白细胞介素-10抑制人肾系膜细胞环氧化酶-2表达及其催化的前列腺素E2释放的实验研究

目的:观察IL-10对IL-1β诱导的人系膜细胞(HMC)前列腺素E₂(PGE₂)释放及环氧化酶-2(cyclooxygenase-2,COX-2)基因和蛋白表达的影响。方法:应用放射免疫测定法检测HMC培养上清中PGE₂,应用RT-PCR和Westernblot分别检测COX-2mRNA和蛋白水平。结果:①IL-1β显著上调PGE₂释放及COX-2基因和蛋白的表达(P均＜0.01);②IL-10对基础状态下PGE₂释放及COX-2基因和蛋白表达无明显影响(P＞0.05);③IL-10可呈剂量依赖性地下调IL-1β诱导的PGE₂释放及COX-2mRNA和蛋白表达(P＜001)。结论:IL-10抑制IL-1β诱导的HMCPGE₂释放及COX-2表达,提示IL-10对HMC具有多方面抗炎作用。     

香烟提取物和脂多糖对人胚肺成纤维细胞TGF-β1 mRNA及蛋白表达的影响

目的:探讨香烟提取物(CSE)和脂多糖(LPS)对体外培养的人胚肺成纤维细胞(HELF)转化生长因子-β₁(TGF-β₁)mRNA及其蛋白表达的影响。方法:应用不同浓度的CSE(1∶50、1∶25和1∶10)、LPS(0.1 mg/L、1 mg/L和10 mg/L)及CSE(1∶25)与LPS(1 mg/L)联合作用于HELF, 37℃作用24 h后, 提取细胞总RNA, 应用逆转录-多聚酶链反应(RT-PCR)及免疫细胞化学技术检测TGF-β₁ mRNA和蛋白表达的变化。结果:CSE低浓度(1∶50和1∶25)时可增加HELF TGF-β₁ mRNA及蛋白的表达(Ｐ＜0.05), 高浓度(1∶10)时未引起TGF-β₁ mRNA及蛋白表达增强(Ｐ＞0.05)。不同浓度的LPS均引起HELF TGF-β₁ mRNA及蛋白表达增强(Ｐ＜0.05)。CSE与LPS联合作用也可增加HELF TGF-β₁ mRNA及蛋白的表达(Ｐ＜0.05)。结论：一定浓度的CSE和LPS可上调肺成纤维细胞TGF-β₁ mRNA及蛋白表达。     

脂氧素A4对肝细胞生长因子诱导HepG2肝癌细胞血管生成相关细胞因子表达的影响

目的: 探讨脂氧素A₄(LXA₄)对肝细胞生长因子(HGF)诱导的HepG2肝癌细胞血管生成相关细胞因子表达的影响。方法: 体外培养HepG2肝癌细胞,实验分为空白组、HGF处理组、HGF+LXA₄处理组、HGF+脂氧素受体激动剂BML-111处理组。RT-PCR检测脂氧素受体(ALX)表达情况,Western blotting检测COX-2、MMP-2、MMP-9、IκBα和NF-κB p65的表达量,ELISA检测TNF-α、IL-1β、VEGF和TGF-β分泌水平, 荧光素酶报告质粒检测NF-κB转录活性。结果: HepG2肝癌细胞表达ALX,LXA₄和BML-111下调COX-2、 MMP-2和MMP-9,抑制TNF-α、IL-1β、VEGF和TGF-β分泌,并且干扰NF-κB转位及其转录活性。结论: 脂氧素抑制HGF诱导HepG2肝癌细胞表达血管生成相关细胞因子,包括VEGF、COX-2、TNF-α、IL-1β、TGF-β、MMP-2及MMP-9,此效应可能通过干扰NF-κB活化实现。     

丹参单体通过下调粘着斑激酶诱导H2O2刺激的肝星状细胞凋亡

目的: 研究丹参单体IH764-3对H₂O₂刺激的肝星状细胞(HSCs)凋亡的诱导作用以及此过程中粘着斑激酶(FAK)的变化。方法: 通过直接细胞计数法测定HSCs增殖;光学显微镜及透射电镜技术观察凋亡细胞的形态学改变,并应用膜联蛋白(Annexin-V)/碘化丙啶(PI)联合标记法检测HSCs的凋亡率;运用RT-PCR方法观察FAKmRNA表达。结果: H₂O₂具有刺激HSCs增殖的作用;丹参单体IH764-3能够诱导H₂O₂刺激的HSCs凋亡,并呈剂量依赖关系:10mg/L、20mg/L、30mg/L及40mg/LIH764-3干预48h后各组凋亡率分别为6.35%、9.28%、15.10%、19.69%,而H₂O₂组为2.30%;30mg/LIH764-3干预HSCs不同时间(12h、24h、48h)的凋亡率分别是6.73%、10.34%、15.10%,呈时间依赖关系;RT-PCR分析表明FAK基因表达强度在加入IH764-3后2h即明显下调,10mg/L,20mg/L,30mg/L及40mg/L组分别比H₂O₂组低了68.71%、71.00%、86.72%、95.16%。结论: 丹参单体IH764-3可以诱导H₂O₂刺激的HSCs凋亡,下调FAKmRNA表达是其诱导HSCs凋亡的机制之一。     

白细胞介素1β刺激对脑微血管内皮细胞COX活性、mRNA表达和PGE2释放的影响

目的： 探讨白细胞介素1β（IL-1β）刺激不同时间对大鼠脑微血管内皮细胞（rCMEC）的环氧合酶（COX）活性及其mRNA表达和PGE₂释放的影响。 方法: 建立rCMEC培养，进行Ⅷ因子相关抗原鉴定。细胞长至融合状态后加入IL-1β（30 μg/L），分别刺激0.5、1、2、4、8、12、24 h，ELISA测定细胞内COX-1、COX-2的活性及细胞外液PGE₂含量，荧光实时定量PCR检测COX-1、COX-2的mRNA表达量，扩增产物进行熔解曲线图及琼脂糖电泳分析，并与未刺激的rCMEC作比较。 结果: ①Ⅷ因子抗体免疫组化染色可见90％以上的培养细胞呈阳性，确认为rCMEC。②IL-1β刺激4 h时细胞培养液PGE₂含量已明显高于未刺激组（P<0.05）；12 h时PGE₂含量达到最大值（P<0.01）；24 h时PGE₂含量有所回降，但与未刺激组比较仍有显著差异（P<0.05）。③IL-1β刺激不同时间rCMEC内COX-1活性与未刺激组相比无统计学差异（P>0.05）；COX-2活性在第8 h时已明显高于未刺激组（P<0.05），12 h活性达峰值（P<0.01），24 h活性有所回降，但仍具显著差异（P<0.05）。④IL-1β刺激不同时间COX-1 mRNA表达与未刺激组比较无明显差异（P>0.05）；未刺激组在本实验条件下未检测到COX-2 mRNA表达，IL-1β刺激1h时可见COX-2 mRNA表达，4 h时COX-2 mRNA表达至峰值，而后开始回降，第12 h时已未见表达。熔解曲线图显示无非特异性扩增；琼脂糖电泳可见扩增基因与目的基因长度相符，结果与荧光定量PCR一致。 结论: IL-1β作用下，rCMEC释放的PGE₂持续增加并于12h达峰值，这一过程主要与COX-2 mRNA表达激活及COX-2活性增加有关。     

幽门螺杆菌对大鼠胃上皮细胞环氧合酶-2表达的影响

目的： 探讨幽门螺杆菌（Hp）水提取物对大鼠胃上皮细胞环氧合酶-2（COX-2）表达及前列腺素E₂（PGE₂）合成的影响。方法：大鼠胃上皮细胞株RGM1体外常规培养，Hp组细胞培养液内Hp水提取物终浓度为2.5 mg/L、5 mg/L、10 mg/L，大肠埃希菌组大肠埃希菌水提取物终浓度10 mg/L。培养 24 h 后收集细胞和上清液分别用于Western blotting分析COX-1、COX-2表达和酶免疫方法测定前列腺素E₂（PGE₂）含量。结果：Hp水提取物剂量依赖地增加RGM1细胞COX-2表达但不影响COX-1表达，而大肠埃希菌水提取物对COX-1和COX-2表达均无明显影响。RGM1细胞培养上清液中PGE₂水平在Hp组（2.5、5、10 mg/L）和大肠埃希菌组分别为（230.70±48.55）ng/g protein、（291.82±33.49）ng/g protein、（342.94±28.70）ng/g protein和（130.54±42.81）ng/g protein。结论：Hp体外诱导大鼠胃上皮细胞COX-2表达而增加PGE₂合成，Hp感染的致癌机制可能与其诱导的COX-2表达有关。     

损害性因素对乳鼠心肌细胞甘氨酸受体α1亚基表达的影响

目的：研究脂多糖（LPS）、缺氧/复氧（H/R）、异丙肾上腺素(ISO)以及高糖（HG）对乳鼠心肌细胞甘氨酸受体α₁亚基（GlyRα₁）表达的影响。方法：体外培养乳鼠心肌细胞，分别用LPS、H/R、ISO以及HG处理，采用CCK-8试剂检测细胞活力，RT-PCR方法检测心肌细胞上GlyRα₁亚基mRNA的表达。结果：LPS（5 mg/L、10 mg/L、20 mg/L、40 mg/L、80mg/L）、ISO（20 μmol/L、100 μmol/L、500 μmol/L）以及HG（25 mmol/L、50 mmol/L）处理心肌细胞24 h与心肌细胞缺氧3 h/复氧3 h、单纯缺氧3 h对心肌细胞存活率无明显影响（P＞0.05）；LPS（5 mg/L、10 mg/L、20 mg/L、40 mg/L、80 mg/L）、缺氧（3 h）/复氧（3 h）、单纯缺氧（3 h）以及ISO（20 μmol/L、100 μmol/L、500 μmol/L）组心肌细胞上GlyRα1亚基mRNA表达均高于对照组（P<0.01），而HG（25 mmol/L、50 mmol/L）组心肌细胞上GlyRα₁亚基mRNA表达均低于对照组（P<0.01）。结论：一定浓度的LPS、ISO与一定时间的H/R、单纯缺氧均可上调乳鼠心肌细胞GlyRα₁亚基mRNA的表达，而HG可下调乳鼠心肌细胞GlyRα₁亚基mRNA的表达。     

H2 S抑制Ang II引起的神经元活性氧水平升高的机制研究

目的: 探讨硫化氢(H₂S)对血管紧张素II(angiotensin II,Ang II)所致延髓神经元活性氧(reactive oxygen species,ROS)水平变化的影响,以及H₂S抗氧化应激的相关机制。方法: 采用原代培养的延髓神经元,分组如下:对照组、Ang II处理组、硫氢化钠(NaHS)处理组、NaHS+Ang II处理组和PD98059(p-ERK1/2蛋白的抑制剂)+ Ang II处理组。选用二氢乙啶(DHE)荧光探针法测定各组神经元胞内的ROS水平,Western blotting检测ERK1/2和p-ERK1/2蛋白的表达量,CCK-8法测定神经元的活性。结果: Ang II(终浓度为100 nmol/L)引起神经元ROS水平升高;NaHS(50~200 μmol/L)明显抑制Ang II引起的神经元ROS水平升高,但单独给予NaHS并不影响神经元ROS水平;p-ERK1/2蛋白的抑制剂PD98059也能明显抑制Ang II引起的神经元ROS水平的升高;适宜作用浓度的NaHS能够显著降低Ang II引起的神经元p-ERK1/2蛋白表达。结论: H₂S能够显著抑制Ang II引起的神经元ROS水平升高,其作用机制与MAPK家族中p-ERK1/2蛋白的表达有关。H₂S可能通过降低p-ERK1/2的表达量而产生抗氧化应激作用。     

汉黄芩素对流感病毒感染肺泡巨噬细胞炎症相关因子的影响

目的: 研究汉黄芩素对甲型流感病毒鼠肺适应株A/FM/1/47(H1N1)感染的大鼠肺泡巨噬细胞(NR8383)产生促炎症细胞因子、炎症介质及氧自由基的影响。 方法: 流感病毒感染 NR8383细胞1 h后,加入含汉黄芩素的培养基(终浓度16 mg/L),药物作用后6 h、12 h和24 h,ELISA法检测细胞上清中肿瘤坏死因子α(TNF-α)和单核细胞趋化蛋白 1(MCP-1)的含量,放射免疫测定法检测细胞上清中前列腺素E₂(PGE₂)、磷脂酸A₂(PLA₂)和白三烯B₄(LTB₄)的含量;药物作用后8 h、24 h、36 h和48 h,生化法检测细胞内一氧化氮(NO)含量和诱导型一氧化氮合酶(iNOS)活性,4 h、8 h、18 h和24 h,生化法检测超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;药物作用后24 h,real-time PCR检测细胞内TNF-α和MCP-1的mRNA水平。 结果: 汉黄芩素抑制了流感病毒感染NR8383细胞后TNF-α、MCP-1的转录和表达(P<0.01),降低了PGE₂、PLA₂、LTB₄和MDA的含量(P<0.05);减少了NO和iNOS的产生(P<0.05),增强了SOD的活性(P<0.05)。 结论: 汉黄芩素明显抑制了流感病毒感染后肺泡巨噬细胞内各种炎症相关因子的产生,具有抗炎作用。     

阿魏酸对H2O2诱导脐静脉内皮细胞表达P-选择素的影响

目的:探讨阿魏酸对H₂O₂刺激脐静脉内皮细胞（HUVECs）表达P-selectin的影响。方法:应用培养的HUVECs经H₂O₂刺激,用FTTC标记抗P-选择素单抗SZ-51-1gG和FCM测定HUVECs表面表达的P-se-lectin,同时观察了阿魏酸对其表达的影响。结果:实验显示正常HUVECs表面表达少量的P-selectin,H₂O₂能刺激培养的HUVECs表面P-selectin表达增高,其中最适的H₂O₂刺激浓度在250-300μmol/L,最适的刺激时间为1.5-2h;随着阿魏酸浓度增加,阿魏酸明显地抑制H₂O₂刺激HUVECs表面P-selectin的表达。结论:H₂O₂能刺激培养的HUVECs表面P-selectin表达增高,阿魏酸明显地抑制其表达。     

COX-2启动子区遗传变异与胃癌发病风险的关系

目的 COX-2的过度表达在肿瘤的发生发展中起重要作用,本文探讨COX-2启动子区单核苷酸多态与胃癌易感性的关系。方法以聚合酶链反应-限制性片段长度多态性对155例胃癌患者和237例正常对照进行基因分型,以logistic回归的比值比（OR）及其置信区间（CI）来评估风险度。结果 COX-2-1290AG及GG基因型与胃癌的发病风险无关,OR为1.24（95%CI=0.66-2.35）。而携带COX-2-1195GA或-1195AA基因型的个体胃癌发病风险显著增加,OR分别为1.91（95%CI=1.05-3.47）,2.71（95%CI=1.40-5.27）。单体型分析发现A_1290-A_1195单体型显著增加了胃癌的发病风险（OR=1.49,95%CI=1.10-2.01）。结论 COX-2启动子区-1195G→A遗传变异与胃癌发病风险相关。     

The effects of vigabatrin on type II spike wave discharges in rats

The phospholipid mediator platelet-activating factor (PAF), and its non-hydrolyzable analog methylcarbamyl-PAF (mc-PAF) increase prostaglandin E₂ (PGE₂) release from astrocyte-enriched cortical cell cultures. Cyclooxygenase (COX) enzymes – of which there are two known isoforms – convert arachidonic acid to prostaglandin (PG) H₂ (PGH₂), which is further metabolized to various PGs, including PGE₂. COX-1 is generally considered to contribute to cell homeostasis, whereas COX-2 is thought to mediate inflammatory/immune PG formation. In this study we examined the involvement of the COX isoforms in PAF-induced PGE₂ release. Treatment of cells with the non-specific COX inhibitor indomethacin, or the specific COX-2 inhibitor NS-398, prior to mc-PAF stimulation completely blocked the PAF-induced release of PGE₂; treatment with more selective COX-1 inhibitors (i.e. piroxicam and SC-560) failed to significantly do so. These data suggest that COX-2 is responsible for PAF-mediated PGE₂ release in primary astrocytes.     

姜黄素对LPS刺激RAW264.7细胞PGE_2和IL-10影响

目的:观察姜黄素对脂多糖刺激小鼠RAW264.7细胞炎症因子的影响。方法:体外培养小鼠RAW264.7细胞,脂多糖刺激小鼠RAW264.7细胞分泌细胞因子,用不同浓度姜黄素进行干预,ELISA法检测培养上清液中抗炎细胞因子IL-10和炎症因子PGE2含量。结果:姜黄素可抑制脂多糖刺激小鼠RAW264.7细胞释放PGE2,促进抗炎因子IL-10表达,并呈一定量效关系,其影响细胞因子释放作用与细胞毒作用无关。结论:姜黄素呈浓度依赖性地抑制脂多糖刺激小鼠RAW264.7细胞释放PGE2,促进IL-10表达。     

精氨酸加压素翻转脂多糖引起大鼠发热及其对痛觉敏感性的影响

目的:研究外周给精氨酸加压素(AVP)对脂多糖(LPS)引起的大鼠发热和痛觉过敏的影响,以及与血清中IL-1β和PGE_2水平变化的关系。方法:实验用成年雄性SD大鼠,在23℃环境温度下,明暗时间各12 h。用无线遥测系统连续测量大鼠体核温度(Tc)、棕色脂肪温度(T_(BAT))和活动。10:00或11:30分别给大鼠腹腔注射LPS(50μg/kg)、AVP(10μg/kg)或V1a受体阻断剂(30μg/kg)。用ELISA法测定血清IL-1β和PGE_2的含量。用足底痛觉测试仪(Hargreaves test)测试大鼠热痛缩爪潜伏期的变化。结果:(1)腹腔注射LPS引起大鼠双相发热过程伴有痛觉过敏现象。(2)AVP能够翻转LPS引起的Tc和T_(BAT)升高反应,降低发热引起的痛觉敏感性。(3)外周给V1a受体阻断剂能提高LPS引起的发热反应,但不影响发热引起的痛觉敏感性变化。(4)AVP能抑制LPS引起的发热大鼠血液中IL-1β和PGE_2水平升高。结论:(1)外周给予AVP可通过抑制棕色脂肪产热以及降低血液中IL-1β和PGE_2的浓度而翻转LPS发热反应并降低发热伴随的痛觉敏感性升高现象。(2)内源性AVP也有限制LPS发热的作用,但可能不影响发热引起的痛觉阈值降低现象。     

Ontogeny of ‘macrophage’ function. V. differential effect of prostaglandin E2 on the activation of newborn and adult mouse macrophages

Our previous papers indicate that peritoneal exudate macrophages (PM) of newborn mice are strongly suppressive for the tumor cell growth in comparison with those of adult mice. Neither newborn PM nor adult PM are cytolytic, but they both can be activated to be cytolytic by being cultured overnight either with a high concentration of bacterial lipopolysaccharide (LPS) or with a low concentration of LPS plus lymphokine. This paper shows that the activation of adult PM was inhibited by prostaglandin E₂ (PGE₂) added simultaneously with LPS, though that of newborn PM was not affected by PGE₂. Adult PM, however, acquired the resistance to PGE₂ by the preculture with LPS and/or lymphokine for 4 hr so as to be activated by LPS in the presence of PGE₂. These results indicate that newborn PM are more activated than adult PM to a state which adult PM attain after moderate activation with LPS or lymphokine.     

Selective inhibition of cyclooxygenase-2 exacerbates methamphetamine-induced dopamine depletion in the striatum in rats

Neuroinflammatory processes associated with induction of cyclooxygenase (COX) have been implicated in the deleterious events resulting in neurodegeneration. The present study was designed to investigate the impact of acute methamphetamine (MA) administration on COX expression and prostaglandin E2 (PGE₂) production, and to evaluate the effect of selective COX-2 inhibition using celecoxib in MA-induced degeneration of dopaminergic terminal and cell apoptosis in the striatum. Male Sprague–Dawley rats were treated with either a neurotoxic regimen of methamphetamine hydrochloride (5 mg/kg, i.p., every 2 h for four injections) with or without celecoxib (7.5 mg/kg) or vehicle. COX-1 expression was not affected by MA, while both COX-2 protein expression and number of COX-2 positive cells in striatum were significantly reduced 24 h after MA treatment. However, after 72 h, a significant upregulation of COX-2 protein was detected. PGE₂ production was correlated with altered COX-2 levels. NFkappa-B (NFκB), a key regulator of COX-2 expression, was activated 72 h after MA administration, and was accompanied by increased Ikappa B (IκB) phosphorylation. Animals receiving MA exhibited an increase in apoptotic cells and notable reductions in dopamine (DA) content (63.9%) in immunoreactivity of tyrosine hydroxylase (TH) and neuron specific microtubule-associated protein 2 (MAP2) in striatum. Administration of celecoxib exacerbated MA-induced DA depletion, and did not affect MA-induced MAP2 damage, apoptosis or proliferation of glial cells. Our findings suggest that COX-2 containing cells are targets of the damage during earlier stages of MA-related neurotoxicity, and that the selective inhibition of COX-2 enzyme is harmful rather than protective. The COX-2 induction appears during the recovery period, and NFκB activation may be an important mechanism.     

Vitamin E Exposure Modulates Prostaglandin and Thromboxane Production by Avian Cells of the Mononuclear Phagocytic System

The production of Prostaglandin E₂ (PGE₂) and Thromboxane B₂ (TXB₂) by turkey blood monocytes and a chicken mononuclear phagocytic cell line MQ-NCSU after exposure to vitamin E (VE) was examined. Turkey embryos were exposed in ovo to 0 and 10 international units (IU) of VE; blood monocytes were collected at 2 weeks of age and cultured. MQ-NCSU macrophage monolayers were exposed to 0, 0.1, 0.25, and 0.5 IU VE. The monocyte/macrophage cultures were exposed to 1 μg/mL bacterial lipopolysaccharide (LPS). Non-stimulated parallel cultures were maintained as controls. The PGE₂ and TXB₂ levels were quantitated in culture supernatants by a competitive ELISA. Blood monocytes from the 10 IU VE poults produced lower PGE₂ levels as compared with the 0 IU VE controls. Upon stimulation with LPS, monocytes from the 10 IU VE group exhibited levels of PGE₂ that were higher than the 0 IU VE group. Levels of TXB₂ were not quantitated in the poult blood monocyte culture supernatants. The PGE₂ and TXB₂ levels in the supernatant of the VE treated MQ-NCSU macrophage cultures were lower than the 0 IU VE controls. Stimulation with LPS resulted in increased PGE₂ and TXB₂ production by the VE-exposed macrophages. The results from this study suggest that in ovo or in vitro exposure with VE may either upregulate or downregulate PGE₂ and TXB₂ production by monocytes/macrophages, and that this production may be dependent upon the exposure to a variety of external stimuli and/or the state of macrophage activation.     

脂氧素对Jurkat T细胞增殖和白细胞介素-2/白细胞介素-2受体表达的影响

目的：研究脂氧素对Jurkat T细胞增殖和白细胞介素-2表达的影响。方法:体外培养Jurkat T细胞，anti-CD3（2 mg/L）和anti-CD28（2 mg/L）单抗刺激Jurkat细胞活化，加入不同浓度脂氧素（0.1 nmol/L-100 nmol/L）共同孵育24h后，加入［3H］-TdR继续孵育6 h，液闪仪测放射性活度；或收集培养上清，ELISA测IL-2水平，收集细胞，流式细胞仪检测细胞表面IL-2受体α亚单位CD25表达，PI染色后经流式细胞仪进行细胞周期分析。结果:脂氧素剂量依赖性抑制anti-CD3和anti-CD28活化的Jurkat T细胞增殖（P<0.05）；细胞周期分析发现脂氧素处理组S期细胞比例减少；脂氧素显著降低培养上清中IL-2 含量（P<0.05）但对CD25表达无明显影响（P>0.05）。结论:脂氧素能抑制活化Jurkat T细胞增殖和白细胞介素-2表达；脂氧素可通过影响T淋巴细胞的活化增殖进而发挥免疫负性调节作用。     

Distribution of adenosine A2 receptor mRNA in the human brain.

The distribution of the messenger RNA coding for the recently cloned adenosine A₂ receptor was studied in the human brain using in situ hybridization histochemistry. A₂ receptor mRNA is exclusively detected in the medium-sized neurons of the caudate, putamen and accumbens nuclei but not elsewhere in the brain. This highly selective distribution of adenosine A₂ receptor mRNA in human dorsal and ventral striatum, similar to that of adenosine A₂ binding sites reported in rodents, suggests a major role in the basal ganglia physiology.