免疫抑制宿主巨噬细胞Toll样受体的表达变化

目的:检测小鼠腹腔巨噬细胞TLRs表达情况及糖皮质激素甲基强的松龙对TLRs mRNA表达的影响.方法:应用RT-PCR检测正常小鼠腹腔巨噬细胞TLR1-13的表达;并以甲基强地松龙为免疫抑制剂,诱导小鼠免疫抑制后检测腹腔巨噬细胞TLRs mRNA表达变化.结果:已发现的TLR1-13在正常小鼠腹腔巨噬细胞均有表达;小鼠腹腔巨噬细胞经糖皮质激素处理后,部分TLRs在mRNA水平上可被上调.结论:Toll样受体是抗真菌先天免疫的重要组成部分.甲基强的松龙对巨噬细胞有毒性作用,甲基强的松龙注射后可导致小鼠腹腔巨噬细胞明显减少.     

抗生素诱导小鼠肠道菌群失调对免疫功能和Toll样受体2、4基因表达的影响

目的:探讨抗生素诱导小鼠肠道菌群失调后对机体免疫功能和Toll样受体2、4的影响.方法:60只BALB/c小鼠分为2组:抗生素组和对照组,抗生素组连续7天口服头孢哌酮,7天后经口给予白色念珠菌.结果:抗生素组小鼠肠道内肠杆菌、肠球菌、双歧杆菌、乳酸杆菌较对照组减少,白色念珠菌增加;脾指数、PHA诱导的淋巴细胞转化率、迟发型变态反应、抗体生成细胞数和脾脏的TLR2和TLR4基因表达水平均低于对照组.结论:抗生素诱导肠道菌群失调后小鼠免疫功能和TLR2、TLR4 mRNA表达均降低.     

Toll样受体4突变对失血性休克致小鼠急性肺损伤的影响

目的：探讨Toll样受体4（TLR4）突变在单纯性失血性休克（无复苏）所致小鼠急性肺损伤（ALI）中的作用及机制。方法：以TLR4基因突变的C3H／HeJ品系及TLR4野生型的CBA品系小鼠为研究对象，每组20只。心脏穿刺复制失血性休克模型，在不同时间点取出肺组织，观察肺组织病理形态变化；通过逆转录一聚合酶链反应（RT-PCR）半定量方法检测肺组织TLR4 mRNA的表达水平；用凝胶电泳迁移（EMSA）法检测肺组织中核因子-kB（NF-kB）的量。结果：失血性休克刺激后，基因突变小鼠肺组织中性粒细胞浸润、红细胞渗出较野生型小鼠明显减轻；基因突变小鼠肺组织TLR4 mRNA表达及NF-kB活性变化不明显，而野生型小鼠TLR4mRNA在2、4和6h表达增加，NF-kB在失血性休克后1和2h活性显著增加。结论：TLR4在失血性休克所致ALI中起重要作用，其突变可减轻失血性休克所致的ALI。     

脾动脉血流减少对门静脉高压大鼠脾脏TLR4及巨噬细胞吞噬活性的影响

目的 观察脾动脉血流减少对肝硬化门静脉高压大鼠脾脏Toll样受体4（Toll-like receptor 4,TLR4）表达和巨噬细胞（Macrophage,Mφ）吞噬活性的影响.方法 肝硬化门静脉高压大鼠模型诱导成功后随机分为肝硬化组（MCG组）、脾动脉缩窄组（SAC组）和脾动脉结扎组（SAL组）,每组10只,另10只正常大鼠为对照组（NCG组）.术后4周处死大鼠,用免疫组化法测定脾脏TLR4表达量,鸡红细胞吞噬实验检测脾脏Mφ吞噬活性,就脾脏TLR4表达量和Mφ吞噬活性进行相关分析.结果 脾脏TLR4表达量：MCG组明显高于NCG组[（210.83±42.57）vs（63.08 ±18.53）],差异有统计学意义（P〈0.01）;SAC组（180.03±29.67）、SAL组（170.83±32.65）,均明显低于MCG组.差异有统计学意义（均P〈0.05）.脾脏Mφ吞噬率和吞噬指数：MCG组明显高于NCG组[（35.83%±4.95%）vs（17.32%±3.51%）]、[（0.63±0.12）vs（0.22±0.08）],差异有统计学意义（均P〈0.05）;SAC组（23.46%±3.31%）、（0.29±0.05）和SAL组（22.34%±2.95%）、（0.23±0.07）,均明显低于MCG组,差异有统计学意义（均P〈0.05）.脾脏Mφ吞噬率、吞噬指数与TLR4表达量呈显著正相关（均P〈0.01）.结论 肝硬化门静脉高压大鼠脾脏TLR4表达量增高,Mφ吞噬活性增强,脾动脉缩窄和结扎能减弱脾脏Mφ吞噬活性,这可能与术后脾脏TLR4表达量减少有关.     

乌司他丁对重症急性胰腺炎患者早期外周血单核细胞Toll样受体4表达的影响

目的:研究乌司他丁对重症急性胰腺炎患者早期外周血单核细胞Toll-样受体4(TLR4)表达的影响.方法:将58例重症急性胰腺炎患者随机分为乌司他丁组(29例,予以常规治疗+乌司他丁)和常规组(29例,予以常规治疗),同时选择20例健康志愿者作为对照组.入院后第1、3、5、7、14天采集静脉血,对照组仅采血1次.用流式细胞仪检测外周血单核细胞TLR4、CD14的表达,用ELISA试剂盒检测血浆TNF-α、IL-6的浓度,并分析与TLR4表达的相关性.结果:与对照组比较,在入院第1天常规组和乌司他丁组患者外周血单核细胞TLR4表达明显增高(P＜0.01),两组在入院后第3、5天TLR4表达逐渐降低,但仍高于对照组(P＜0.05).乌司他丁组与常规组相比,入院第1天治疗前,外周血单核细胞TLR4表达差异无统计学意义(P＞0.05),而在入院后第3、5、7天乌司他丁组外周血单核细胞TLR4的表达均明显低于常规组(P＜0.05).血浆TNF-a、IL-6浓度的变化与TLR4变化一致.结论:重症急性胰腺炎患者早期外周血单核细胞表面TLR4的表达明显增高,乌司他丁对重症急性胰腺炎引起的全身炎症反应的拮抗和抑制作用与其抑制单核细胞表面Toll样受体4的表达从而抑制炎症反应有关.     

阿托伐他汀对慢性充血性心力衰竭患者外周血Toll样受体表达的影响

目的探讨慢性充血性心力衰竭(CCHF)患者外周血单个核细胞Toll样受体4(TLR4)表达改变情况及阿托伐他汀对其的干预效应。方法选取80例CCHF住院患者,其中40例按照标准治疗方案进行治疗(标准治疗组);另外40例在标准治疗的基础上口服阿托伐他汀20mg/d(阿托伐他汀组)。40名健康者为对照组。在治疗前和治疗后1个月,应用流式细胞仪检测外周血单个核细胞表面TLR4的表达,采用酶联免疫吸附试验检测血清肿瘤坏死因子(TNF)-α水平;并对TLR4的表达与血清TNF-α水平进行相关性分析。结果标准治疗组、阿托伐他汀组的外周血单个核细胞表面TLR4表达水平均较对照组显著升高(P值均<0.05),并且与血清TNF-α水平呈正相关(r=0.076,P<0.05)。治疗后1个月,标准治疗组和阿托伐他汀组的外周血单个核细胞表面TLR4的表达和血清TNF-α水平均较本组治疗前显著降低(P值分别<0.05、0.01),阿托伐他汀组的下降值均显著高于标准治疗组(P值均<0.05)。结论 TLR4表达可能与CCHF发生有关,阿托伐他汀可显著降低外周血单个核细胞表面TLR4的表达,提示阿托伐他汀的抗炎性免疫作用与抑制TLR4介导的炎性免疫反应有关。     

Impact of Toll-like Receptor 4 Deficiency on Cerebrocardiac Syndrome

In order to investigate the role of Toll-like receptor 4 （TLR4） in cerebrocardiac syndrome （CCS）, the partial cerebral ischemia/reperfusion （I/R） models in mice with different TLR4 genotypes were established in the present study. TLR4 wild-type （C3H/HeN） and mutant （C3H/HeJ） mice of 6-8 weeks of age were divided into 4 groups at random： C3H/HeN sham group （n=10）, C3H/HeJ sham group （n=10）, C3H/HeN model group （n=10） and C3H/HeJ model group （n=10）. Partial cerebral I/R was caused by the middle cerebral artery occlusion （MCAO） to duplicate CCS models in mice. After the operation, the electrocardiogram （ECG）, the level of tumor necrosis factor-alpha （TNF-c0 in myocardial tissue and the cardiac pathological changes were observed in each group. It was shown that the brain infarct volume in C3H/HeN model group was larger than that in C3H/HeJ model group （P〈0.01）. The ST segment change and T wave inversion occurred frequently in model groups. Moreover, the TNF-ct level in C3H/HeN model group was higher than that in C3H/HeJ model group （P〈0.01）. The myocar- dial injury was aggravated in C3H/HeN group as compared with C3H/HeJ group. It was concluded that TLR4 was implicated in the development of CCS.     

Susceptibility to Vaginal Candidiasis under Different Conditions in Mice

In order to study the susceptibility of murine vaginal mucosa to Candida albicans under different conditions, vaginal lavage fluid and vaginal tissue of mice were observed and compared between murine models with normal immune system （estrogen-treated mice） and immunosuppressed murine model, and between primary infection model of vaginal candidiasis and secondary infection one. The average level of colony forming unit （CFU） from the immuosuppressed group was higher than that from estrogen-treated group at each time point and the peak time was delayed. The differences between the two groups were statistically significant （P〈0.05） from the fourth day after inoculation. A significant difference existed in the average level of CFU between the control group and the estrogen-treated group （P〈0.05）, and between the control group and the immuosuppressed group （P〈0.01）. It was concluded that the vaginal mucosa from the immunosuppressed mice is more susceptible to Candida albicans and no difference is found in susceptibility between mice with primary infection and secondary infection.     

TLR-2和TLR-4在不同疾病胸腔积液中的水平及临床意义

目的探讨不同疾病胸腔积液中Toll样受体2（TLR-2）及Toll样受体4（TLR4）的水平及其临床意义。方法收集81例胸腔积液及其同源外周血,其中结核性胸腔积液组32例,肿瘤性胸腔积液组28例,细菌性胸腔积液组21例。应用酶联免疫吸附（ELISA）法测定胸水上清液和血清中TLR-2和TLR-4的浓度,并对结果及意义进行分析。结果（1）胸腔积液间：结核组TLR-2浓度（22．19±2．12）ng／L分别与肿瘤性组（14．50±1．93）ng／L、细菌组（13．99±2．36）ng／L相比差异均有统计学意义（P〈0．05）;肿瘤性组、细菌组间相比差异均无统计学意义（P〉0．05）。而TLR4在三组间比较差异均无统计学意义（P〉0．05）。（2）血清中：细菌组TLR-4浓度（5．34±2．13）ng／L,分别与结核组（1．54±1．09）ng/L、肿瘤性组（0．10±0．06）ng／L相比差异均有统计学意义（P〈0．05）。（3）胸腔积液与血清中：TLR-2和TLR-4比较差异均有统计学意义（P〈0．05）。（4）结核性胸腔积液中：TLR-2和TLR-4具有相关性（P〈0．05）。结论TLR-2可有助于结核性与非结核性胸腔积液的鉴别。TLR-2和TLR-4在结核性胸膜炎发病中可能具有共同作用。     

尿毒症患者单个核细胞Toll样受体4表达及意义

目的 探讨尿毒症患者外周血单个核细胞(PBMC)表面Toll样受体4(TLR4)表达的临床意义.方法 用FITC荧光标记的抗TLR4单抗对80例维持性血液透析患者[(随机分为普通透析液组(CD)和超纯透析液组(UPD)]、40例尿毒症非透析患者和40例正常人(对照组)的外周血单个核细胞表面TLR4分子进行免疫荧光染色,应用流式细胞仪检测.结果 (1)普通血液透析组、非透析组、对照组3组间TLR4表达分别为(18.1±3.7)%、(24.5±4.6)%、(31.6±5.8)%差异有统计学意义(P<0.05);(2)使用超纯透析液(UPD)与普通透析液(CD)透析6个月时TLR4表达(23.1±3.2)%与(18.1±3.7)%差异有统计学意义(P<0.05);普通血液透析3、6、12个月TLR4表达为(22.6±3.9)%、(18.1±3.7)%、(14.7±2.6)%差异有统计学意义(P<0.05),使用超纯透析液治疗后TLR4表达略有下降,但差异无统计学意义(P>0.05).结论 在尿毒症非透析及透析患者中TLR4表达均降低;使用超纯透析液较普通透析液TLR4表达增多;随普通透析时间延长,TLR4表达减少,使用超纯透析液对TLR4表达无明显影响.     

TLR4在乳腺癌组织中的表达与微血管密度的关系及临床意义

目的：研究Toll样受体4（TLR4）在乳腺癌组织中的表达及其与肿瘤微血管生成的关系,并探讨其临床意义。方法：采用免疫组织化学法对58例乳腺癌组织及30例正常乳腺组织中TLR4表达进行检测;用CD105标记肿瘤新生血管,计数乳腺癌及正常乳腺组织中微血管密度（MVD）。结果：TLR4在乳腺癌和正常乳腺组织中的阳性表达率分别为69.0%和13.3%,差异有统计学意义（P〈0.05）;乳腺癌组织平均MVD值明显高于正常乳腺组织,差异有统计学意义（P〈0.05）;乳腺癌组织中TLR4的表达与MVD呈正相关（r=0.581,P〈0.05）;TLR4的表达和MVD计数与乳腺癌患者年龄、肿瘤大小、组织分级等因素无关（P〉0.05）,但与淋巴结转移及TNM分期关系密切（P〈0.05）。结论：TLR4在乳腺癌组织中表达增高,其异常表达与肿瘤MVD有关,促进肿瘤新生血管形成可能是其参与乳腺癌的发生发展机制之一。     

Lipopolysaccharide upregulates the expression of Toll-like receptor 4 in human vascular endothelial cells

目的：探讨内皮细胞TLR2和TLR4的表达以及内毒素对其表达的影响。方法：按RNA提取试剂盒说明书提取内毒素作用后的ECV-304(人脐静脉内皮细胞株)和人脐静脉内皮细胞总RNA,运用逆转录PCR(RT-PCR)检测细胞TLR2和TLR4mRNA的表达。结果：人脐静脉内皮细胞和ECV304细胞株均能表达TLR2和TLR4mRNA,但是,TLR4的表达水平明显强于TLR2的表达,LPS能明显上调TLR4的表达,但对TLR2的表达无影响。结论：本研究提示TLR4能明显上调LPS作用的信号转导分子,在LPS对内皮细胞的激活效应中具有重要的作用;ECV-304是内皮细胞研究的良好的实验模型,可代替人脐静脉内皮细胞而较为广泛应用。     

Relationship between the expression of Toll-like receptor 2 and 4 in mononuclear cells and postoperative acute lung injury in orthotopic liver transplantation

Background The aim of this study was to investigate the potential relationship between the dynamic expression of Toll-like receptor 2 and 4 （TLR2/4） in peripheral blood mononuclear cells as well as changes in serum concentration of inflammatory factors and acute lung injury （ALl） in patients after orthotopic liver transplantation （OLT）. Methods The peripheral blood samples of 27 patients （23 men and 4 women with ASA Ⅲ to Ⅳ） who received OLT were collected for measurement of TLR2/4 at T1 （after induction of anesthesia）, T2 （25 minutes after anhepatic phase）, T3 （3 hours after graft reperfusion） and T4 （24 hours after graft reperfusion）. The expression of TLR2/4 in mononuclear cells was measured by flow cytometry. The serum concentrations of tumor necrosis factor （TNF）-α, interleukin （IL）-1β and IL-8 were measured by enzyme-linked immunosorbent assay （ELISA）. Twenty-seven patients were assigned to ALl group （n=-9） and non-ALl group （n=-18） according to the diagnostic criteria of ALl. The expression of TLR2/4 in the ALl group or non-ALl group was analyzed. Results Compared to the non-ALl group, the volumes of blood loss, ascites, total output and transfused red blood cells were higher in the ALl group, and the anhepatic phase lasted longer （P 〈0.05, P 〈0.01）. The expression of TLR2/4 in mononuclear cells increased significantly at T3 and T4, and serum concentrations of TNF-α, IL-1β and IL-8 increased significantly too. There was no significant difference in Child-Turcotte-Pugh （CTP） scores between the ALl group and non-ALl group （P 〉0.05）. The expression of TLR2/4 in mononuclear cells increased significantly at T3 and T4 in the ALl group （P 〈0.05, P〈0.01）. A positive correlation was noted between the expression of TLR4 in mononuclear cells and the serum concentrations of TNF-α, IL-1β （P=0.041, P=0.046） in the ALl group. In the non-ALl group, statistical results showed that the expression level of TLR2/4 in mononuclear cells was not significantly different during the peri-operative period of OLT （besides TLR4 expression at T4）. Compared to expression in mononuclear cells was more significant in the mononuclear cells exceeded that at T1 by one time were more 16. the non-ALl group, the increasing amplitude of TLR2/4 ALl group. The patients whose TLR2/4 expression in likely to suffer from ALl （P=0.013）, with a relative risk of Conclusion The expression level of TLR2/4 in mononuclear cells increases significantly in the peri-operative period of OLT, and it may be a high risk factor for occurrence of postoperative ALl.     

Expression and Significance of Toll-like Receptor 2, 4 of Peripheral Blood Mononuclear Cells in Acute Abdomen Patients Associated with Systemic Inflammatory Response Syndrome

The changes of Toll-like receptor (TLR) 2, 4 of peripheral blood mononuclear cells (PBMCs) in the acute abdomen patients associated with systemic inflammatory response syndrome (SIRS) and their potential significance were explored. A clinical study was performed on 103 acute abdomen patients in whom 65 were associated with SIRS. Forty healthy individuals served as normal controls. The mRNA expression of TLR2, 4 was detected by RT-PCR, and the expression of TNF-αand EL-6 by ELISA. The level of plasma endotoxin, hospital stay and mortality were measured. It was found that the endotoxin level was increased to varying degrees in all the acute abdomen patients, and the endotoxin level was and hospital stay longer in SIRS group than in non-SIRS group (P<0.01). TLR2 mRNA, TLR4 mRNA, IL-6 and TNF-αcould be detected with low value in normal controls, but they were up-regulated markedly on the 1st day after admission. Then TLR4 mRNA, IL-6 and TNF-αwere decreased gradually, but TLR2 mRNA maintained at a high level till the 5th day. These indexes above in SIRS group were higher than those in non-SIRS group (P<0.01). The results of correlation analysis revealed the expression of TLR2, 4 mRNA was positively correlated with the levels of TNF-αand IL-6, and the hospital stay. The results of Logistic regression demonstrated that over-expression of TLR2, 4 mRNA might result in higher risk of multiple organ dysfunction syndrome (MODS). It was concluded that in the acute abdomen patients associated with SIRS, the expression of TLR2, 4 in PBMCs was increased markedly, suggesting that TLR might play an important role in the pathogenesis of acute abdomen associated with SIRS.     

耐药白色念珠菌弥散性感染小鼠模型的建立

目的为新药筛选建立耐药白色念珠菌弥散性感染小鼠模型。方法对免疫抑制ICR小鼠静脉注射临床耐氟康唑白色念珠菌CaR,通过临床症状、生存情况、组织载菌量、组织病理、细胞因子分析以及药物治疗等方法对模型评价。结果 CaR感染小鼠在接种后第1天出现死亡,相比临床药敏菌株Ca S感染组,16 d观察期内动物死亡率无显著性差异(CaR,90.7%;Ca S,86.2%,P=0.158),但观察早期CaR组死亡比Ca S组快。感染第4天,不同组织均可检测到念珠菌,并发现与Ca S组相比,肾、脑组织载菌量具有显著性差异。真菌引起的典型肉芽肿样是肾、脑、心的主要组织病理学特征。利用流式细胞仪检测肾组织细胞因子,IL-1α、IL-6、TNF-α、IFN-γ等细胞因子变化显著,与Ca S组比较,IL-1α和IFN-γ显著升高,TNF-α显著下降。CaR和Ca S感染小鼠给予10 mg/kg氟康唑治疗,死亡率分别为83.3%、37.5%,具有显著性差异。结论本研究成功建立了耐药白色念珠菌弥散性感染小鼠模型,有望成为抗感染新药研发的重要工具。     

右美托咪啶对老年患者全麻术后炎症因子水平及单核细胞TLR2和TLR4表达的影响

目的观察右美托咪啶对老年腹部手术患者围术期炎症因子水平及单核细胞TLR2和TLR4表达的影响。方法选择老年全麻患者91例,采用随机数字表法将患者分为：右美托咪啶组（D组）46例和对照组（C组）45例。两组均常规咪达唑仑、顺苯磺酸阿曲库铵、舒芬太尼、依托咪酯行麻醉诱导,诱导前10min,D组静脉输注右美托咪啶针,负荷量0.5滋g/kg,再分别恒速静脉输注右美托咪啶针0.5滋g/（kg·h）至术毕前30min;C组采用同样方法静脉输注等容量0.9%氯化钠溶液。记录舒芬太尼、瑞芬太尼和异丙酚用量。于麻醉前（T0）、手术开始1h（T1）、手术结束后12h（T2）及24h（T3）时各采集右颈内静脉血5ml,采用ELISA测定HMGBl、TNF-α、IL-6水平,并采用流式细胞仪检测单核细胞TLR2和TLR4的表达水平。结果与C组比较,D组瑞芬太尼针和丙泊酚针用量明显减少（P＜0.01）,舒芬太尼用量差异无统计学意义（P＞0.05）。与C组比较,D组TNF-α、IL-6和HMGB1水平在T1、T2、T33个时点均明显降低（P＜0.05）;D组单核细胞TLR2和TLR4的表达水平在T1、T2、T33个时点均明显降低（均P＜0.05）。结论右美托咪啶可以降低老年腹部手术患者围术期炎症因子水平及抑制单核细胞TLR2和TLR4表达。     

白念珠菌磷脂甘露聚糖对单核细胞产生白介素-6、白介素-8的影响

目的 研究白念珠菌磷脂甘露聚糖( PLM)诱导人急性单核细胞白血病细胞系细胞(THP-1)产生的炎症反应是否依赖Toll样受体(TLR)2.方法 实时荧光定量逆转录PCR分析PLM体外刺激THP-1细胞TLR2、TLR4、前炎症因子[白介素(IL)-6]和趋化因子(IL-8)的mRNA表达水平.酶联免疫吸附法检测IL-6、IL-8分泌含量.免疫印迹法分析TLR2的蛋白表达.结果 PLM可升高THP-1细胞的IL-6和IL-8 mRNA表达和分泌水平(均P＝0.0000).PLM上调THP-1细胞的TLR2 mRNA和蛋白表达水平(P＝0.0000),但对TLR4 mRNA表达无影响.PLM经β-D-甘露糖苷水解酶处理后,不能诱导上述受体及因子的表达.TLR2中和抗体能抑制PLM诱导的IL-6和IL-8产生(P ＝0.0003,P＝0.0010).结论 白念珠菌胞壁PLM依赖TLP2介导激活人THP-1细胞产生炎症反应.