LPS及TGF-β对巨噬细胞中Tim-3表达的影响及其机制的初步探索

目的 探讨促炎因子脂多糖(lipopolysaccharide,LPS)和抑炎因子转化生长因子-β(transforming growth factor-beta,TGF-β)对巨噬细胞中T细胞免疫球蛋白黏蛋白-3(T-cell immunoglobulin and mucin domain-containing protein3,Tim-3)表达的影响,并初步探索其影响机制.方法 使用LPS和TGF-β分别刺激小鼠源巨噬细胞系RAW264.7、人源巨噬细胞系U937,在基因水平和蛋白水平分别检测巨噬细胞中Tim-3的表达情况,同时观察巨噬细胞中金属蛋白酶ADAM10/17在基因水平的变化.结果 LPS可抑制巨噬细胞中Tim-3在基因和蛋白水平的表达,而TGF-β作用相反.LPS促进巨噬细胞中ADAM10/17在基因水平的表达,TGF-β作用正相反.结论 炎症微环境中促炎介质LPS和抑炎介质TGF-β分别抑制和促进巨噬细胞中Tim-3的表达,同时,金属蛋白酶ADAM10/17可能参与LPS、TGF-β对巨噬细胞中Tim-3在蛋白水平表达的调控.     

Tim-3抗体的生物学活性及应用研究

目的：制备抗人Tim-3单抗并评价其生物学活性,为干预Tim-3表达失调引发的疾病提供实验基础。方法制备重组人Tim-3蛋白,常规方法免疫BALB/c小鼠,筛选出阳性克隆,并对其识别、中和活性进行体内外验证,探讨其作为免疫干预手段的可行性。结果①获得一株能够分泌具有较好结合活性的抗人Tim-3单克隆抗体细胞株（克隆号L3D）,其分泌的免疫球蛋白亚型为IgG2a;②流式实验结果表明,该抗体能够结合人U937细胞上的Tim-3分子,且与小鼠RAW 264．7细胞上Tim-3有一定的交叉结合活性;③该抗体能够阻断Gal-9分子诱导的人THP1细胞凋亡,具有良好的体外中和活性;④体内注射L3D,显示该抗体能够显著加重脓毒血症模型小鼠病情,提高炎性因子的表达水平,显示出其良好的体内中和活性。结论成功制备一株分泌抗人Tim-3抗体的细胞株,其良好的结合及中和活性为基于Tim-3表达异常相关疾病的干预提供了实验基础。     

辐射对巨噬细胞极化和功能的调节作用

巨噬细胞是存在于所有组织中的调节性细胞,它们对辐射刺激的反应机制复杂,既有共同的应对感染、损伤的变化过程,也有自己独特的极化转变方式。辐射相关的极化转变决定了巨噬细胞的功能类型。巨噬细胞受到不同的照射后,受到不同细胞因子的调节,涉及多个信号通路,极化程度也有所不同。本文综述辐射对巨噬细胞极化和功能的调节作用,以期对免疫基础研究和临床放射治疗提供参考。     

创面巨噬细胞分泌物对成纤维细胞的调节作用

采用体外培养体系研究家兔不同时期皮肤创面巨噬细胞上清液和冻融液对创面成纤维细胞增殖和胶原蛋白合成的调节作用。用皮下埋置自制不锈钢管法，直接获取手术后７、１４、２１、２８天活化的创面巨噬细胞，贴壁纯化后培养４８小时收集上清液；细胞经－３０℃与３７℃反复冻融３次收集冻融液。从兔皮肤３～５天刨面取新生肉芽组织，经组织培养获取创面成纤维细胞。测定成纤维细胞中３Ｈ－胸腺嘧啶和３Ｈ－脯氨酸的掺入量。结果上清液对成纤维细胞增殖和胶原蛋白合成具有不同程度的促进作用；冻融液主要表现为抑制作用。说明巨噬细胞能合成或分泌某些生物活性物质调节成纤维细胞的活动，从而控制创伤愈合的进程。     

巨噬细胞在脓毒症中的免疫调节作用研究进展

巨噬细胞作为脓毒症中天然免疫反应中的抗感染细胞及特异性免疫中的关键抗原呈递细胞,其功能变化在脓毒症中的作用受到人们的特别关注.该文综述了巨噬细胞在脓毒症中的免疫调节作用研究进展.     

Tim-3通过Nrf2/COX-2通路抑制巨噬细胞抗李斯特菌的机制研究

目的 探讨T细胞免疫球蛋白和黏蛋白结构域分子-3(Tim-3)在李斯特菌(Listeria)感染中通过核因子相关因子2(Nrf2)/环氧化酶2(COX-2)通路对巨噬细胞的影响及可能机制.方法 提取Tim-3转基因小鼠和野生小鼠腹腔巨噬细胞,利用RT-PCR检测COX-2的表达;Tim-3融合蛋白阻断RAW264.7巨...     

利用RNA干扰技术研究14-3-3ζ对肺癌细胞转移的抑制作用

目的:探讨14-3-3ζ对肺癌细胞转移的抑制作用.方法:以人肺巨细胞癌低转移细胞株为模型,利用RNAi技术研究14-3-3ζ被干涉后肿瘤细胞恶性表型的变化,探讨14-3-3ζ与肿瘤转移的相关性.结果:本研究发现14-3-3ζ被干涉后细胞的集落形成能力明显提高,黏附能力明显降低,细胞迁移、侵袭能力明显提高.结论:14-3-3ζ可能抑制肺癌细胞的转移.     

小鼠腹腔巨噬细胞趋化和吞噬功能中SRC-3的作用研究

目的 探讨类固醇受体辅活化子（SRC）-3蛋白在脂多糖诱导小鼠腹腔巨噬细胞趋化和吞噬功能中的作用.方法 健康SPF级雌性野生型（SRC-3＋/＋）小鼠、SRC-3基因敲除（SRC-3-/-）小鼠各5只,分别分离和原代培养腹腔巨噬细胞,并相应分为SRC-3＋/＋组和SRC-3-/-组.收集并调整细胞浓度为1×106/ml,接种于6孔板,1 ml/孔,给予10 μg/ml LPS刺激,分别在刺激前（0 h）、刺激后4 h和12 h收集腹腔巨噬细胞.通过Western blot测定腹腔巨噬细胞Toll样受体4（TLR4）的表达,并通过transwell趋化实验和中性红吞噬实验,分别测定腹腔巨噬细胞的趋化指数（CI）和吞噬功能.结果 LPS诱导前两组腹腔巨噬细胞TLR4的蛋白表达和CI差别无统计学意义,但SRC-3-/-组的吞噬功能显著低于SRC-3＋/＋组（P＜0.01）;无论是SRC-3＋/＋组,还是SRC-3-/-组,LPS诱导4 h和12 h后,两组腹腔巨噬细胞TLR4的表达和CI均显著增高（P＜0.01）,但在相应时间点,SRC-3-/-组与SRC-3＋/＋组相比差别无统计学意义.LPS刺激4 h后,两组吞噬功能均显著增加（P＜0.01）,但SRC-3-/-组增加的程度显著低于SRC-3＋/＋组（P＜0.01）;相反,LPS刺激12 h后,两组吞噬功能均受到不同程度的抑制（P＜0.05或P＜0.01）,且SRC-3-/-组较SRC-3＋/＋组抑制的程度更低（P＜0.01）.结论 SRC-3调节腹腔巨噬细胞的先天性免疫功能可能与吞噬功能有关,而与其趋化功能无关.     

低剂量辐射减轻化疗对巨噬细胞功能抑制作用的实验研究

有的研究证实,低剂量辐射（ＬＤＲ）可明显增强荷瘤小鼠的免疫功能并提高化疗的抑瘤作用［１,２］,但在此过程中巨噬细胞（Ｍ）功能的变化还不清楚。本实验以荷有Ｌｅｗｉｓ肺癌的Ｃ５７ＢＬ／６Ｊ小鼠作为实验动物模型,探讨ＬＤＲ与化疗联合应用对荷瘤小鼠Ｍ功能...     

游泳训练对小鼠巨噬细胞功能影响的实验研究

对游泳训练3个月的C_(57)B L/6小鼠巨噬细胞功能进行了研究。结果每日运动30分钟组巨噬细胞吞噬功能和抑瘤活性增强,运动60分钟组巨噬细胞IL-1分泌水平和吞噬功能增高,运动120分钟组的巨噬细胞IL-1分泌水平和吞噬功能则明显降低。提示不同运动量的运动训练引起巨噬细胞功能变化有差异,适量运动可以增强巨噬细胞功能,过大的运动则对巨噬细胞功能有损害或抑制作用。     

低氧预处理的人脐带间充质干细胞对巨噬细胞极化的调节作用观察

目的 探讨低氧培养的人脐带间充质干细胞对巨噬细胞极化的影响.方法 采用组织块贴壁法获取人脐带来源的间充质干细胞(hUC-MSCs),分别将细胞置入常氧(氧浓度21％)和低氧(氧浓度5％)条件下培养,通过细胞成脂、成骨分化诱导观察其多向分化能力,Live/death染色检测细胞活性,ELISA法检测其细胞因子蛋白含量.利用Transwell将常氧和低氧培养的hUC-MSCs与脂多糖(IPS)刺激后的巨噬细胞(THP-1)共培养,免疫荧光检测THP-1的极化情况,ELISA检测THP-1分泌炎症因子以及抗炎因子的情况.结果 低氧条件下培养的hUC-MSCs具有成脂、成骨的多向分化潜能;Live/death染色结果显示常氧和低氧培养下的hUC MSCs均具有较高的细胞活性;低氧条件下培养的hUC-MSCs中前列腺素E2(PGE2)、吲哚胺2,3-双加氧酶(IDO)表达水平明显高于常氧培养的hUC-MSCs;低氧条件下培养的hUC-MSCs可促进THP-1向M2型巨噬细胞转化,同时炎症因子肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)的表达明显降低,而抗炎因子IL-10的表达明显增强.结论 低氧培养的hUC-MSCs可促使THP-1 向M2巨噬细胞转化,提高其抗炎能力.     

Effects of IL-4 on pulmonary fibrosis and the accumulation and phenotype of macrophage subpopulations following thoracic irradiation

Purpose: Thoracic irradiation injures lung parenchyma, triggering inflammation and immune cell activation, leading to pneumonitis and fibrosis. Macrophage polarization contributes to these processes. Since IL-4 promotes pro-fibrotic macrophage activation, its role in radiation-induced lung injury was investigated.

Materials and methods: Lung macrophage subpopulations were characterized from 3–26 weeks following exposure of WT and IL-4?/? mice to 0 or 12.5 Gray single dose thoracic irradiation.

Results: Loss of IL-4 did not prevent fibrosis, but blunted macrophage accumulation within the parenchyma. At 3 weeks following exposure, cell numbers and expression of F4/80 and CD206, an alternative activation marker, decreased in alveolar macrophages but increased in infiltrating macrophages in WT mice. Loss of IL-4 impaired recovery of these markers in alveolar macrophages and blunted expansion of these populations in infiltrating macrophages. CD206+ cells were evident in fibrotic regions of WT mice only, however Arg-1+ cells increased in fibrotic regions in IL-4?/? mice only. Radiation-induced proinflammatory Ly6C expression was more apparent in alveolar and interstitial macrophages from IL-4?/? mice.

Conclusions: IL-4 loss did not prevent alternative macrophage activation and fibrosis in irradiated mice. Instead, a role is indicated for IL-4 in maintenance of macrophage populations in the lung following high single dose thoracic irradiation.     

Assessment of macrophage infiltration in a Murine model of abdominal aortic aneurysm

Purpose

To evaluate the use of an ultrasmall superparamagnetic iron oxide (USPIO) contrast agent as a marker for the detection of macrophage in a preclinical abdominal aortic aneurysm animal (AAA) model.

Materials and Methods

Osmotic pumps were implanted subcutaneously in apoE^?/? mice for continuous infusion of Angiotensin II (Ang‐II). Weekly bright‐blood gradient echo scans were performed on the suprarenal abdominal aorta to evaluate aneurysm development. Once an AAA was detected, animals were administered 1000 μmol/kg of the USPIO contrast agent ferumoxtran‐10 (Combidex®) followed by in vivo scanning 24 h post‐USPIO administration. After in vivo imaging, aortas were harvested for ex vivo imaging, histology, iron quantification, and gene expression analysis.

Results

Reduced signal intensity was evident in the post‐USPIO transverse images of the abdominal aorta. The areas of reduced signal were primarily along the aneurysm shoulder and outer perianeurysm areas and corresponded to regions of macrophage infiltration and colocalized USPIO determination by means of histological staining. The absolute iron content measured significantly correlated to the area of signal reduction in the ex vivo images (r = 0.9; P < 0.01). In the AAA tissue, the macrophage‐driven cytokine gene expression was up‐regulated along with a matrix metalloproteinase known to mediate extracellular matrix breakdown in this disease model.

Conclusion

These results demonstrate the feasibility of using an USPIO contrast agent as a surrogate for detecting the acute inflammatory process involved in the development of abdominal aneurysms. J. Magn. Reson. Imaging 2009;30:455–460. Published by Wiley‐Liss, Inc.

     

Exposure to radiation from single or combined radio frequencies provokes macrophage dysfunction in the RAW 264.7 cell line

Purpose: The aim of this study was to determine whether exposure to radiation from single or multiple radio-frequency (RF) signals at 900 and 2450?MHz would induce effects in the RAW 264.7 cell line.

Materials and methods: Cell cultures were exposed to single or combined RF for 4, 24, 48, or 72?h in a GTEM electromagnetic test chamber. At the end of the radiation exposure time, viability and cell growth were analyzed by flow cytometry, nitric oxide (NO) production was measured by colorimetry, the expression of HSP70 and TNF-α was ascertained by qPCR, and the phagocytic activity was observed by microscopy.

Results: NO production increased after 48?h exposure at 2450?MHz, compared with controls. The group subjected to the combined interaction of two RFs showed an increase of HSP70 after 48?h exposure and a significant increase of NO and TNF-α after 72?h. The phagocytic activity of macrophages decreased in all groups as exposure time increased.

Conclusions: Our results indicated a decrease in phagocytic activity and an increase in inflammatory, cytoprotective, and cytotoxic responses in macrophages after continuous and combined exposure of multiple RF signals. Multiple RF interact in everyday life, the immune response in humans is unknown.     

1,25-二羟维生素D3对Th1优势应答大鼠免疫功能的影响

目的研究1,25-二羟维生素D31,25-(OH)2D3对内毒素(LPS)诱导的Th1优势应答大鼠T细胞免疫功能的影响。方法以1,25-(OH)2D3喂饲Lewis大鼠,1μg/d,共14天,对照组给予同体积的赋形剂,第15天大鼠腹腔注射LPS10ng/kg。每组25只大鼠,各留10只观察死亡率,其余大鼠6h后处死,留取标本,观察1,25-(OH)2D3对大鼠免疫细胞的细胞因子分泌、表型分化和细胞增殖的影响。结果注射LPS后观察96h,对照组大鼠24h内死亡5只(5/10),其余5只大鼠96h内均存活,实验组大鼠无死亡(0/10)。与对照组比较,实验组大鼠外周血IL-12和IFN-γ水平显著降低(3986±328pg/mlvs4160±289pg/ml,4840pg/ml±802pg/mlvs5264pg/ml±524pg/ml,P<0·05),IL-4水平显著升高(5·57±1·75pg/mlvs3·72±1·6pg/ml,P<0·05)。脾脏HE及PCNA免疫组化染色显示实验组大鼠白髓中淋巴细胞显著减少,PCNA阳性细胞呈局灶性或散在分布。流式细胞仪检测显示,实验组大鼠外周血CD4 CD25 淋巴细胞百分率(%)显著高于对照组(1·09±0·29vs0·73±0·00,P<0·01)。结论1,25-(OH)2D3能减轻致死剂量的LPS对大鼠的损伤,对Th1优势应答大鼠免疫细胞的细胞因子分泌、淋巴细胞增殖和细胞亚群分化具有调控作用。     

FOXO3a介导PTEN对DNA修复基因RAD51表达的调节作用

目的探讨转录因子FOXO3a在PTEN调节RAD51表达中的作用。方法利用Western印迹技术分析PTEN缺陷与否时,总FOXO3a以及细胞核内FOXO3a磷酸化的情况;转染外源AKT到PTEN野生型细胞或外源PTEN和失去激酶活性的AKT（AKT-DN）到PTEN缺陷型细胞,Western印迹检测核内FOXO3a的磷酸化情况。结果 PTEN缺失导致细胞核内FOXO3a磷酸化水平增高;外源PTEN可以降低PTEN缺陷型细胞核内FOXO3a的磷酸化水平,外源AKT可以增加PTEN野生型细胞核内FOXO3a的磷酸化水平;沉默FOXO3a在一定程度上导致RAD51表达水平下降。结论 FOXO3a可以结合到RAD51基因的启动子区,PI3K/AKT/FOXO3a信号通路是PTEN调节RAD51表达的重要方式之一。     

In vivo MR evaluation of the effect of the CCR2 antagonist on macrophage migration

The CCR2 antagonist is a receptor antagonist for monocyte chemoattractant protein‐1 and is known to be a potential anti‐inflammatory therapeutic agent. Recently used optimized labeling techniques for superparamagnetic iron oxide, macrophage homing, and recruitment toward the infection site can be observed on in vivo MRI. This study details the effect of the CCR2 antagonist on the macrophage migration and the feasibility of in vivo MRI for assessing the inhibition of chemotactic activity by the CCR2 antagonist. On binding assay, the CCR2 antagonist inhibits the binding affinity of monocyte chemoattractant protein‐1 to CCR2. Increased expression of messenger ribonucleic acid (mRNA) and expression of CCR2 and CD11b on the cellular surface, as induced by monocyte chemoattractant protein‐1, was shown, and the effect of monocyte chemoattractant protein‐1 on CCR2 and CD11b was restricted by the CCR2 antagonist. In a migration test using the transwell system, macrophages treated with the CCR2 antagonist showed significantly decreased chemotactic migration compared to that of wild‐type macrophages. MR images of infected left calf muscles in 12 mice were obtained 24 h after administration of macrophages labeled with superparamagnetic iron oxide. MRI successfully demonstrated the effect of the CCR2 antagonist on the directional migration of macrophages. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

细胞因子及异丙酚对中性粒细胞内caspase-3活性的影响

 目的观察细胞因子白介素-6(IL-6)和白介素-10(IL-10)及异丙酚对离体的健康人中性粒细胞内caspase-3活性的影响,了解细胞因子及异丙酚影响中性粒细胞凋亡的机制.方法选择12例健康志愿者,采静脉血并分离中性粒细胞,将每例分离的中性粒细胞分为8组,分别加入生理盐水(对照组)、IL-6、IL-10、IL-6+IL-10、异丙酚、异丙酚的脂质溶剂intralipid、IL-6+异丙酚、IL-6+intralipid孵育,18 h后利用专用的试剂盒用流式细胞仪检测每个样本caspase-3激活的程度,并进行组间比较.结果与对照组相比,IL-6显著抑制中性粒细胞caspase-3的激活(P＜0.05),而IL-10、异丙酚和intralipid对caspase-3的激活则无显著影响;但IL-10和异丙酚均能够对抗IL-6对caspase-3激活的抑制(P＜0.05),intralipid则未表现对抗IL-6的作用.结论IL-6可抑制中性粒细胞内caspase-3的激活,而IL-10和异丙酚可对抗IL-6的这一作用,提示IL-6、IL-10和异丙酚对中性粒细胞凋亡的影响至少部分是通过影响caspase-3的激活来实现的.