吸入低浓度异氟烷对肺感染大鼠气血屏障的影响

目的探讨低浓度异氟烷对肺感染大鼠气血屏障的影响。方法健康清洁级Wistar大鼠32只,体重240～300g,随机分为4组：盐水正常组（C组）、异氟烷正常组（Ⅰ组）、内毒素组（L组）和给内毒素后吸入异氟烷组（T组）。气管内注入内毒素（LPS,10mg/kg溶于0．5ml生理盐水）或等量生理盐水15min后,T组、I组吸入0．68%异氟烷2h后再机械通气2h；L组、C组单纯机械通气4h。监测平均动脉压（MAP）,分别于机械通气20min（T0）、气管内注入内毒素或生理盐水后15min（T1）、吸入异氟烷1h（T2）、2h（T3）、停止异氟烷吸入1h（T4）、2h（T5）采集动脉血行血气分析；于T2、T3、T4时点进行气管内吸引,抽取气道内液体并计算其总重量,随后处死动物,测定肺湿/干重比（W/D）、支气管肺泡灌洗液（BALF）中性粒细胞百分比和蛋白浓度,并行肺组织病理学检查。结果与L组相比,T组在T2、T5时点动脉血氧分压升高（P＜0．05）,在T4、T5时点动脉血二氧化碳分压降低（P＜0．05）；T组气道内液体总重量、BALF中性粒细胞百分比和蛋白浓度均降低（P＜0．05）；T组肺脏病理改变减轻。虽然T组指标有改善,但尚未恢复到C组水平。结论吸入低浓度异氟烷对肺感染大鼠气血屏障具有一定的保护作用,可能与抑制中性粒细胞在肺内积聚有关。     

内毒素诱导急性肺损伤小鼠基因表达的综合分析

目的利用改良的基因表达综合分析(SAGE)技术监测内毒素诱发小鼠急性肺损伤的基因表达状况，探讨急性肺损伤的分子机制。方法气管内注射内毒素(25mg／kg)复制急性肺损伤模型。正常对照小鼠气管内注射等体积等渗盐水。内毒素注射24h后以氯胺酮深度麻醉后处死小鼠，整块取出肺脏用于SAGE研究。建立正常和急性肺损伤两个SAGE标记物文库。结果正常文库包括24670个SAGE标记物，代表12168个基因。急性肺损伤文库包括26378个SAGE标记物，代表13：397个基因。急性肺损伤组有11种基因升高10倍以上，107种基因升高5～10倍，2121种基因升高2—5倍。7种基因表达降低大于10倍，87种基因降低5～10倍，1571种基因降低2～5倍。结论证实了内毒素诱导的急性肺损伤发生机制中各种基因表达的变化，并发现了以往没有报道的基因。对这些基因功能的进一步研究有助于阐明急性肺损伤的发生机制，并为临床诊断提供有参考价值的血清标记物。     

Is endotoxin and cytokine release related to a decrease in gastric intramucosal pH after hemorrhagic shock?

Objectives: (a) To investigate the relationship between gut ischemia parameters (gastric intramucosal pH [pHi], mucosal–arterial carbon dioxide difference [PCO₂-gap]), and endotoxin or cytokine release during hemorrhagic shock; (b) to compare the predictive value of pHi, PCO₂-gap and arterial lactate concentrations. Design: Prospective study. Setting: Surgical intensive care unit of a university hospital. Patients: 20 multiple trauma patients with severe hemorrhagic shock. Interventions: Intramucosal measurements and blood samples were obtained on admission to the emergency room and repeatedly over 48 h. Measurements and results: Endotoxin was measured using a chromogenic limulus amoebocyte assay. Cytokine [tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6)] values were evaluated by immunoradiometric assays. Only 3 patients had positive blood cultures but endotoxins were detected at least once in all patients. Endotoxin levels were similar in survivors and nonsurvivors over the study period and were not related to pHi or PCO₂-gap. Initially, high levels of IL-6 were observed in both nonsurvivors and survivors [median 1778 pg/ml (range 435–44 540) vs 2068 pg/ml (range 996–92 300)]. IL-6 levels progressively decreased in the survivors but not significantly. On admission, TNFα concentrations were similar in nonsurvivors and survivors (42 ± 35 vs 46 ± 27 pg/ml). From the 24th h, TNFα values were higher in the nonsurvivors than in the survivors (24 h: 72 ± 38 vs 34 ± 17 pg/ml, p < 0.05). The greatest IL-6 levels were found for a pHi < 7.20 (28.5 ± 36.5 vs 1.8 ± 1.3 ng/ml, p < 0.05) or a PCO₂-gap > 7.5 mmHg (1 kPa) (32.5 ± 37.5 vs 1.7 ± 1.3 ng/ml, p < 0.01). With the same pHi threshold, no difference was found in endotoxin levels. The lactate concentrations were predictive for outcome from the 12th h (9.5 ± 5.9 vs 3.6 ± 2.3 mmol/l, p < 0.05). Conclusions: During severe hemorrhagic shock, endotoxin translocation from the gut was a common phenomenon that seemed independent of both pHi values and outcome. It could not explain IL-6 and TNFα release. In severe hemorrhagic shock, neither pHi nor PCO₂-gap provides additional information to the lactate measurements. Received: 17 February 1997 Accepted: 7 August 1997     

内毒素对大鼠淋巴细胞表达TLR4及NF-κB与分泌IL-6的影响

目的研究内毒素干预离体大鼠淋巴细胞后TLR4 mRNA、NF-κB mRNA的表达及IL-6分泌的变化,探讨淋巴细胞在全身炎症反应综合征(SIRS)发生及进展中的作用机制。方法制备健康雄性Wistar大鼠脾脏淋巴细胞,培养至对数期,随机分为对照组和实验组。对照组不做处理。实验组加入不等量内毒素,调整浓度为低浓度组(10 ng/ml)、高浓度组(100 ng/ml),培养3、6、12 h。RT-PCR技术检测TLR4及NF-κB的mRNA表达水平,ELISA技术测定IL-6的分泌量。结果干预3 h后,低浓度组:TLR4及NF-κB的mRNA表达与对照组比较无显著差异(P>0.05),IL-6的分泌与对照相比差异有统计学意义(P<0.05)。高浓度组:TLR4 mRNA的表达及IL-6的分泌与对照组比较差异有统计学意义(P<0.05),NF-κB的mRNA表达与对照组比较无显著差异(P>0.05)。干预6、12 h后,低、高浓度组:TLR4及NF-κB的mRNA表达与IL-6的分泌分别与对照组比较差异均有统计学意义(P<0.05)。此外,TLR4及NF-κB的mRNA表达水平及IL-6的分泌量随时间延长增加。结论内毒素能刺激大鼠淋巴细胞高表达TLR4、NF-κB及分泌包括IL-6在内的各种细胞因子与炎症介质,促进SIRS的发生及发展。     

脑源性多器官功能障碍综合征模型血清内毒素及其受体基因表达

目的探讨急性前脑缺血致多器官功能障碍综合征(MODS)模型血清内毒素含量及其受体在各脏器基因表达的规律,分析脑原性多器官功能障碍综合征(CMODS)的发生机制。方法随机将54只Wistar大鼠分为正常对照组(6只)、假手术组(8只)和前脑缺血组(40只),后者又被随机分为12h、24h、36h、48h、和72h时相点的5个亚组,每亚组各8只;建立大鼠急性前脑缺血模型;分别采用偶氮显色法鲎试验定量法测定血清内毒素和原位杂交技术测定肺、肝、肠和肾组织CD14mRNA水平;使用CMIA真彩色医学图像分析系统,检测CD14mRNA的相对含量。结果大鼠急性前脑缺血后12h血清内毒素升高(0184±0055)Eu/L,24h达高峰(0639±0064)Eu/L,72h基本恢复至正常水平(0117±0024)Eu/L;肺、肝、肠和肾组织CD14mRNA的表达也在缺血后12h升高,24~36h达高峰,48h后下降,并以肺脏变化最显著(P<0001);正常对照组和假手术组CD14mRNA在各脏器均有不同程度的表达,其中两组肺脏的表达有显著性差异(P<001)。内毒素与其受体在各脏器的表达均存在显著相关性(均为P<001),其中与肠、肺组织CDmRNA表达相关最显著(P<0001)。结论急性前脑缺血致MODS大鼠存在内毒素血症;急性脑血管病→应激反应→肠道黏膜屏障损害→内毒素易位→内毒素血症→内脏器官功能障碍→MODS是CMODS发生的     

白细胞介素-1受体相关激酶-4短发夹RNA阻断库普弗细胞激活效应的实验研究

目的 探讨以白细胞介素-1受体相关激酶-4（IRAK-4）特异性短发夹RNA（shRNA）阻断内毒素诱导的库普弗细胞（KCs）激活效应的可行性。方法 构建两对表达IRAK-4-shRNA的阳性载体质粒（pSfIRAK-4-A，pSfIRAK-4-B）及一对阴性载体质粒（pSⅡRAK-4-C）。分离培养小鼠KCs，分为正常对照组，RNA干扰（RNAi）对照组（转染pSⅡRAK-4-C）与RNAi抑制组（转染pSⅡRAK-4-A，pSⅡRAK-4-B）。质粒转染后24h，加入0．1μg／ml脂多糖（LPS）。6h后，蛋白免疫印迹法及逆转录聚合酶链反应测定IRAK-4蛋白和mRNA表达水平；酶联免疫吸附法检测0、1、3、6、12h后KCs的核因子-κB（NFKB）活性及培养上清液中肿瘤坏死因子α含量。结果 RNAi抑制组IRAK-4表达水平，以及LPS刺激后NFKB活性、肿瘤坏死因子α峰值均明显低于正常对照组和RNAi对照组，t值分别为22．50，4．18及958．49，P〈0．01；尤其是pSⅡRAK-4-A组，抑制效果明显优于pSⅡRAK-4-B，t值分别为12．60，3．36及256．39，P〈0．01。结论 以IRAK-4为靶点的shRNA能有效的阻断内毒素诱导的KCs激活效应。     

异丙酚对内毒素诱导大鼠急性肺损伤的保护作用

目的 探讨不同剂量异丙酚对内毒素(LPS)诱导大鼠急性肺损伤(ALI)的保护作用。方法 股静脉注射内毒素(LPS)5mg/kg,建立大鼠ALI模型。24只健康雄性Wistar大鼠随机分为四组:对照组(C组,输注生理盐水),LPS对照组(L组,股静脉注射LPS后输注生理盐水),低剂量异丙酚治疗组(Lp1组,股静脉注射LPS后立即输注异丙酚5mg/kg,随后5mg·kg~(-1)·h~(-1)维持),高剂量异丙酚治疗组(Lp2组,股静脉注射LPS后立即输注异丙酚10mg/kg,随后10mg·kg~(-1)·h~(-1)维持),每组6只。于注射LPS后1、2、3、4h抽血并于4h时处死大鼠,酶联免疫吸附法(ELISA)测定血清和支气管肺泡灌洗液(BALF)中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-10(IL-10)水平;测肺湿/干重比;并观察BALF中性粒细胞计数比、蛋白浓度。结果 L组大鼠肺湿/干重、BALF中性粒细胞计数比及蛋白浓度均明显增加(P<0.05),血清及BALF中TNF-α、IL-1β、IL-10水平显著性升高(P<0.01),而异丙酚治疗组的各项指标均较内毒素组减轻,大剂量作用更明显(P<0.01)。结论 异丙酚对内毒素诱导的大鼠急性肺损伤有保护作用,大剂量作用较明显。     

Identification, cloning and sequencing of two major venom proteins from the box jellyfish, Chironex fleckeri.

Two of the most abundant proteins found in the nematocysts of the box jellyfish Chironex fleckeri have been identified as C. fleckeri toxin-1 (CfTX-1) and toxin-2 (CfTX-2). The molecular masses of CfTX-1 and CfTX-2, as determined by SDS-PAGE, are approximately 43 and 45 kDa, respectively, and both proteins are strongly antigenic to commercially available box jellyfish antivenom and rabbit polyclonal antibodies raised against C. fleckeri nematocyst extracts. The amino acid sequences of mature CfTX-1 and CfTX-2 (436 and 445 residues, respectively) share significant homology with three known proteins: CqTX-A from Chiropsalmus quadrigatus, CrTXs from Carybdea rastoni and CaTX-A from Carybdea alata, all of which are lethal, haemolytic box jellyfish toxins. Multiple sequence alignment of the five jellyfish proteins has identified several short, but highly conserved regions of amino acids that coincide with a predicted transmembrane spanning region, referred to as TSR1, which may be involved in a pore-forming mechanism of action. Furthermore, remote protein homology predictions for CfTX-2 and CaTX-A suggest weak structural similarities to pore-forming insecticidal delta-endotoxins Cry1Aa, Cry3Bb and Cry3A.     

热限及热限加致热原负荷时脑脊液及血浆中cAMP含量变化的研究

为探讨内毒素(ET)和内生致热原(EP)热限的机制,在家兔静脉注射非热限与热限剂量的EF或EP后及热限加不同致热原负荷时,用竟争性蛋白结合法检测脑脊液和血浆样品中cAMP的含量。结果发现:(1).脑脊液中cAMP含量随EF性发热效应的增强而升高,到达EP热限后则不再上升,而血浆cAMP含量无类似变化。(2).脑脊液和血浆中cAMP含量均随ET性发热效应的增强而上升,到达ET热限后均不再升高。(3).虽然ET热限的发热水平明显高于EP热限,且EP热限加ET负荷使发热水平超过EP热限;但各组之间脑脊液中cAMP含量却无明显差异。上述结果表明,中枢cAMP生成受限可能既是构成EP热限的重要因素,又是ET热限的一个重要成因。但是除cAMP外,ET热限的构成还与其它因素有关。     

急性坏死性胰腺炎大鼠的炎性介质变化和生长抑素的作用

探讨急性坏死性胰腺炎（ＡＮＰ）大鼠的炎性介质变化和生长抑素（ｓｔｉｌａｍｉｎ）的治疗作用。方法将ＳＤ大鼠随机分为ＡＮＰ组，ＡＮＰ＋生理盐水治疗组（ＡＮＰ＋ＮＳ），ＡＮＰ＋Ｓｔｉｌａｍｉｎ治疗组（ＡＮＰ＋Ｓ）和正常对照组（ＮＣ）。经胰胆管逆行注射３．５％牛磺胆酸建立大鼠ＡＮＰ模型。测定血的ＩＬ－１β，ＩＬ－６，ＩＬ－１２，ＴＮＦα和内毒素，淀粉酶，磷脂酶Ａ２，并对内脏标本行病理检查及电镜分析。结果在ＡＮＰ组和ＡＮＰ＋ＮＳ组的内毒素为２５０±１３ｐｇ／ｍｌ，２３０±１６ｐｇ／ｍｌ；淀粉酶３００００±８００Ｕ／Ｌ，２８０００±５２Ｕ／Ｌ；磷脂酶Ａ２２３０±３４Ｕ／Ｌ，２２４±２８Ｕ／Ｌ及细胞因子的水平与ＡＮＰ＋Ｓ和ＮＣ组比较，均有统计学意义。ＡＮ＋ＮＳ和ＡＮＰ组可引起心、肝、肾、肺的严重损伤，而ＡＮＰ＋Ｓ组的多器官损伤则明显减轻。结论ＡＮＰ引起多器官损伤与炎性介质过度表达有关，生长抑素对ＡＮＰ的治疗有效。