趋化因子及受体介导CD4~+CD25~+Foxp3~+Treg迁移研究进展

CD4~+CD25~+Foxp3~+调节性T细胞可以有效抑制同种异体反应时细胞介导的免疫反应,近年来是器官移植领域学者关注热点之一。既往研究表明,Tregs既存在于移植器官受体的各级淋巴组织中,而且也分布在移植物内部中。因为Tregs的活化位置不同,其免疫抑制功能也不同,如果Tregs用来预防免疫排斥反应的话,那么Tregs在体定位和迁移信息特别关键。趋化因子受体是CD4~+CD25~+Foxp3~+Treg进行靶向迁移、在特定部位发挥免疫调节作用的结构基础。Tregs在体迁移是一个由多个细胞、多种趋化因子及其受体参与的运动过程。随着淋巴组织中新的趋化因子受体和Tregs特异性趋化因子的发现,提示Tregs在体复杂的迁移过程和活化主要是由趋化因子及受体介导。因此,趋化因子领域的研究将会为器官移植病人提供新的治疗思路。     

天然调节T 细胞与诱导调节T 细胞的生物学特性

CD4+CD25+FOXP3+的调节T细胞(regulatory T cells,Treg)在维持机体免疫平衡方面起着重要的作用。体外扩增Treg细胞用于治疗自身免疫病、哮喘及诱导器官移植免疫耐受引起人们极大的兴趣。Treg细胞可分为2个亚群,分别为nTreg和iTreg,两者有不同的生物学特性。nTreg在特定条件下,可以分泌具有促进炎症的IL-17;iTreg在体内可丢失FOXP3,失去其免疫抑制功能。Treg细胞用于临床治疗,还有许多问题需要研究解决。     

体外扩增后脐血来源Treg细胞的有效性及安全性研究

通过检测扩增后调节性T细胞(regulatory T cells,Tregs)的有效性和安全性,研究脐血来源的CD4~+CD25~+Foxp3~+Treg细胞的临床运用前景。实验中,新鲜分离的Treg细胞通过CD3/CD28抗体包被微磁珠进行体外扩增,以检测脐血来源Treg细胞的体外增殖能力,同时利用流式细胞术检测新鲜分离的Treg细胞与扩增后细胞的表型。随后,采用混合淋巴实验(mixed lymphocyte reaction,MLR)检测Treg细胞的功能,利用刺激实验检测扩增后Treg细胞的免疫原性,并且利用NOD-Prkdc~(scid)IL2rg~(null)免疫缺陷小鼠进行体内安全性检测。结果显示,4周后脐血来源的Treg细胞能够扩增到1 000倍以上,与新鲜分离的Treg细胞相比,扩增后的细胞CD45RA表达降低,CD45RO和CD39表达升高;同时,MLR结果显示扩增后的Treg细胞具有较强的抑制能力,并且免疫原性低;此外,体内结果显示扩增后的Treg细胞不会引起移植物抗宿主反应。实验结果初步表明,脐血来源体外扩增后的Treg细胞不仅在数量和功能上能够满足临床移植的需要,并且免疫原性低安全性高。     

山萘酚通过增强Treg细胞免疫抑制功能延长移植物生存时间

目的:探讨应用山萘酚增强Treg细胞免疫抑制功能,从而抑制大鼠移植物排斥反应并改善移植物生存的作用和机制。方法:以Wister大鼠和SD大鼠分别为供、受体,建立同种异体皮肤移植排斥反应动物模型。观察受体老鼠皮肤移植物的情况,记录移植物失功时间(移植物皮片80%面积发生排斥)。RT-PCR检测移植7天后脾细胞、淋巴细胞FOXP3、CTLA-4和IL-10的mRNA水平,用HE染色组织病理学观察术后7天移植皮片的淋巴细胞浸润程度。体外实验T细胞增殖抑制试验加入山萘酚作为对照,观察Treg功能情况。结果:1.山萘酚能增强移植后同种异体移植物的生存时间(DMSO组6.3±0.3天,山萘酚组13.7±0.39天,P<0.01);2.RT-PCR显示山萘酚可增强细胞CTLA-4(对照组9.24±0.17,山萘酚组12.48±0.145,P<0.05)、FOXP3(对照组0.96±0.07,山萘酚组1.41±0.07,P<0.01)和IL-10(对照组0.95±0.12,山萘酚组1.50±0.16,P<0.05)的mRNA水平;3.体外T细胞增殖抑制实验中,山萘酚可增强Treg细胞的免疫抑制功能。结论:在大鼠皮肤移植模型中,山萘酚可延长皮肤移植物的生存时间,提高Treg细胞相关IL-10、FOXP3和CTLA-4的mRNA水平;体外实验中,能抑制效应T细胞的增殖,表明山萘酚在提高移植物生存方面存在一定的价值。     

CD4~+CD25~+ Treg细胞与移植免疫耐受

诱导器官移植受者对供者抗原的免疫耐受是防治同种异型移植排斥反应的最理想途径。目前认为,免疫耐受形成的主要机制包括:胸腺及骨髓阴性选择引起的克隆清除(Clonal deletion)、组织特异性自身抗原低表达引起的克隆忽视(Clonal ignorance)、阻断T细胞共刺激信号引起的克隆无能(Clonal anergy)、嵌合体(Chimerism)的形成、调节性T细胞(Regulatory Tcell,Treg)介导的克隆抑制(Clonal suppression)等。近年来CD4+CD25+ Treg细胞的研究已成为免疫学界的热门课题之一。已知CD4+CD25+ Treg细胞存在于小鼠、大鼠和人体中,是机体自然存在的具有主动调节活性的T细胞,对维持自我耐受和控制自身免疫病发挥着重要作用。本文着重就CD4+CD25+ Treg细胞的免疫调节机制及其在诱导移植免疫耐受方面的研究进展做一综述。     

比较体外扩增调节性T细胞的两种方法

利用免疫磁珠法(immunomagnetic beads,MACS)从BALB/c小鼠脾脏中分别分离出CD4+CD25+Tregs(regulatory T cells)和CD4+T细胞,CD4+CD25+Tregs加入anti-CD3/CD28磁珠与1 000 U/m L IL-2体外扩增14 d;CD4+T细胞加入anti-CD3/CD28磁珠、100 U/m L IL-2和5 ng/m L重组人源性转化生长因子-β(transforming growth factor-β,TGF-β)体外扩增5 d。分析两种方法扩增后Tregs免疫抑制功能及数量。结果显示,两种方法扩增的Tregs免疫抑制功能无明显差别(P0.05),但是分别从大小相当的BALB/c小鼠脾脏所提取出的细胞中,通过CD4+T细胞扩增5 d可以得到(4.9±0.4)×107 Tregs,而通过CD4+CD25+Tregs扩增14 d只能得到(1.9±0.1)×107 Tregs(P0.05)。结果发现,通过两种方法扩增得到的Tregs免疫抑制功能无明显差异,但是通过CD4+T细胞扩增Tregs方法所需时间短,而且得到的细胞数量多。     

高剂量热休克蛋白gp96通过激活调节性T细胞预防1型糖尿病

旨在以非肥胖糖尿病(Non-obese diabetic,NOD)小鼠为动物模型,研究高剂量昆虫细胞表达的重组热休克蛋白gp96(Recombinant gp96,rgp96)对1型糖尿病(Type 1 diabetes,T1D)的预防作用。以高剂量rgp96免疫NOD小鼠,用血糖仪监测小鼠血糖值,用流式细胞术检测小鼠脾脏CD4~+CD25~+Foxp3~+调节性T细胞(Regulatory T cells,Tregs)亚群频率,然后用一系列体外实验探究高剂量rgp96对Tregs的影响。结果显示高剂量rgp96免疫有效地预防或延缓小鼠T1D发病,免疫诱导Tregs数量明显增加。体外实验发现rgp96蛋白促进Tregs增殖,诱导Foxp3表达上调和IL-10分泌增加。研究结果为开发基于rgp96的新型T1D预防和治疗性疫苗提供了依据。     

黄芪多糖对免疫抑制模型小鼠Treg细胞及Th17细胞亚群的影响

本研究旨在探讨黄芪多糖对免疫抑制模型小鼠CD4+CD25+调节性T细胞(Treg细胞)和CD4+Th17细胞亚群功能的影响。通过小鼠腹腔注射环磷酰胺建立免疫抑制动物模型,应用黄芪多糖对免疫抑制小鼠进行治疗,采用流式细胞仪分析脾脏中CD4+T细胞、CD4+CD25+Treg细胞的比例,ELISA检测血清IL-17的水平。结果表明:与模型对照组比较,黄芪多糖高、中剂量明显降低小鼠脾脏CD4+T细胞数的比例(P0.05),同时黄芪多糖上调小鼠脾脏CD4+CD25+Treg细胞数的比例(P0.05),降低免疫抑制小鼠血清IL-17的水平,尤其1.0 g/(kg·d)剂量组的IL-17水平下降较为明显(P0.01)。提示黄芪多糖具有提高免疫抑制小鼠CD4+CD25+Treg细胞数的比例、降低CD4+T细胞的数量及抑制IL-17分泌活性的作用。     

砂生槐多糖提取物对免疫抑制模型小鼠Treg和Th17的影响

为了开发藏药砂生槐的药用价值,本文拟研究其多糖提取物对免疫抑制小鼠调节性T细胞(Treg)和Th17的影响。水提醇沉法提取砂生槐枝条多糖物质,腹腔注射环磷酰胺建立免疫抑制小鼠模型,模型小鼠经砂生槐多糖灌胃干预后,分离脾细胞,流式细胞术检测CD4+T细胞比例和CD4+CD25+Treg细胞比例;收集血清,ELISA检测IL-17水平。结果显示,各剂量组砂生槐多糖均能够引起免疫抑制小鼠的脾脏CD4+CD25+Treg细胞比例明显增多(P0.05),同时明显抑制IL-17的产生(P0.05)。以上结果提示砂生槐多糖可能通过上调Treg而抑制免疫功能,同时抑制IL-17的产生,发挥免疫抑制作用。     

Toll样受体4结合脂多糖对天然调节性T细胞抑制功能的影响及其机制

探讨Toll样受体4结合脂多糖(lipopolysaccharide,LPS)对天然调节性T细胞(natural regulatory T cells,n Tregs)抑制功能的影响及其机制。采用磁珠分选健康人外周血中n Tregs,并用流式细胞术检测分选纯度后进行原代细胞培养。将不同浓度的LPS(10μg/m L,1μg/m L,0.1μg/m L)分别加入抗TLR4单抗封闭的n Tregs(即anti-TLR4+LPS组)和正常n Tregs(即Non anti-TLR4组)两组细胞、同时设定不加LPS作为对照组。采用实时定量PCR和酶联免疫吸附(ELISA)检测各组n Tregs中IL-10、TGF-β的m RNA表达水平和细胞培养上清液中蛋白含量。采用CFSE法流式细胞术检测不同浓度LPS处理的n Tregs与CD4+T效应细胞混合培养后CD4+T细胞的增殖水平。Western blotting检测LPS刺激后n Tregs的胞浆及胞核中NF-κBP65蛋白表达水平。结果分选得到的n Tregs纯度82%(84.52±2.10)%。Non anti-TLR4组n Treg细胞中IL-10的m RNA表达水平和培养上清液中蛋白含量均较对照组和anti-TLR4组显著增加(p0.05),且anti-TLR4组n Treg细胞IL-10的m RNA和培养上清液蛋白含量均较对照组显著降低(p0.05);Non anti-TLR4组n Treg细胞中TGF-β的的m RNA表达水平和培养上清液中蛋白含量均较对照组和anti-TLR4组均显著降低(p0.05),且anti-TLR4组n Treg细胞中TGF-β的m RNA表达水平和培养上清液中蛋白含量较对照组显著增高(p0.05)。n Tregs可显著抑制CD4+T细胞的增殖,且与CD3/CD28抗体活化的阳性对照组相比有差异(p0.05)。Non anti-TLR4组中,CD4+T细胞增殖指数较对照组显著降低(p0.05);anti-TLR4组中,CD4+T细胞增殖指数较阴性对照组显著增加(p0.05),较阳性对照组亦明显增加(p0.05)。1μg/m L LPS刺激后,Non anti-TLR4组n Tregs胞浆蛋白中NF-κBp65的含量较对照组降低,而胞核中则相应增加;anti-TLR4组则无明显变化。因此本研究认为,n Tregs膜型TLR4与LPS相互结合,可促使n Tregs抑炎性因子(IL-10,TGF-β)m RNA表达水平和分泌含量产生变化,可增强对效应性CD4+T细胞增殖的抑制功能,其变化机制可能与NF-κB信号分子活化相关。     

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.     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

Identification and Characterization of the First Equine Parainfluenza Virus 5

正Dear Editor,Parainfluenza virus 5 (PIV5), known as canine parainfluenza virus in the veterinary field, is a negative-sense,nonsegmented, single-stranded RNA virus belonging to the Paramyxoviridae family (Chen 2018). The virus was first reported in primary monkey kidney cells in 1954 (Hsiung1972), then it has been frequently discovered in various