多发性骨髓瘤患者骨髓单个核细胞Treg、Th17和血清IL-6、IL-10与临床分期以及治疗效果的关系分析

摘要 目的：探讨多发性骨髓瘤（MM）患者骨髓单个核细胞调节性T细胞（Treg）、辅助性T细胞（Th17）和血清白细胞介素-6（IL-6）、白细胞介素-10（IL-10）与临床分期以及治疗效果的关系。方法：选择2016年3月至2020年12月河北医科大学第一医院收治的MM患者60例为研究对象,检测并对比不同Durie-Salmon分期患者的骨髓单个核细胞Treg、Th17、Treg/Th17及血清IL-6、IL-10水平;患者入院后均给予常规治疗,根据疗效分为有效组和无效组,比较两组治疗前后骨髓单个核细胞Treg、Th17、Treg/Th17及血清IL-6、IL-10水平;分析Treg、Th17、Treg/Th17及血清IL-6、IL-10与MM患者Durie-Salmon分期、治疗效果的相关性。结果：MM患者骨髓单个核细胞Treg、Treg/Th17及血清IL-6、IL-10水平III期组高于II期组,II期组高于I期组（P＜0.05）。有效组治疗后骨髓单个核细胞Treg、Treg/Th17水平及血清IL-6、IL-10水平较治疗前明显降低（P＜0.05）;治疗后,骨髓单个核细胞Treg、Treg/Th17及血清IL-6、IL-10水平无效组高于有效组（P＜0.05）。骨髓单个核细胞Treg、Treg/Th17及血清IL-6、IL-10水平与MM患者Durie-Salmon分期呈正相关,与治疗效果呈负相关（P＜0.05）;骨髓单个核细胞Th17水平与MM患者的Durie-Salmon分期、治疗效果无明显的相关性（P＞0.05）。结论：骨髓单个核细胞Treg、Treg/Th17水平及血清IL-6、IL-10水平与MM患者肿瘤临床分期、治疗效果密切相关,检测其水平可对MM的临床治疗及预后起到一定评估作用。     

抗人IL-6单克隆抗体的制备及鉴定

用基因重组人IL-6免疫Balb/c小鼠,采用小鼠杂交瘤技术,筛选克隆到分泌抗人重组IL-6单克隆抗体的杂交瘤细胞株,并对其中2H₂、 1D₂ 和4B₄瘤细胞株进行了鉴定.其抗体类别均为IgG,亚类分别为IgG1和IgG2a.用多种细胞因子和无关蛋白的鉴别试验结果证实它们均特异地识别rhIL-6.免疫转染结果显示,该单抗识别分子质量为21 ku的IL-6单一条带.IL-6单克隆抗体的亲和常数K_aff= 1.62×10⁹ (mol/L)^-1.     

辅助性T细胞17的研究进展

CD4 T细胞根据释放细胞因子不同可分为不同的亚型:辅助性T细胞(T helper cells, Th)1、Th2和调节性T细胞(regulatory T cells, Treg).最近发现了一种新的CD4 T细胞亚型:以分泌白细胞介素-17为主要特征的Th17细胞.Th17细胞的发现对CD4 T细胞分化、Th细胞漂移等传统理论提出了挑战,同样也为感染、免疫性疾病提供了新的研究思路,尤其是Treg、Th1、Th2和Th17细胞在体内的平衡对于维持正常免疫应答反应有重要意义.     

TGF-β与IL-6介导的骨髓基质干细胞对EAE的双向调节作用研究

目的:探讨在实验性自身免疫性脑脊髓炎(EAE)治疗中TGF-β与IL-6介导的骨髓基质干细胞(BMSCs)的双向调节作用与机制。方法:分离纯化BMSCs,并与EAE大鼠动物模型淋巴细胞共培养,应用抗TGF-β和IL-6单克隆抗体封闭TGF-β或IL-6通路,ELISA检测不同比例MSC与淋巴细胞共培养中对细胞因子的作用,流式细胞仪检测MSC对Th细胞亚群分化的影响,过继免疫临床评分检测BMSC治疗中的关键通路。结果:1:10比例BMSCs共培养组与对照组相比,IL-17分泌量下降(P0.05),Treg细胞显著增高(P0.05)而Th17细胞显著降低(P0.05),共培养后的淋巴细胞进行过继免疫回输后,1:10干细胞共培养组临床评分显著降低(P0.05),而1:100比例共培养组与上述结果相反,中和TGF-β和IL-6抗体可以调节此免疫作用。结论:BMSCs通过TGF-β和IL-6通路调节免疫系统调节性T细胞(Treg)和Th17的细胞平衡,此过程与BMSCs剂量密切相关,本研究可为干细胞更好的临床应用提供理论基础。     

MiRNA在自身免疫性心肌炎中的研究进展

自身免疫性心肌炎(autoimmune myocarditis,AMC)常因机体的细胞和体液免疫功能障碍，引发心肌组织周围炎症细胞浸润，诱发自身免疫反应，进而逐渐发展为慢性损伤和扩张性心肌病，严重影响患者的预后。微小核糖核酸(microRNA,miRNA)常在自身免疫性心肌炎中表达失调，调控基因表达或免疫细胞激活与分化，参与调节心肌细胞的生长和凋亡。本文分别从miRNA在自身免疫性心肌炎中的潜在作用机制、诊断及预后的生物标志物及治疗方面，对miRNA在自身免疫性心肌炎中的研究进展及存在的问题进行简要阐述。     

HPV感染患者阴道微生态、Th17/Treg细胞及相关细胞因子表达分析CSCD

目的 分析人乳头瘤病毒（HPV）感染患者阴道微生态、辅助性T细胞17(Th17)/调节性T细胞（Treg）及相关细胞因子表达情况,为该类患者的治疗提供参考。方法 选取2020年5月至2022年6月厦门大学附属妇女儿童医院收治的HPV感染患者108例作为研究组,另选取我院同期健康体检者108例作为对照组。两组受试者均进行HPV筛查、阴道菌群检测以及阴道微生态检测,采用流式细胞仪检测Th17/Treg细胞;采用酶联免疫吸附法（ELISA）检测白细胞介素-6(IL-6)、 IL-10和IL-17水平;采用Spearman法分析HPV感染患者阴道菌群和Th17/Treg细胞及相关细胞因子的相关性。结果 HPV共检出9种亚型,主要以HPV 16和HPV 18为主。研究组患者阴道乳杆菌阳性率显著低于对照组,衣原体、解脲支原体、滴虫和细菌性阴道病阳性率均显著高于对照组（均P<0.05）。研究组患者阴道pH>4.5、阴道菌群密集度Ⅱ～Ⅳ级、阴道菌群多样性Ⅱ～Ⅳ级和微生态失调率均显著高于对照组（均P<0.05）。研究组患者Th17/Treg、IL-6、IL-10和IL-17水平显著高于对照组（均P<0.05）。Spearman相关性分析显示,阴道中乳杆菌与Th17/Treg、IL-6、IL-10和IL-17水平均呈负相关;衣原体、解脲支原体、滴虫和细菌性阴道病与Th17/Treg、IL-6、IL-10和IL-17水平均呈正相关（均P<0.05）。结论 HPV感染患者存在阴道微生态失调,而且Th17/Treg细胞、IL-6、IL-10和IL-17水平均异常升高。     

白细胞介素-18在部分自身免疫性疾病中的作用

白细胞介素-18(interleukin-18,IL-18)是一个多肽片段,具有促进T细胞增殖、增强细胞毒性T细胞和自然杀伤细胞活性等功能。自身免疫性疾病是指机体对自身抗原发生免疫反应而导致自身组织损害所引起的疾病。IL-18在免疫调节中有着高效、广泛的作用,部分自身免疫性疾病发病与免疫系统的异常会伴随IL-18的高表达,且不同病程IL-18表达量会有规律性变化,因此IL-18可作为一种自身免疫性疾病较敏感的标志物。现对IL-18在常见自身免疫性疾病如系统性红斑狼疮、成人Still病、自身免疫性心肌炎和类风湿关节炎中的作用作一综述。     

Th17/Treg细胞及其失衡在乙型肝炎致病机制中的作用

我国是乙型肝炎病毒(HBV)高感染率国家,乙型肝炎发病机制十分复杂,宿主免疫调节紊乱是导致不能有效清除病毒、病情迁延不愈的重要原因,其中CD4+T淋巴细胞发挥主要作用。最近,新发现的CD4+T细胞的几种亚群为乙型肝炎致病机制的研究提供了新思路。这些新的T细胞亚群中,有一种被称为Th17细胞,表达转录因子ROR-γt,并分泌各种IL-17因子参与免疫反应。另一种为Treg细胞,表达转录因子Fox P3,主要分泌TGF-β因子,当TGF-β单独存在时,初始的效应T细胞分化为Treg细胞。辅助性Th17细胞(Th17)和调节性T细胞(regulatory T cell,Treg)在分化发育、增殖及功能上有着密切的联系,并参与乙型肝炎的致病过程,对乙型肝炎的发生、发展、及愈后有一定影响。最近的研究表明,Th17/Treg的失调可能参与了乙型肝炎的异常免疫反应,从而导致慢性炎症的形成和HBV的持续感染。本文就Th17细胞和Treg细胞及其失衡在乙型肝炎致病机制中的作用予以综述,为乙型肝炎的免疫学治疗提供理论基础。     

IL-37对粉尘螨诱导气道上皮及固有免疫细胞产生IL-6的作用研究

目的探讨气道上皮细胞及固有免疫细胞经粉尘螨刺激后,白细胞介素37(interleukin 37,IL-37)对其产生细胞因子IL-6的影响。方法体外培养人肺泡上皮细胞系A549、小鼠肺上皮细胞系MLE-12、小鼠巨噬细胞系RAW264.7和原代小鼠固有淋巴样2型细胞(ILC2细胞),当细胞融合度达70%时,用IL-37预处理2 h,再给予粉尘螨粗提物刺激细胞,每种细胞均设PBS、IL-37及粉尘螨对照,并分别于不同时间点收集细胞沉淀和细胞培养液上清;用实时聚合酶链反应(real-time polymerase chain reaction,real-time PCR)法检测细胞因子IL-6在mRNA水平上的变化,用双抗体夹心ELISA检测细胞因子IL-6在蛋白水平的变化,用流式细胞术分选ILC2细胞并检测ILC2细胞表面IL-37受体的表达情况。结果粉尘螨粗提物可促进A549细胞、MLE-12细胞、RAW264.7细胞和ILC2细胞表达I L-6,且呈时间依赖的方式(P0.05)。IL-37可抑制粉尘螨粗提物所诱导的IL-6在A549细胞、MLE-12细胞和RAW264.7细胞表达(P0.05);但IL-37对粉尘螨粗提物刺激ILC2细胞分泌IL-6无明显抑制作用(P0.05)。结论IL-37可抑制粉尘螨诱导的气道上皮细胞和巨噬细胞产生IL-6,并可通过负向调控下调气道炎症反应,为IL-37在哮喘治疗中的潜在应用提供了实验依据。     

益生菌对自身免疫病的作用及机制研究现状

自身免疫病是机体免疫功能紊乱而导致组织器官受损的一类疾病,包括类风湿关节炎、系统性红斑狼疮、多发性硬化症、自身免疫性肝炎等。糖皮质激素及免疫抑制剂是治疗自身免疫病的常用药物,但长期使用会产生代谢紊乱、免疫低下、继发感染等副作用。随着肠道菌群与自身免疫病相关研究的进展,益生菌干预自身免疫病成为一大研究热点。研究证实,益生菌缓解自身免疫病安全有效,有望成为辅助疗法甚至替代疗法。本文就益生菌缓解类风湿关节炎、系统性红斑狼疮、多发性硬化症、自身免疫性肝炎等的作用及相关机制进行综述。     

人IL17-RD胞外区真核表达及其单克隆抗体的制备

为了进一步研究白介素17受体D (IL-17RD) 在IL-17信号的调节作用,探索是否可以通过单克隆抗体阻断IL-17RD介导的IL-17信号通路而缓解自身免疫疾病,利用昆虫表达载体从Sf9细胞中表达纯化人IL-17RD-ECD蛋白,免疫Balb/C小鼠30 d,取小鼠脾脏细胞并与小鼠骨髓瘤细胞SP2/0进行融合,应用有限稀释法进行筛选,经过克隆化后筛选到一株能稳定分泌抗IL-17RD-ECD的杂交瘤细胞株1F8。经过初步鉴定,该细胞株分泌的抗体类型为IgG1+kappa类,经过Western blot     

Atorvastatin ameliorates experimental autoimmune neuritis by decreased Th1/Th17 cytokines and up-regulated T regulatory cells

Statins have anti-inflammatory and immune-regulating properties. To investigate the effects of atorvastatin on experimental autoimmune neuritis (EAN), an animal model of Guillain–Barré syndrome (GBS), atorvastatin was administered to Lewis rats immunized with bovine peripheral myelin in complete Freund’s adjuvant. We found that atorvastatin ameliorated the clinical symptoms of EAN, decreased the numbers of inflammatory cells as well as IFN-γ⁺ and IL-17⁺ cells in sciatic nerves, decreased the CD80 expression and increased the number of CD25⁺Foxp3⁺ cells in mononuclear cells (MNC), and decreased the levels of IFN-γ in MNC culture supernatants. These data provide strong evidence that atorvastatin can act as an inhibitor in EAN by inhibiting the immune response of Th1 and Th17, decreasing the expression of co-stimulatory molecule, and up-regulating the number of T regulatory cells. These data demonstrated that statins could be used as a therapeutic strategy in human GBS in future.     

Cardioprotective effects of recombinant human erythropoietin in rats with experimental autoimmune myocarditis

Erythropoietin (EPO) has been known to have cytoprotective effects on several types of tissues, presumably through modulation of apoptosis and inflammation. The effect of EPO on myocardial inflammation, however, has not yet been clarified. We investigated the cardioprotective effects of EPO in rats with experimental autoimmune myocarditis (EAM). Seven-week-old Lewis rats immunized with cardiac myosin were treated either with EPO or vehicle and were examined on day 22. EPO attenuated the functional and histological severity of EAM along with suppression of mRNAs of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 in the hearts as well as a reduction of apoptotic cardiomyocytes. The EPO receptor (EPO-R) was upregulated in the myocardium of EAM compared with that of healthy rats. These results may suggest that EPO ameliorated the progression of EAM by modulating myocardial inflammation and apoptosis.     

Probing binding mechanism of interleukin-6 and olokizumab: in silico design of potential lead antibodies for autoimmune and inflammatory diseases

Computer-aided antibody engineering has been successful in the design of new biologics for disease diagnosis and therapeutic interventions. Interleukin-6 (IL-6), a well-recognized drug target for various autoimmune and inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, and psoriasis, was investigated in silico to design potential lead antibodies. Here, crystal structure of IL-6 along with monoclonal antibody olokizumab was explored to predict antigen–antibody (Ag???Ab)-interacting residues using DiscoTope, Paratome, and PyMOL. Tyr56, Tyr103 in heavy chain and Gly30, Ile31 in light chain of olokizumab were mutated with residues Ser, Thr, Tyr, Trp, and Phe. A set of 899 mutant macromolecules were designed, and binding affinity of these macromolecules to IL-6 was evaluated through Ag???Ab docking (ZDOCK, ClusPro, and Rosetta server), binding free-energy calculations using Molecular Mechanics/Poisson Boltzman Surface Area (MM/PBSA) method, and interaction energy estimation. In comparison to olokizumab, eight newly designed theoretical antibodies demonstrated better result in all assessments. Therefore, these newly designed macromolecules were proposed as potential lead antibodies to serve as a therapeutics option for IL-6-mediated diseases.     

A critical role of LFA-1 in the development of Th17 cells and induction of experimental autoimmune encephalomyelytis

Snu13p is a Saccharomyces cerevisiae protein essential for pre-messenger RNA splicing and pre-ribosomal RNA processing. Snu13p binds U4 snRNA of the spliceosome and box C/D snoRNAs of the pre-ribosomal RNA processing machinery to induce assembly of each ribonucleoprotein complex. Here, we present structural and biochemical analysis of Snu13p. The crystal structure of Snu13p reveals a region of the protein which could be important for protein interaction during ribonucleoprotein assembly. Using the structure of Snu13p we have designed the first temperature-sensitive mutants in Snu13p, L67W and I102A. Wild-type and mutant Snu13p proteins were assayed for binding to U4 snRNA and U3 snoRNA. Both temperature-sensitive mutants displayed significantly reduced RNA binding compared to wild-type protein. As the temperature-sensitive mutations are not in the known RNA binding region of Snu13p this indicates that these mutants indirectly influence the RNA binding properties of Snu13p. This work provides insight into Snu13p function during ribonucleoprotein assembly.     

Anti-IL-17A blocking antibody reduces cyclosporin A-induced relapse in experimental autoimmune encephalomyelitis mice

Cyclosporin A (CsA) is effective at reducing pathogenic immune responses, but upon withdrawal of CsA the immune response often “rebounds” resulting in a relapse or exacerbation of disease. The mechanisms, cells and cytokines involved in the relapse or exacerbation after CsA withdrawal are unknown. We hypothesized that CsA withdrawal induces IL-17 production that could be responsible for relapse, and examined the effect of anti-IL-17A antibody on relapse induced after CsA withdrawal in mouse experimental autoimmune encephalomyelitis (EAE). CsA treatment markedly decreased the EAE disease score during the first episode, but augmented disease severity after CsA withdrawal, compared to untreated mice. After discontinuation of CsA the production of IL-17A was increased and the severity of relapse in EAE was reduced by treatment with anti-IL-17A antibody. These results suggest that the resumption of T cell immune responses after CsA withdrawal leads to a burst of IL-17A production that is at least partially responsible for relapse in EAE mice.     

IL-21单克隆抗体对MRL/lpr狼疮小鼠的治疗作用*

目的: 探讨IL-21单克隆抗体对MRL/lpr狼疮小鼠的免疫治疗作用。方法: 将20只MRL/lpr狼疮小鼠随机分为模型组和治疗组,每组10只;同年龄同性别C57BL/6小鼠10只作为正常组。治疗组小鼠每周腹腔注射IL-21单克隆抗体(100 μg),正常组及模型组小鼠每周腹腔注射等量生理盐水(100 μg),连续干预8周。干预结束后观察小鼠皮毛、活动等一般性状及浅表淋巴结大小,并采集小鼠血液、尿液及肾脏标本。采用Western blot法检测三组小鼠肾组织中IL-21蛋白表达情况;采用ELISA法比较三组小鼠血清抗ds-DNA抗体、ANA抗体、血尿素氮、肌酐和炎症因子IL-17A、TGF-β1水平;采用生物化学法比较三组小鼠24 h尿蛋白水平。结果: 与正常组比较,模型组小鼠肾组织IL-21、血清抗ds-DNA、ANA抗体水平、尿素氮、肌酐、IL-17A浓度及24 h尿蛋白水平均升高(P＜0.05),TGF-β1浓度降低(P＜0.01);与模型组比较,治疗组小鼠肾组织IL-21、血清抗ds-DNA、ANA抗体水平、尿素氮、肌酐、IL-17A浓度、24 h尿蛋白水平均降低(P＜0.05),TGF-β1浓度升高(P＜0.01)。结论: 腹腔注射IL-21单克隆抗体可改善MRL/lpr狼疮小鼠免疫功能与肾损害,提示治疗机制可能与重塑Th17/Treg相关细胞因子平衡有关。     

Successful selection of an infection‐protective anti‐Staphylococcus aureus monoclonal antibody and its protective activity in murine infection models

Recent clinical trials to develop anti‐methicillin‐resistant Staphylococcus aureus (MRSA) therapeutic antibodies have met unsuccessful sequels. To develop more effective antibodies against MRSA infection, a panel of mAbs against S. aureus cell wall was generated and then screened for the most protective mAb in mouse infection models. Twenty‐two anti‐S. aureus IgG mAbs were obtained from mice that had been immunized with alkali‐processed, deacetylated cell walls of S. aureus. One of these mAbs, ZBIA5H, exhibited life‐saving effects in mouse models of sepsis caused by community‐acquired MRSA strain MW2 and vancomycin‐resistant S. aureus strain VRS1. It also had a curative effect in a MW2‐caused pneumonia model. Curiously, the target of ZBIA5H was considered to be a conformational epitope of either the 1,4‐β‐linkage between N‐acetylmuramic acid and N‐acetyl‐D‐glucosamine or the peptidoglycan per se. Reactivity of ZBIA5H to S. aureus whole cells or purified peptidoglycan was weaker than that of most of the other mAbs generated in this study. However, the latter mAbs did not have the protective activities against S. aureus that ZBIA5H did. These data indicate that the epitopes that trigger production of high‐yield and/or high‐affinity antibodies may not be the most suitable epitopes for developing anti‐infective antibodies. ZBIA5H or its humanized form may find a future clinical application, and its target epitope may be used for the production of vaccines against S. aureus infection.     

Production and characterization of a monoclonal antibody for Pefloxacin and mechanism study of antibody recognition

In this report, an artificial antigen (PFLX–BSA: Pefloxacin connected bovine serum albumin) was successfully prepared. The monoclonal antibody against pefloxacin was produced and characterized using a direct competitive ELISA. The linear range of detection was 0.115–6.564 µg/L. The limit of detection defined as IC₁₅ was 0.170 ± 0.05 µg/L and the IC₅₀ was 0.902 ± 0.03 µg/L. The antibody variable region genes were amplified, assembled, and sequenced. A three–dimensional structural model of the variable region was constructed to study the mechanism of antibody recognition using molecular docking analysis. Three predicted essential amino acids, Thr53, Arg97 of heavy chain and Thr52 of light chain, were mutated to verify the theoretical model. Three mutants lost binding activity signi?cantly against pefloxacin as predicted. These may provide useful insights for studying antigen–antibody interaction mechanisms to improve antibody affinity maturation in vitro.