M-CSF与L929细胞培养上清诱导分化骨髓巨噬细胞的差异分析

[目的]通过比较常用的M-CSF与L929细胞培养上清两种诱导方式获得骨髓巨噬细胞(BMDM)的形态及基本生物学功能,为选择制备BMDM的方法提供实验依据。[方法]用两种方式诱导小鼠骨髓获取BMDM,然后分别利用Zeiss荧光显微镜和流式细胞仪比较BMDM的细胞形态及纯度;通过吞噬实验、杀菌实验以及液相芯片技术(liquid chip)比较两组BMDM的生物学功能;同时将上述结果与体内分化成熟的腹腔巨噬细胞相比较。[结果]两组诱导分化成熟的BMDM均呈现不规则形或梭形;BMDM的纯度分别为M-CSF组98.6%,L929上清组99.6%;腹腔细胞贴壁后巨噬细胞的纯度是98.8%;吞噬实验、杀菌实验两组之间及其与腹腔巨噬细胞相比无显著差异;炎症因子释放水平检测中3组细胞存在IL-6或TNF-α某些时间点释放水平差异。[结论]M-CS与L929上清诱导方式所获得BMDM的形态相似,纯度相近(98.6%比99.6%),吞噬细菌能力无显著统计学差异(21.31±5.83比26.10±6.11,t=-0.745,P0.05),杀菌能力无显著差异(杀菌30min:36.41%±4.21%比39.53%±6.75%;60min:44.35%±6.75%比45.27%±1.96%,两者均P0.05),但是炎症因子释放的某些时间点有差异(TNF-α的释放在LPS刺激后24 h M-CSF组高于L929组:549.92±412比271.47±432,t=-0.34,P0.05),应根据实验目的选择获取BMDM的诱导方式。     

DUSP8过表达通过JNK/p38 MAPK通路来改善LPS诱导的巨噬细胞炎症反应

双特异性磷酸酶8（dual-specificity phosphatase 8, DUSP8）是双特异性蛋白磷酸酶家族的成员之一,被报道参与多个疾病发生过程。然而,DUSP8是否参与巨噬细胞等免疫细胞炎性应答过程,目前仍未有研究证实。本研究旨在检测DUSP8在脂多糖(LPS)诱导的巨噬细胞炎症反应中的表达,并探讨过表达DUSP8在巨噬细胞炎症反应的作用。利用100 ng/mL LPS刺激野生型C57BL/6小鼠骨髓来源巨噬细胞（bone marrow derived macrophage,BMDM）,分别在不同时间点收取细胞,实时PCR和Western 印迹检测发现LPS处理后,BMDM中DUSP8的表达水平明显降低（P<0.05）,且在12 h达到最低值;随后,分别转染DUSP8过表达载体（DUSP8-EGFP）和对照载体（EGFP）于BMDM,Western 印迹检测发现DUSP8-EGFP转染能够显著上调DUSP8的表达水平（P<0.05）;进一步用流式细胞术(flow cytometry, FCM)检测发现DUSP8过表达使巨噬细胞表面分子CD80和CD86的表达显著下调（P<0.05）;同时,中性红吞噬实验结果显示,DUSP8过表达后巨噬细胞的吞噬能力明显降低（P<0.05）;此外,ELISA (enzyme linked immunosorbent assay)检测结果显示,过表达DUSP8显著降低IL-1β,IL-6的表达水平（P<0.05）;最后,Western 印迹结果显示,JNK和p38 MAPK的磷酸化水平在DUSP8过表达组中明显降低（P<0.05）。以上表明,DUSP8过表达可显著改善LPS诱导的巨噬细胞炎症反应,其机制主要通过抑制JNK和p38 MAPK的活化。     

姜黄素诱导Nrf2 核转位对脂多糖引起库普弗细胞分泌炎症 细胞因子的影响

目的:研究姜黄素诱导大鼠Kupffer细胞Nrf2核转位对脂多糖(LPS)引起的炎症细胞因子分泌的影响。方法:分别用10μM、20μM和30μM干预Kupffer细胞8h,诱导Nrf2核转位水平;将Kupffer细胞随机分为对照组、LPS组和干预组,对照组正常培养未加姜黄素和LPS,LPS组用10μg/mL的LPS加入Kupffer细胞培养液共同培养2 h;干预组用30μM姜黄素干预8h后,余处理同LPS组。Western blot检测Nrf2核转位水平,分光光度法检测细胞MDA、GSH水平,ELISA法检测上清液TNF-α和IL-6,放免法检测IL-1β。结果:①姜黄素诱导Kupffer细胞Nrf2核转位,核转位水平随浓度增加而增高。②LPS组MDA水平较对照组显著升高(P<0.01),干预组MDA水平较LPS组显著降低(P<0.01),仍显著高于对照组(P<0.01)。LPS组GSH水平较对照组显著降低(P<0.01),干预组GSH水平较LPS组显著升高(P<0.01),仍显著低于对照组(P<0.01)。③LPS组上清液TNF-α,IL-1β和IL-6显著高于对照组(P<0.01),干预组均显著低于模型组(P<0.01),但显著高于对照组(P<0.01)。结论:姜黄素通过诱导Kupffer细胞Nrf2核转位,降低LPS诱导的氧化应激损伤,抑制Kupffer细胞分泌炎症细胞因子。     

微进化对阿萨希毛孢子菌与小鼠巨噬细胞RAW264.7相互作用的影响

目的研究微进化对阿萨希毛孢子菌(T.asahii)与巨噬细胞RAW264.7相互作用的影响。方法将微进化前后的T.asahii与RAW264.7细胞共培养,检测RAW264.7对两株菌的吞噬和杀伤能力以及两株菌对RAW264.7产生的细胞毒性的差异,同时分析RAW264.7自身细胞因子分泌情况的变化。结果巨噬细胞对原代菌株(TO)的吞噬能力以及杀伤率均明显高于微进化株(TEVO);TO菌株对巨噬细胞的细胞毒性要强于TEVO;当巨噬细胞与菌株按1∶3或1∶6共培养24 h时,与TO共培养的巨噬细胞TNF-α和IL-6的分泌量要高于TEVO组,而按1∶9共培养时TEVO组细胞因子的分泌量却高于TO组。结论微进化后的TEVO菌株与巨噬细胞的相互作用明显弱于原代株TO,这也为TEVO菌株在宿主体内长期共存提供了良好的基础。     

阻断Kv1.3通道可增强RAW264.7巨噬细胞的吞噬功能

本研究旨在阐明电压门控性钾通道1.3(Kv1.3)在巨噬细胞吞噬功能中的作用.利用RAW264.7巨噬细胞吞噬鸡红细胞的半定量检测系统及吞噬异硫氰酸荧光素标记的大肠杆菌(E.coli)k-12的流式细胞术定量检测系统测定巨噬细胞的吞噬功能.研究发现,用海葵神经毒素(Sh K)(100 pmol/L)选择性阻断Kv1.3通道能显著增强处于静息状态的和被脂多糖(LPS)激活的RAW264.7巨噬细胞吞噬鸡红细胞的能力;Sh K也可增强静息RAW264.7细胞吞噬大肠杆菌的能力,但由于LPS刺激吞噬的效应近乎饱和,Sh K并不能进一步增加被LPS激活的RAW264.7细胞吞噬大肠杆菌的数量.Sh K促进LPS激活的RAW264.7细胞释放一氧化氮(NO),但并不增加静息RAW264.7细胞的NO释放.Sh K(100 pmol/L)自身并不影响静息RAW264.7细胞释放细胞因子,但能抑制LPS激活的RAW264.7细胞释放白细胞介素-1?.Sh K(100 pmol/L)对RAW264.7细胞的活力无明显影响.RAW264.7细胞表达Kv1.3通道蛋白;LPS使RAW264.7细胞的Kv1.3蛋白表达下调,菲律宾菌素Ⅲ(小凹蛋白依赖性内吞途径抑制剂)使Kv1.3蛋白表达上调,细胞松弛素D对Kv1.3蛋白表达无明显影响.研究表明,RAW264.7细胞表达Kv1.3蛋白;阻断Kv1.3通道可增强RAW264.7细胞的吞噬能力和NO生成.结果提示,Kv1.3通道可能是RAW264.7细胞吞噬活动的负调节因子,有可能成为治疗巨噬细胞吞噬功能异常相关疾病的一个靶点.     

大黄鱼头肾巨噬细胞体外模型的构建

通过优化Percoll密度梯度离心技术,从大黄鱼 (Larmichthys crocea) 头肾组织中分离纯化巨噬细胞,并优化细胞培养条件。对所分离细胞进行形态学分析、普通光镜和电镜超微结构观察以及细胞功能验证实验。实验结果表明:细胞单层置于22℃无CO2恒温培养箱中,于L15培养基中培养5d后仍保持较高的活力。普通光镜和电镜观察到大黄鱼头肾巨噬细胞具有伪足发生和较强的吞噬能力、胞浆中富含线粒体以及吞噬小泡。巨噬细胞与不同浓度(0、0.1、1、20 μg/mL)的脂多糖(LPS,Escherichia coli 055:B5)分别孵育3h和24h。施加LPS刺激后,细胞呼吸暴发活性随LPS浓度以及孵育时间变化而变化。孵育3h后,0.1 μg/mL LPS协同PMA可显著诱导巨噬细胞活性氧中间体(ROI)的产生。孵育24h后,所有处理组LPS协同PMA显著抑制细胞ROI的生成(P 0.05)。未添加PMA时, 经1 μg/mL LPS孵育3h和24h后的巨噬细胞细胞ROI的生成量显著降低(P 0.05), 其他LPS处理组间细胞ROI的生成量无显著差异。       

转移因子对小鼠腹腔巨噬细胞吞噬功能的影响和E玫瑰花形成的作用

本文报道了转移因子对小鼠腹腔巨噬细胞吞噬功能的影响和对脾细胞E玫瑰花形成的作用。转移因子为本单位从健康猪脾细胞提取的针剂,含多核苷酸和多肽等低分子生物活性物质,每支含量为3×10~3个脾细胞提取物,每天一次0.5ml剂量注入小鼠体内,连续5次后取动物腹腔巨噬细胞和脾细胞悬液,测定其吞噬功能并观察E玫瑰花形成作用。结果表明,转移因子对小鼠腹腔巨噬细胞吞噬的百分率和吞噬指数与对照组比有明显差异(P<0.01),对小鼠脾细胞E玫瑰花形成作用与对照组比差异也极显著(P<0.01)。从而看出,转移因子能使小鼠腹腔巨噬细胞吞噬功能增强,亦能使特异的玫瑰花形成细胞中T淋巴细胞增多,增强了机体的免疫功能。     

Toll样受体抗体抑制脂多糖激活巨噬细胞的吞噬活行

应用脂多糖(LPS)激活巨噬细胞后其吞噬能力大大增强,以此为模型发现TLR2的多抗能部分抑制LPS激活巨噬细胞吞噬金葡萄球菌的能力,也能部分阻断对U937细胞的吞噬活性.TRAIL或TNFα多克隆抗体同样能起到类似作用.实验还发现低浓度血清培养亦在一定程度上抑制LPS激活巨噬细胞的吞噬作用.结果证实TLR2能介导LPS的功能,而某些血清因子参与了介导LPS的信号转导过程,提示LPS激活巨噬细胞吞噬能力的提高与诱导表达TRAIL等细胞因子有关.     

Toll样受体抗体抑制脂多糖激活巨噬细胞的吞噬活性

应用脂多糖(LPS)激活巨噬细胞后其吞噬能力大大增强, 以此为模型发现TLR2的多抗能部分抑制LPS激活巨噬细胞吞噬金葡萄球菌的能力, 也能部分阻断对U937细胞的吞噬活性. TRAIL或TNFα多克隆抗体同样能起到类似作用. 实验还发现低浓度血清培养亦在一定程度上抑制LPS激活巨噬细胞的吞噬作用. 结果证实TLR2能介导LPS的功能, 而某些血清因子参与了介导LPS的信号转导过程, 提示LPS激活巨噬细胞吞噬能力的提高与诱导表达TRAIL等细胞因子有关.     

维生素D3对黄颡鱼头肾极化分型的巨噬细胞影响

研究以黄颡鱼(Pelteobagrus fulvidraco)头肾巨噬细胞为研究对象,通过细菌脂多糖(LPS)和环磷酸腺苷(cAMP)分别诱导M1型和M2型极化,200 pmol/L维生素D₃孵育后对其形态学特征、生物学功能及极化相关基因的表达进行分析鉴定来确定维生素D₃在巨噬细胞极化中的调节作用。结果表明,维生素D₃能降低诱导后M1型和M2型巨噬细胞的死亡率,并增强巨噬细胞的吞噬活性。在M1型巨噬细胞中维生素D₃能够抑制活性氧(ROS)和炎症介质一氧化氮(NO)的产生,降低超氧阴离子自由基的活力,白介素1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的表达水平显著降低(P<0.05);在M2型细胞中能够增加精氨酸酶的活性,显著增加白介素10(IL-10)和转化生长因子(TGF-β)的表达水平(P<0.05),最终抑制巨噬细胞向M1表型极化,促进巨噬细胞向M2表型极化,发挥抗炎作用;黄颡鱼头肾巨噬细胞中Nos-2和Arg-2分别是M1和M2巨噬细胞的生物标记基因。研究结果为进一步研究鱼...     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

鸡传染性法氏囊病病毒研究进展

传染性法氏囊病(Infection bursal disease, IBD)是由鸡传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的鸡和火鸡的一种高度接触性传染病,给世界各国的禽养殖业带来了巨大损失.自IBDV发现至今新的变异株不断出现,分子结构的改变导致病毒致病力的改变及宿主对疫苗应答的改变,使得传统的疫苗已不能控制其流行,因此各国学者对其基因组结构和功能进行了广泛深入的研究,并积极研制新型有效的疫苗以达到防治的目的.     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.