细胞染色阳性;而淀粉样β蛋白组多数细胞胞浆被染成黄褐色。各剂量组的阿魏酸钠可以不同程度地抑制由淀粉样β蛋白激活巨噬细胞造成的诱导型一氧化氮合酶的表达,该抑制作用呈明显剂量依赖性。结论:①淀粉样β蛋白能激活巨噬细胞,使其分泌大量的肿瘤细胞坏死因子α和一氧化氮,诱导型一氧化氮合酶表达也相应增加。②阿魏酸钠通过其抗炎作用呈明显剂量依赖性抑制淀粉样β蛋白对巨噬细胞的激活作用,从而使巨噬细胞生成肿瘤细胞坏死因子α、诱导型一氧化氮合酶、一氧化氮水平降低。     

CD14对胃癌SGC-7901细胞TNF-α、IL-1β、IL-6和IL-12表达的影响

目的 研究胃癌细胞中CD14的过表达对TNF-α、IL-1β、IL-6、IL-12的影响,探讨CD14在胃癌发生发展中的作用.方法 在本实验室前期建立的胃癌SGC-7901 CD14稳定转染细胞系的基础上,利用CD14蛋白受体胞壁酰二肽（MDP）刺激细胞,RT-PCR检测各细胞因子mRNA的表达,Western blot检测各细胞因子蛋白的表达,以空质粒转染的胃癌SGC-7901细胞为对照,探讨CD14强制表达对TNF-α、IL-1β、IL-6、IL-12表达的影响.结果 转染并稳定表达CD14的细胞中TNF-α、IL-13、IL-6、IL-12在mRNA及蛋白表达水平上都有不同程度的升高,与空质粒转染的细胞相比,差异均有统计学意义（P均＜0.05）.结论 CD14对TNF-α、IL-1β、IL-6、IL-12的表达具有一定的促进作用.     

核因子-κB对胰腺AR42J细胞肿瘤坏死因子-α表达的调控

目的 探讨脂多糖(lipopolysaccharide,LPS)对胰腺腺泡AR42J细胞细胞因子表达的影响及其可能机制,进一步阐明急性胰腺炎的发病机制.方法 用不同质量浓度的LPS(0.001,0.01,0.1,1,10,100 mg/L)刺激AR42J细胞18 h,同时用10mg/L的LPS刺激AR42J细胞不同时间(2,6,12,18,24h),RT-PCR检测核因子-κB(NF-κB)P65和肿瘤坏死因子-α(TNF-α)mRNA表达的变化,放射免疫法(RIA)检测培养液上清TNF-α蛋白浓度的改变.对NF-κB(P65)mRNA的表达与TNF-α mRNA的表达进行相关和回归分析.结果 RT-PCR结果表明0.001 mg/L的LPS处理AR42J细胞时即可出现TNF-α及NF-κB(P65)mRNA表达的明显上调,且呈量效关系;用10 mg/L的IPS处理后,在2 h后即可出现TNF-α及NF-κB(P65)mRNA表达的明显上调,并且呈时效关系.RIA结果表明用0.01 mg/L的LPS处理AR42J细胞后即可出现TNF-α蛋白表达的明显上调,且呈量效关系;用10mg/L的LPS处理后,在6 h后即可出现TNF-α蛋白表达的明显上调,并且呈时效关系.TNF-α mRNA的表达与P65 mRNA的表达呈正相关.结论 LPS可以以时间剂量依赖方式地刺激AR42J细胞NF-κB(P65)和TNF-α的表达,NF-κB(P65)mRNA的表达与TNF-αmRNA的表达呈正相关.针对NF-κB靶点,抑制其活性,可为包括AP的治疗提供一条新的途径.     

脂多糖对血管平滑肌细胞表达MMP-9的影响及可能机制

目的探讨脂多糖(lipopolysaccharides,LPS)对培养的大鼠血管平滑肌细胞(vascular smooth musch cell,VSMC)表达基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)的影响及可能的分子机制。方法贴块法进行血管平滑肌细胞培养,细胞免疫化学检测MMP-9的蛋白表达及核因子NF-κB激活情况,原位分子杂交分析MMP-9mRNA表达。结果LPS是MMP-9的强诱导物,可在蛋白及mRNA水平诱导MMP-9的表达,LPS对其诱导作用强度与LPS的浓度呈正相关。与正常组比较LPS刺激15min即有NF-κB p65核转移,30min达高峰,1h后减弱。结论LPS诱导平滑肌细胞MMP-9表达,其作用呈浓度时间依赖性,MMP-9表达升高可能与LPS激活NF-κB有关。MMP-9表达升高和NF-κB激活提示LPS在动脉粥样硬化发病过程中可能起一定作用。     

CD14抑制肽的体外生物活性

目的研究CD14抑制肽(CD14inhibitory peptide,CD14-IP)与CD14的结合活性及对内毒素(LPS)和脂多糖结合蛋白(LBP)诱导的人单核巨噬细胞株U937表达肿瘤坏死因子-α(TNF-α)的影响。方法采用酶联免疫吸附(ELISA)法进行CD14-IP与CD14的结合实验。U937细胞用佛波脂(PMA)诱导成熟后分为五组:正常对照组、LPS组(100ng/mLLPS 100ng/mLrhLBP)、高剂量多肽组、中剂量多肽组及低剂量多肽组,后三组分别给予10μg/mL、1.0μg/mL和0.1μg/mL的CD14-IP。用ELISA测定培养细胞上清TNF-α的含量,用RT-PCR测定U937细胞TNF-αmRNA的表达水平。结果CD14-IP有较强的与CD14结合的能力;CD14-IP组TNF-α和TNF-αmRNA水平均较LPS组低,较正常组高。结论CD14-IP能与CD14结合,并能降低培养细胞TNF-α和TNF-αmRNA的表达,具有抑制CD14生物活性的作用。     

miRNA-146a-5p对癫痫大鼠海马神经元NF-κB信号转导通路的影响

目的观察miRNA-146a-5p(miR-146a-5p)对癫痫大鼠海马神经元核因子κB(NF-κB)信号转导通路的影响。方法体外培养新生SD大鼠海马神经元,随后制备癫痫海马神经元模型,随机分为三组,空白对照组(control组):转染时添加等体积opti-MEM培养基;阴性对照组(NC组):转染80 n M浓度NC至癫痫大鼠海马神经元; miR-146a-5p组:转染80 n M浓度miR-146a-5p mimics至癫痫大鼠海马神经元。转染后培养24 h,再用200 ng/ml脂多糖(LPS)诱导6 h,采用免疫荧光双染法鉴定体外培养海马神经元,采用膜片钳技术检测正常大鼠海马神经元和癫痫大鼠海马神经元自发性放电频率,采用逆转录-聚合酶链反应(RT-PCR)检测未经LPS诱导各组海马神经元miR-146a-5p表达水平及经LPS诱导后各组海马神经元核因子κB抑制蛋白α(IκBα)、磷酸化IκBα(p IκBα)、NF-κB P65、IKB激酶-β(IKKβ) mRNA表达水平,采用Western blot法检测经LPS诱导后各组海马神经元IκBα、p IκBα、NF-κB P65、IKKβ蛋白表达水平,采用酶联免疫吸附(ELISA)法检测经LPS诱导后各组细胞培养液中肿瘤坏死因子α(TNF-α)、白细胞介素1(IL-1)、白细胞介素6(IL-6)、细胞间黏附分子1(ICAM-1)表达水平。结果体外培养7 d的癫痫大鼠海马神经元与正常大鼠海马神经元相比较形态未见明显异常;癫痫大鼠海马神经元自发性放电频率明显较正常大鼠海马神经元明显增加(P 0. 05); miR-146a-5p组海马神经元miR-146a-5p表达水平明显高于control组和NC组(P 0. 05);经LPS诱导后miR-146a-5p组海马神经元p IκBα、NF-κB P65、IKKβmRNA及蛋白表达水平明显低于control组和NC组(P 0. 05),而IκBαmRNA及蛋白表达水平明显高于control组和NC组(P 0. 05);经LPS诱导后miR-146a-5p组细胞培养液中TNF-α、IL-1、IL-6、ICAM-1表达水平明显低于control组和NC组(P 0. 05)。结论miR-146a-5p可抑制LPS诱导的癫痫大鼠海马神经元NF-κB信号转导通路中p IκBα、NF-κB P65、IKKβmRNA及蛋白表达,促进IκBαmRNA及蛋白表达,减少TNF-α、IL-1、IL-6、ICAM-1等炎症因子的释放。     

白细胞介素6对小鼠肺泡巨噬细胞CD14、清道夫受体表达的调节功能

目的:阐明白细胞介素(IL-6)对小鼠肺泡巨噬细胞(alveolarmacrophage,AM)清道夫受体(scavengerreceptor,SR)及CD14表达的直接调节作用及探讨IL-6提高细胞免疫功能的作用。方法:分离培养小鼠AM,以不同剂量(0,0.01,0.1,1,10,100μg/L)IL-6刺激细胞16h或以100μg/LIL-6在不同时间(0,2,4,8,12,16h)刺激细胞,采用免疫细胞化学及RT-PCR方法观察SR,CD14表达变化。结果:IL-6刺激AM能增强CD14蛋白表达并抑制SR蛋白表达,低至0.01μg/LIL-6刺激16h或100g/LIL-6刺激6h后就能显著增强CD14mRNA并明显抑制SRmRNA表达,与此同时,CD14蛋白表达也明显增强而SR蛋白表达显著下降。结论:IL-6刺激AM能在mRNA及蛋白水平显著增强CD14表达并抑制SR表达。     

Upregulation of mouse CD14 expression in Kupffer cells by lipopolysaccharide

Western blot analysis showed that a monoclonal antibody against recombinant mouse CD14 (mCD14), designated rmC5-3, specifically reacted with mouse macrophage cell line J774, but not myeloma cell line NS1. Fluorographic and immunocytochemical analysis demonstrated specific binding of rmC5-3 with mouse resident macrophages, inflammatory monocytes and neutrophils, and macrophage cell lines. Immunohistochemical staining using rmC5-3 showed that CD14-positive Kupffer cells (KC) were small in number in the liver in nonstimulated mice. The number of stained KC, which were rich in the midzonal and periportal regions, gradually increased with time after intraperitoneal injection of lipopolysaccharide (LPS), peaked 6 h after injection, and returned to normal by 20 h after injection. Staining intensity over time was proportional to the number of KC. A slight increase in mCD14 expression was observed in peritoneal macrophages 2 h after LPS administration in vivo using flow cytometric analysis. mCD14 mRNA became detectable at 1 h after the intraperitoneal injection of LPS (20 micrograms/mice), and the level dramatically increased with time, peaking at 3 h, and sharply dropped at 6 h. The resident peritoneal macrophages demonstrated a constitutively high mCD14 mRNA expression, which slightly increased 2 h after LPS (100 ng/ml) stimulation in vitro. The level of mCD14 expression in macrophages did not increase after intraperitoneal injection of LPS (20 micrograms/mice).     

唾液酸结合免疫球蛋白样凝集素1在冠状动脉粥样硬化性心脏病患者外周血单个核细胞的表达及临床意义

目的 观察唾液酸结合免疫球蛋白样凝集素1(sialic acid-binding immunoglobulin-likelectin 1,Siglec-1,也称CD169)在冠状动脉粥样硬化性心脏病(CHD)患者外周血淋巴细胞、单核细胞和中性粒细胞上的表达水平,并探讨其与冠状动脉粥样硬化发生发展的关系.方法 流式细胞术检测57例CHD患者及38名健康对照者外周血CD14CD169双阳性细胞的表达率;生化常规测定所有入选者血脂水平;实时荧光相对定量逆转录(FQ-RT)-PCR方法检测入选对象外周血单个核细胞(PBMCs)中CD169 mRNA的含量.结果 流式细胞仪检测发现,CD169在健康对照组及CHD组淋巴细胞和中性粒细胞上均无表达;CHD组单核细胞CD14CD169双阳性率为(12.7±2.4)%,显著高于健康对照组[(1.0±0.3)%,t=23.2,P<0.01];CHD组CD169 mRNA的拷贝数显著高于健康对照组拷贝数(t=6.59,P<0.01),为健康对照组的3..倍;而CHD血脂正常组和血脂异常组的CD169阳性率[分别为(12.2±2.3)%和(13.4±2.5)%,t=1.87,P>0.05]和mRNA平均拷贝数(分别为健康对照组的3.64和2.79倍,t=0.98,P>0.05)差异均无统计学意义.结论 CD169组成性表达于特定组织巨噬细胞上,健康人外周血单核细胞上并不表达,单核巨噬细胞受到炎症刺激时,CD169表达迅速上调.CD169蛋白及mRNA含量在CHD患者外周血单核细胞上表达显著升高,CHD患者外周血单核细胞已发生巨噬细胞化,单核巨噬细胞介导的免疫炎症反应在CHD发生发展过程中起重要作用.     

Murine CD14 gene expression in vivo: extramyeloid synthesis and regulation by lipopolysaccharide

A murine model system was used to study the distribution and regulation of CD14 gene expression in vivo. Western blot analysis failed to detect CD14 in plasma from untreated CB6 (BALB/c x C57Bl6) mice, but showed markedly increased levels of CD14 in plasma from mice treated with lipopolysaccharide (LPS). Plasma levels of CD14 increased in a time- and dose-dependent manner, reaching a maximum between 8 and 16 h. Northern blot analysis of total RNA extracted from mouse tissues revealed low, but significant, levels of CD14 mRNA in many tissues of untreated animals with the highest levels in uterus, adipose tissue, and lung. After intraperitoneal injection of LPS, induction of CD14 gene expression was detected in all organs examined with the extent of induction varying between organs. Induction of CD14 mRNA was both time and dose dependent. Maximum induction in the heart and lung was observed 2-4 h after injection of LPS, while liver and kidney showed maximal induction between 8 and 16 h. In situ hybridization showed that CD14 mRNA was expressed in myeloid cells in many tissues, and that expression in these cells was upregulated by LPS. Unexpectedly, CD14 mRNA was also detected in other cells within tissues, including epithelial cells, and expression in these cell types also was upregulated by LPS. Immunochemical analysis revealed that CD14 antigen colocalized to the cytoplasm of cells expressing CD14 mRNA. These studies demonstrate that CD14 gene expression is not restricted to myeloid cells, and that the level of expression of CD14 is influenced by exposure to LPS.     

脂多糖诱导血管平滑肌细胞分泌白细胞介素-6及p38丝裂素活化蛋白激酶的调控作用

目的 探讨p38丝裂素活化蛋白激酶(MAPK)信号转导通路在脂多糖(LPS)作用下血管平滑肌细胞(VSMC)分泌白细胞介素-6(IL-6)中的调节作用.方法 将体外培养的大鼠胸主动脉VSMC分为LPS刺激组、p38MAPK抑制剂SB203580干预组、SB203580对照组和溶液对照组.LPS组以终浓度100μg/L的LPS与VSMC共同孵育;干预组VSMC以p38MAPK抑制剂SB203580 10μmol/L预处理2 h,再加入终浓度100 9g/L的LPS共同孵育;对照组仅以SB203580 10 μmol/L预处理2 h;溶液组仅加入去血清培养液培养.各组于培养0、3、6、12、24 h后采用实时聚合酶链反应(real-time PCR)和酶联免疫吸附法(ELISA)分别检测细胞IL-6 mRNA和上清液中IL-6蛋白表达.结果 LPS刺激3 h,VSMC中IL-6 mRNA和蛋白表达即出现明显增高CmRNA(21.3±3.2)×104,蛋白(296.2±19.6)ng/L],12 h达高峰CmRNA(131.4±11.2)×104,蛋白(897.7±34.0)ng/L],24 h有所降低[mRNA(15.3±4.7)×104,蛋白(194.3±24.0)ng/L],但仍显著高于溶液组(mRNA(9.4±1.9)×104,蛋白(29.4±4.4)ng/L,均P<0.05].干预组3、6、12 h可明显抑制LPS诱导VSMC中IL-6的分泌[mRNA(15.4±3.6)×104、(43.2±6.6)X 104、(56.2±5.5)×104,蛋白(180.3±23.6)、(432.2±56.8)、(546.2±57.9)ng/L,均P<0.05].结论 LPS诱导VSMC可明显增加IL-6的mRNA和蛋白表达,p38MAPK抑制剂SB203580可显著抑制IL-6转录和蛋白合成,表明p38MAPK信号转导通路可能通过直接或间接作用参与了IL-6的分泌调节作用.     

Expression patterns of the lipopolysaccharide receptor CD14, and the FCgamma receptors CD16 and CD64 on polymorphonuclear neutrophils: data from patients with severe bacterial infections and lipopolysaccharide-exposed cells

In polymorphonuclear neutrophils (PMN) CD14, one of the receptors for lipopolysaccharides (LPS) is stored intracellularly as a preformed protein, with only few receptors expressed on the surface. We now report that in patients with severe bacterial infections, CD14 expression is profoundly upregulated, as is CD64 (FcgammaRI), the high-affinity receptor for IgG, whereas CD16 (FcgammaRIII) was partly lost from the surface. To further analyze regulation of these receptors, PMN of healthy donors were exposed to low doses of LPS. By brief exposure (10-120 min) to LPS, CD14 was transferred to the surface in a cytochalasin B-sensitive manner, as were CD16 and CD64. Prolonged culture (up to 48 h) resulted in a further upregulation of CD14, sustained expression of CD64, and profound decline of CD16, yielding a similar pattern of receptor expression as seen in the patients. Subsequent studies revealed that LPS induced de novo synthesis of CD14: the increase of surface expression could be inhibited by cycloheximide and by interfering with a known LPS-induced signaling event, the translocation of NFkappaB. Moreover, an up to 10-fold increase of specific mRNA was seen, as was incorporation into CD14 of 35S-methionine. The de novo synthesis prolonged expression of CD14, whereas the CD16 expression declined, generating a PMN phenotype characteristic for severe infection and indicative of escape from apoptosis of a PMN subpopulation.     

慢性血吸虫感染对脓毒症小鼠保护作用的研究

目的 初步探讨慢性血吸虫(SJ)感染对脓毒症小鼠的保护作用及其机制.方法 选择BALB/c雄性小鼠,按随机数字表法分组进行三部分实验.实验1:经腹部皮肤接种SJ尾蚴感染8周建立慢性SJ感染模型,分为正常组和SJ组,每组10只;用酶联免疫吸附法(ELISA)检测血清白细胞介素(IL-4和IL-10)、肿瘤坏死因子-α(TNF-α)、γ-干扰素(IFN-γ)水平,实时荧光定量聚合酶链反应(PCR)检测腹腔巨噬细胞IL-10和TNF-α的mRNA表达,了解慢性SJ感染小鼠免疫状态.实验2:以脂多糖(LPS)腹腔注射诱导小鼠脓毒症模型,分为LPS组和SJ-LPS组,每组15只;用ELISA法动态观察注射LPS后0、24、48和72 h细胞因子的变化,0 h的水平相当于正常小鼠和SJ感染8周水平,观察慢性SJ感染对脓毒症过程的影响.实验3:分别以盲肠结扎穿孔术(CLP)和LPS诱导两种不同的脓毒症模型,评价慢性SJ感染对脓毒症小鼠72 h存活率的影响.结果 实验1:SJ组血清抗炎因子IL-4[(151.35±12.24)ng/L]和IL-10[(133.22±11.09)ng/L]水平较正常组[IL-4(56.32±8.66)ng/L,IL-10(48.17±7.23)ng/L]显著升高(均P＜0.05),并可使巨噬细胞向替代活化性巨噬细胞分化,慢性SJ感染使腹腔巨噬细胞高表达IL-10 mRNA(SJ组4.46±1.82,正常组1.52±0.60),抑制TNF-α mRNA表达(SJ组1.61±0.93,正常组2.32±1.03,均P＜0.05).实验2、3:慢性SJ感染小鼠血清IL-4、IL-10于注射LPS后0 h即显著升高,随后下降,至72 h仍明显高于LPS组[IL-4(ng/L):92.2±7.6比41.5±4.5;IL-10(ng/L):92.1±7.8比35.6±4.0,均P＜0.05];TNF-α、IFN-γ均于24 h达峰值后逐渐下降,至72 h SJ-LPS组仍显著低于LPS组[TNF-α(ng/L):82.9±5.6比91.5±5.2;IFN-γ(ng/L):44.1±4.8比52.6±4.0,均P＜0.05].慢性SJ感染可明显改善CLP或LPS所致脓毒症小鼠的存活率(CLP:80%比20%,LPS:70%比30%,均P＜0.05).结论 慢性SJ感染可使脓毒症小鼠血清抗炎因子升高,存活率上升,从而起到保护作用.

Abstract：

Objective To preliminarily study the protective effect of chronic schistosoma japonica (SJ)infestation against sepsis in mice and its mechanism. Methods BALB/c male mice were used, and the experiment was divided into three parts. Experiment 1: chronic SJ infestation model was reproduced by SJ cercaria inoculation through abdominal skin for 8 weeks. Twenty mice were randomly grouped into normal group (n=10) and SJ group (n=10). The levels of interleukins (IL-4, IL-10), tumor necrosis factor-α(TNF-α) and interferon-γ (IFN-γ) in serum were detected by enzyme linked immunosorbent assay (ELISA).Real-time polymerase chain reaction (PCR) was employed to detect the levels of IL-10 mRNA and TNF-αmRNA in abdominal macrophages. This experiment was meant to evaluate immune state in mice with chronic SJ infestation. Experiment 2: lipopolysaccharide (LPS) was intraperitoneally injected to reproduce sepsis model. Thirty mice were randomly grouped into LPS group (n=15) and SJ-LPS group (n=15). The levels of cytokines were determined by ELISA at 0, 24, 48 and 72 hours after LPS injection. This experiment was meant to detect the effect of chronic SJ infestation in mice during the septic process. Experiment 3 : two types of sepsis model were reproduced by cecal ligation and puncture (CLP) and LPS injection, respectively. The survival rate of mice with chronic SJ infestation in 72 hours in either type of sepsis was evaluated. Results Experiment 1, compared with normal group [IL-4 (56.32±8.66) ng/L, IL-10 (48.17±7.23) ng/L],chronic SJ infestation showed an increase in serum IL-4 [(151. 35 ± 12. 24) ng/L] and IL-10 [(133. 22 ±11. 09) ng/L, both P＜0. 05]. Chronic SJ infestation also resulted in an increase in IL-10 mRNA expression (SJ group 4. 46±1. 82, normal group 1. 52±0. 60) and inhibited TNF-α mRNA expression (SJ group 1. 61±0.93, normal group 2. 32±1.03) in abdominal macrophages (both P＜0. 05), indicating that macrophages could be differentiated into alternative activated macrophages. Experiments 2 and 3 showed that the levels of serum IL-4 and IL-10 were increased at 0 hour after LPS injection, and then gradually decreased in SJ-LPS group, but the levels were still higher than those in LPS group at 72 hours [IL-4 (ng/L): 92. 2±7. 6 vs.41.5±4. 5; IL-10 (ng/L): 92. 1±7. 8 vs. 35. 6±4. 0, both P＜0. 05]; the levels of TNF-α and IFN-γ were increased at 24 hours, and then decreased in SJ-LPS group, and the levels were lower than those in LPSgroup at 72 hours [TNF-α (ng/L): 82. 9±5. 6 vs. 91. 5±5. 2; IFN-γ (ng/L): 44.1±4. 8 vs. 52. 6±4. 0,both P＜0. 05]. Therefore, chronic SJ infestation could improve the survival rate of mice with sepsis induced by CLP or LPS (CLP: 80% vs. 20%, LPS: 70% vs. 30%, both P＜0.05). Conclusion Chronic SJ infestation could elevate anti-inflammatory factors in septic mice, thus ameliorating the survival rate, so it has protective effect on mice with sepsis.     

高容量血液滤过对多器官功能障碍犬内毒素及CD14的影响

目的 观察高容量血液滤过(HVHF)治疗多脏器功能障碍综合征(MODS)犬中血浆内毒素(LPS)及其受体CD14的变化,探讨HVHF防治MODS的作用机制。方法 12只Beagle 犬采用失血性休克+复苏灌注+内毒素血症建立MODS模型,随机(随机数字法)分为HVHF组(n=6)和MODS组(n=6),HVHF组在LPS注射完毕后给于HVHF治疗24h。分别于术前(T1)、LPS注射前(T2)、LPS注射完后0 h(T3),3 h(T4),6 h(T5),9 h(T6),12 h(T7),15 h(T8)及24 h(T9),观察血浆LPS及其受体CD14、器官功能指标变化。LPS采用MB-80微生物快速动态检测系统测定。CD14采用ELISA测定。统计学方法各时间点前后采用重复测量资料方差分析。结果 HVHF治疗后血浆LPS及CD14明显下降,在T4-9时间点明显低于MODS组(P＜0.01)。超滤液中能检测出CD14,SC为(0.81±0.09)。超滤液中未能检测出LPS。HVHF组主要器官功能明显改善,平均动脉压在T4-9时间点显著高于MODS组(P＜0.0l)。动脉血氧分压逐渐升高,与MODS组在T4-9时间点比较差异有统计学意义(P＜0.01)。HVHF治疗后肌酐、尿素氮逐渐下降,与MODS组在T4-9时间点比较差异有统计学意义(P＜0.0l)。结论 HVHF能改善主要器官功能、降低血浆LPS及CD14峰值,有效遏制过度炎症反应。     

内毒素休克小鼠脂钙蛋白2及其相关细胞因子的表达

摘要：目的 探讨内毒素对小鼠脂钙蛋白2及其相关细胞因子表达水平的影响。方法 C57BL/6小鼠予内毒素腹腔内注射，10mg/kg、20 mg/kg、30 mg/kg各为1组，对照组仅注射生理盐水（n=8）。观察24小时后留取盲肠和血清，ELISA和Western blot测定IL-1β、IL-6、IL-17A、LCN2，实时PCR相对定量肠组织中LCN2 mRNA。结果 与非造模组比较，大剂量内毒素组（20 mg /kg和30 mg /kg）的血清IL-6、LCN2与肠组织IL-1β、IL-6、IL-17A、LCN2显著增高。与10 mg /kg组比较，30 mg /kg组的血清IL-6、LCN2水平以及肠组织IL-17A、LCN2、IL-1β显著增高。各组2-ΔΔCT以均值±标准差表示，对照组1.548±0.417，10mg/kg 组3.184±0.716，20mg/kg组 4.875±0.547, 30mg/kg组7.489±0.820，各组两两比较均显示统计学差异。结论 内毒素剂量依赖性地引起小鼠肠组织LCN2表达水平增高；血清IL-6和 LCN2水平以及肠组织IL-1β、IL-17A与LCN2水平之间存在一定的相关性。     

iPSC-MSC来源的外泌体对LPS刺激肺泡巨噬细胞产生炎性因子的影响

目的探讨i PSC-MSC来源的外泌体(exosome,Exo)对LPS刺激的肺泡巨噬细胞释放炎性因子的作用。方法采用旋转超滤法提纯i PSC-MSC外泌体,以无外泌体培养基培养肺泡巨噬细胞NR8383,分别给予Exo(50μg/ml)、LPS(50ng/ml)、LPS(50ng/ml)+Exo(50μg/ml)培养24h,以不含Exo和LPS培养为对照组,利用酶联免疫标记法(ELISA)检测各组上清液中TNF-α、IL-1β、IL-6蛋白浓度。结果提取物经透射电镜观察为圆形或半圆形囊泡,直径40~100nm,表达Exo标志物CD9和CD63;LPS组上清液中TNF-α、IL-1β和IL-6的浓度(435.38±36.31pg/ml、319.76±39.14pg/ml和408.33±43.44pg/ml)与空白对照组(37.48±8.75pg/ml、33.51±7.88pg/ml和37.73±8.46pg/ml)和Exo组(38.71±9.14pg/ml、32.05±6.81pg/ml和42.84±6.54pg/ml)比较显著上调(P0.01);LPS+Exo组TNF-α、IL-1β和IL-6的浓度(369.30±32.74pg/ml、249.23±36.77pg/ml和328.91±46.45pg/ml)与LPS组相比,明显减少(P0.05);空白对照组与Exo组的TNF-α、IL-1β和IL-6的浓度无显著性差异。结论成功富集Exo;i PSC-MSC来源的Exo可抑制LPS诱导的肺泡巨噬细胞表达炎性因子。