硫酸化人参总皂苷对人工感染MDV鸡淋巴细胞活性的调节

鸡马立克氏病病毒（MDV）经腹腔注射感染8日龄雏鸡,攻毒15 d后检测硫酸化人参总皂苷对鸡外周血白细胞数和淋巴细胞增殖活性的影响,并利用半定量RT-PCR方法分析外周血淋巴细胞IFN-γmRNA的表达水平。结果表明,MDV感染能引起鸡淋巴细胞百分比相对增多（P〈0.01）,人参总皂苷及其硫酸化人参总皂苷不能改变MDV所致变化（P〉0.05）,且硫酸化人参总皂苷与人参总皂苷之间没有差异（P〉0.05）;MDV感染极显著抑制淋巴细胞增殖（P〈0.01）,人参总皂苷能明显改善MDV对感染鸡淋巴细胞增殖的抑制（P〈0.01）,硫酸化人参总皂苷促进了MDV所致的抑制状态（P〈0.01）;人参总皂苷及其硫酸化人参总皂苷均能增强MDV所致鸡淋巴细胞IFN-γmRNA的表达,与健康组相比,硫酸化人参总皂苷极显著增加IFN-γmRNA表达（P〈0.01）。结果提示,硫酸化人参总皂苷对人工感染MDV鸡的淋巴细胞活性具有调节作用,其作用比人参总皂苷的作用更强。     

表达鸡IL-12重组乳酸乳球菌的构建及其生物学活性检测

鸡白细胞介素12(ChIL-12)是由IL-12p40和IL-12p35两个亚基组成的一个异源二聚体,具有促进T淋巴细胞和自然杀伤细胞(NK cell)成熟的作用,能够作为黏膜疫苗佐剂。本研究中采用公认的食品级乳酸乳球菌表达系统构建表达目的融合蛋白Ch IL-12p40-(G_4S)_3ChIL-12p35(rChIL-12),并经western blot检测。结果显示:目的融合蛋白呈可溶性状态表达。体外鸡淋巴细胞刺激试验显示,经rChIL-12处理的鸡脾淋巴细胞能够产生干扰素γ(IFN-γ),表明该融合蛋白具有诱导IFN-γ产生的生物学活性。本研究结果为进一步利用rChIL-12作为粘膜疫苗佐剂的研发奠定了基础。     

海兰鸡IL-18全基因的克隆与序列分析

IL-18是一种能诱导产生IFN-γ的新型细胞因子,在调节Th1型细胞免疫应答中起重要作用。根据GenBank发表的鸡IL-18cDNA基因序列,自行设计一对引物,经植物血凝素(PHA)活化60日龄海兰鸡的脾淋巴细胞后,提取其总RNA,经反转录-聚合酶链反应(RT-PCR)扩增,扩增产物进行了T-A克隆、测序,获得了中国海兰鸡IL-18基因全序列,其大小为597 bp,与GenBank中查得的鸡IL-18基因进行比较发现,中国海兰鸡IL-18基因与Schneider报道的鸡IL-18基因序列完全一致,为进一步研究鸡IL-18基因表达、生物学活性和应用奠定了基础。     

禽网状内皮组织增生病病毒和呼肠孤病毒感染SPF鸡对IFN-γ产生的影响

1日龄SPF鸡分别接种禽网状内皮组织增生病病毒（REV）、禽呼肠孤病毒（ARV）和REV＋ARV,于感染后3、5、46 d采集脾细胞做脾淋巴细胞培养并采用双抗体夹心ELISA法检测IFN-γ水平。结果表明,与对照相比,鸡感染REV后脾淋巴细胞IFN-γ的分泌水平显著升高。感染后5 d,REV单独感染引起IFN-γ8倍的增长,达到峰值,然后逐渐下降。ARV单独感染引起IFN-γ2倍的增长。REV和ARV共感染鸡脾淋巴细胞IFN-γ的分泌量增长了3倍。至感染后46 d,共感染和REV单独感染的IFN-γ水平与对照组相比仍然差异显著,ARV单独感染与对照组相比差异不再显著。     

混饮低剂量ChlFN-α对肉鸡细胞免疫功能及细胞因子水平的影响

本实验研究低剂量鸡α-干扰素(ChtFN-α)对内鸡细胞免疫及细胞因子水平的影响.15日龄肉鸡连续混饮0.1 IU/mL、1 IU/mL和10 IU/mL剂量的ChIFN-α,分别在混饮后14 d和21 d翼静脉采取血样,采用中性红.结晶紫联合测定白细胞总数,经典方法进行白细胞分类计数,MTT比色分析法测定淋巴细胞转化能力,LDH释放法测定NK细胞的杀伤活性,双夹心ELISA法测定血清中IFN-γ、IL-4、IL-12水平.结果显示肉鸡连续混饮ChIFN-α后,能显著提高白细胞总数、淋巴细胞百分比和血清中IFN-γ水平,对IL-4、IL-12的浓度水平影响不显著,但提高IFN-γ/IL-4的比例,对淋巴细胞增殖和NK细胞的杀伤活性影响不明显.试验表明,混饮低剂量ChlFN-α后可调节肉鸡的细胞免疫功能及细胞因子水平,增强肉鸡的抗病能力.     

G2系和G5系鸡感染马立克氏病毒早期细胞因子转录水平的变化

为研究主要组织相容性抗原(MHC)不同的G2系和G5系鸡在感染马立克氏病毒(MDV)后立即早期病毒复制及细胞因子转录水平的动态变化,本实验对14日龄G2和G5系SPF鸡滴鼻接种超强毒(vv MDV)Md5株,以28S r RNA基因为内参,利用双重荧光定量RT-PCR(dq RT-PCR)方法对接种后4 h、24 h、48 h和96 h肺淋巴细胞、脾脏和外周血淋巴细胞(PBL)中MDV meq、IFN-γ、IL-18、IL-4和IL-10的m RNA含量进行检测。结果显示,接种后96 h仅在两品系感染鸡的肺淋巴细胞和脾脏中检测到了meq m RNA,G2系鸡群肺淋巴细胞中病毒m RNA含量高于G5系,但脾脏中较低。96 h肺淋巴细胞和脾脏中IFN-γ、IL-4和IL-10转录水平大幅上调,并且G2系鸡群均略高于G5。本研究结果提示G2系对MDV的抵抗能力强于G5,这两种品系鸡感染MDV后可能主要启动以Th2型为主的细胞免疫反应,为研究MDV对不同MHC单倍型鸡的致病性提供了实验依据。     

海兰鸡白细胞介素-18全基因的克隆与序列分析

白细胞介素-18（Interleukin-18，IL-18）是一种能诱导产生IFN-γ的新型细胞因子，在调节Th1型细胞免疫应答中起重要作用。根据GenBank发表的鸡IL-18cDNA基因序列，自行设计一对引物，经植物血凝素（PHA）活化60日龄海兰鸡的脾淋巴细胞后，提取其总RNA，经反转录-聚合酶链反应（RT—PCR）扩增，扩增产物进行了T-A克隆、测序，获得了中国海兰鸡IL-18基因全序列，其大小为597bp，与在GenBank中查得的鸡IL-18基因进行比较发现，中国海兰鸡IL-18基因与Schneider报道的鸡IL-18基因序列完全一致，为进一步研究鸡IL-18基因表达、生物学活性和应用奠定了基础。     

1日龄雏鸡接种超抗原SEB和MDV后脾与胸腺淋巴细胞IFN-γ的体外诱生

为研究超抗原(SAg)在禽类诱导的免疫机理和进一步揭示MDV(马立克氏病病毒)感染的免疫学规律,采用NO-释放法,设计用雏鸡骨髓巨噬细胞为靶细胞检测1日龄雏鸡接种超抗原SEB和MDV后脾与胸腺淋巴细胞IFN-γ体外诱生的动态变化.结果发现,超抗原SEB(SAG-SEB)可提高脾与胸腺淋巴细胞IFN-γ体外诱生水平,其中,高剂量(1 ng/只)引起短时加强后下降,而低剂量(0.01 ng)则出现缓升缓降现象;而MDV接种则抑制脾与胸腺淋巴细胞IFN-γ体外诱生; SAg-SEB能增强雏鸡细胞免疫,主要表现于接种初期(1-10 d PI),而MDV却在接种后1-25 d间降低细胞免疫;说明超抗原SEB和MDV虽然均可引起鸡T细胞增殖,但对鸡免疫细胞IFN-γ的体外诱生作用不同.     

鸡白细胞介素18(IL-18)与临床疾病相关性的研究进展

白细胞介素18(IL-18)是一个系统的、多功能细胞因子,其能高水平诱生IFN-γ,增强NK细胞活性,增强Th1型细胞的免疫应答,在抗感染、抗肿瘤和免疫调节等方面起重要作用,且可作为免疫佐剂。文章就其与临床疾病相关的研究进展作一简要综述。     

禽呼肠病毒感染对SPF鸡外周血T细胞亚型变化和细胞因子转录的影响

为探讨禽呼肠病毒(ARV)对SPF鸡外周血淋巴细胞中CD4+、CD8+T细胞数量变化和细胞因子mRNA转录水平的影响,利用流式细胞术和实时荧光定量PCR方法分别测定了ARV感染后1、7、14、21、28、35d感染组和对照组SPF鸡外周血淋巴细胞中CD4+、CD8+T细胞含量和细胞因子IL-1β、IL-6、IL-17、IL-18、IFN-γ、TNF-α基因mRNA相对转录时相。流式细胞术检测结果表明,SPF鸡感染ARV后7d和14dCD4+、CD8+T细胞比值高于对照组,其中感染7d,CD8+T细胞含量差异显著(P0.05);感染后1、21、28、35d感染组CD4+、CD8+T细胞比值均低于对照组,感染1d后CD4+、CD8+T细胞含量均差异显著(P0.05),说明外周血T细胞亚型变化是ARV感染的重要表现之一。实时荧光定量PCR结果表明,与对照组相比,感染组外周血淋巴细胞中IL-1β、IL-6(除7d外)、IL-18(除14d外)和TNF-α在整个感染过程中表达上调,IL-17和IFN-γ除感染1d外,均表达下调,说明IL-1β、IL-6、IL-17、IL-18、IFN-γ和TNF-α均参与了ARV的感染进程。     

不同感染量REV对鸡免疫反应和细胞毒性作用的影响

用不同剂量网状内皮增生症病毒（REV）感染肉鸡和SPF鸡后，检测血液中T淋巴细胞对ConA的反应和NK细胞、细胞毒T细胞（CTL）的细胞毒性作用以及NDV抗体生成变化等，观察REV感染对机体非特异性、特异性细胞免疫反应和体液免疫反应的影响。结果表明无论高剂量还是低剂量REV感染均造成体液免疫和非特异性细胞免疫抑制，而且高剂量比低剂量对免疫功能的抑制作用更强，但对NK细胞和细胞毒T细胞的细胞杀伤活性却有升高趋势，在抗肿瘤方面发挥一定的作用。这种结果说明REV感染对机体的免疫抑制是有选择性的，且抑制程度与感染病毒量有关。     

不同感染剂量MDRV对番鸭免疫反应和细胞毒性作用的影响

用不同剂量番鸭呼肠孤病毒(MDRV MW9710株)感染8日龄番鸭后,通过检测血液中淋巴细胞对ConA、LPS的反应和NK细胞、细胞毒T细胞(CTL)的细胞毒性作用,探讨MDRV感染对番鸭免疫细胞功能和细胞毒性作用的影响。结果显示,不同感染剂量MDRV均会抑制番鸭血液淋巴细胞对ConA、LPS的增殖反应,降低NK细胞和CTL细胞的杀伤活性,且影响程度与剂量相关;同时感染番鸭生长缓慢,脾脏肿大,胸腺和法氏囊缩小。上述结果表明,MDRV感染能导致番鸭免疫抑制,且细胞免疫抑制程度与感染病毒量有关。     

Early cell-mediated immune responses to Marek's disease virus in two chicken lines with defined major histocompatibility complex antigens

N2a and P2a chickens, resistant and susceptible to Marek's disease (MD), respectively, were used to examine relationships between major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) and natural killer (NK)-like cell activity with resistance to infection with Marek's disease virus (MDV). Ten-day-old chickens were infected with MDV and euthanatized at selected times to evaluate for NK cell and MHC-restricted cytotoxicity. The N2a MDV-infected chickens had an early cell-mediated immune response characterized by a sustained NK-like cytotoxicity that coincided with a measurable MHC-cytotoxicity that was lower than controls. Although MHC-restricted and NK cell cytotoxicity was demonstrated in P2a MDV-infected chickens at 8 dpi, both abruptly decreased and remained low for the remainder of the 20-day experiment. The critical time point that may determine the resistance to MD appears to be within the first 2 weeks post-infection. Improvement of the chicken NK cell activity may be a good candidate for both selection and immunomodulation MD control programs.     

毒害艾美耳球虫初次感染对雏鸡外周血液免疫功能的影响

应用免疫SPA菌体花环、间接ELISA及细胞培养技术和四甲基偶氮唑盐(MTT)测定法对毒害艾美耳球虫(Eimerianecatrix)初次感染雏鸡外周血液的T、B细胞数量及其对ConA或PMA的增殖功能和IgG、IgM、IgA含量的动态变化进行了较全面系统的研究。结果发现,E.necatrix初次感染雏鸡外周血液的T、B细胞数量及其相应增殖功能、血清中上述三种免疫球蛋白含量均不同程度地高于未感染的对照雏鸡,表明E.necatrix初次感染雏鸡外周血液的细胞免疫和体液免疫功能均明显提高。     

Characterization of porcine xenoreactive cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) against mouse P815 cells were detected after stimulation of porcine peripheral blood mononuclear cells (PBMC) with irradiated Balb/c splenocytes. In vivo priming prior to in vitro stimulation slightly enhanced CTL activity, but lysis of targets was undetectable from lymphocytes from non-immune or immune animals that were not cultured with mouse splenocytes. After primary culture with Balb/c (H-2d) splenocytes, specific killing of P815 (H-2d) targets and not L929 (H-2k) targets indicated that recognition was specific for the H-2 locus. Similarly, CTL primed by mouse cells from either of two congenic strains recognized targets with alleles homologous to the stimulating cells. The anti-murine CTL was confirmed to be a CD8+ T cell based on studies using specific monoclonal antibodies to the porcine CD4 or CD8 cells. The cells responsible for the cytotoxicity of P815 targets lacked the characteristics of non-specific NK cells because (1) naive PBMC were unable to lyse NK targets (K562 cells) during the 4 h cytotoxic assay and (2) CTL killing of P815 targets increased with time after primary stimulation, whereas killing of K562 cells remained low at all times. These results suggest that porcine CTL can be readily generated against the xenogeneic mouse major histocompatibility complex.     

重组鸡白细胞介素-6/2融合蛋白对新城疫(LaSota株)活疫苗免疫增强作用研究

为探究重组鸡白细胞介素-6/2融合蛋白(rChIL-6-Linker-ChIL-2,重组融合蛋白)对新城疫病毒(NDV)活疫苗(LaSota株)的免疫增强作用,本研究将90只SPF鸡随机分为6组,分别为PBS对照组、NDV弱毒苗对照组、rChIL-6-Linker-ChIL-2免疫增强组、rChIL-6蛋白免疫增强组、rChIL-2蛋白免疫增强组及rChIL-6+rChIL-2混合蛋白免疫增强组,将鸡白细胞介素重组融合蛋白水剂与LaSota株联合接种于SPF鸡,分别采用MTS法、流式细胞术、ELISA和HA/HI法检测接种前后不同时间各组鸡外周血及脾淋巴细胞增殖活性、鸡外周血中CD3+CD4+/CD3+CD8+T淋巴细胞的百分含量、血清中Th1/Th2型细胞因子表达水平及免疫抗体水平等指标。结果显示,接种后7～21d时,与NDV弱毒苗对照组相比,同时接种重组融合蛋白组鸡淋巴细胞增殖活性、外周血CD3+CD4+/CD3+CD8+T淋巴细胞的百分含量比值及血清中重组鸡IL-4蛋白(rChIL-4)、ChIL-6、ChIL-2、重组鸡IFN-α蛋白(ChIFN-α)、ChIFN-γTh1/Th2型细胞因子表达水平明显提升;HI免疫抗体较NDV弱毒苗对照组提高了1.0～1.9个滴度,较单一rChIL-6、rChIL-2对照组分别提高了0.2～0.7、0.2～1.1个抗体滴度。综上所述,rChIL-6-Lin-ker-ChIL-2融合蛋白对NDV(LaSota株)活疫苗在鸡体内具有明显的免疫增强效果。     

Natural killer cell activity in chickens exposed to Marek's disease virus: inhibition of activity in susceptible chickens and enhancement of activity in resistant and vaccinated chickens

Chickens of 2 genetic lines (lines P and N) were inoculated with a pathogenic strain of Marek's disease (MD) virus (MDV) and chronologically examined for disease response and natural killer (NK) cell expression. The NK cell reactivity was assayed in an in vitro cytotoxicity assay in which effector cells from the spleen of test chickens were reacted with 51Cr-labeled LSCC-RP9 target cells. Chickens of line P developed progressive debilitating disease and a high incidence of gross tumors and death. The NK cell reactivity of line-P chickens infected with MDV was significantly lower than that of uninfected control hatchmates. In contrast, NK cell levels were significantly elevated in MDV-inoculated line-N chickens that were resistant to MD and in chickens of lines P or N that had been inoculated with herpesvirus of turkeys (HVT). NK cell levels were also elevated in line P if chickens were vaccinated with HVT before infection with MDV. Inhibition of NK reactivity in susceptible chickens and elevation of reactivity in naturally resistant or vaccinated chickens may indicate a role for the NK cell system in regulating resistance to MD.     

Induction of proinflammatory cytokines and caspase-1 by leptin in monocyte/macrophages from holstein cows

The proliferation of peripheral blood mononuclear cells (PBMC) containing both monocyte/macrophages and T lymphocytes increased after treatment with T-cell mitogen (concanavalin A: Con A). PBMC treated with either leptin alone or combination of leptin and ConA showed enhanced proliferative activity by 10-40%, compared with those treated with ConA alone. In contrast, isolated T lymphocytes treated with leptin and ConA showed lowered proliferative activity than the ConA-treated alone, indicating that leptin induced production of some cytokines from monocyte/macrophages, that subsequently resulted in enhancement of T lymphocytes proliferation in PBMC. Among the cytokines examined, monocyte/monocytes constitutively expressed interleukin (IL)-1beta, IL-12p35, IL-18 mRNA, and faintly expressed tumor necrosis factor (TNF)-alpha and IL-12p40 mRNA. Leptin treatment augmented the monocyte/macrophages mRNA expression of only TNF-alpha and IL-12p40 to comparable levels of cells treated with lipopolysaccharide (LPS). However, leptin treatment increased monocyte/macrophages production of IL-1beta as well as TNF-alpha, and induced the mRNA expression of caspase-1, which is shown to mediate the conversion of latent pro-IL-1beta and pro-IL-18 to active forms. These results suggest that leptin directly acts on monocyte/macrophages to produce factors that induce T lymphocytes proliferation such as IL-12p35/p40 complex through IL-12p40 induction and IL-1beta/IL-18 production through caspase-1 induction.     

AIV感染雏鸡免疫器官T细胞免疫功能及IL-2诱生活性变化

应用组织培养技术及MTT法,对鹅源H5N1型AIV感染雏鸡胸腺、脾脏T细胞增殖功能及IL-2诱生活性进行检测,结果发现:雏鸡感染AIV后,免疫器官T细胞增殖功能和IL-2诱生活性在感染初期较对照雏鸡明显降低,后期有所恢复,表明AIV感染雏鸡细胞免疫受到抑制,中枢与外周免疫器官分子免疫调节功能障碍.7和21日龄AIV感染雏鸡均发病,但21日龄感染雏鸡上述指标的下降时间较7日龄感染雏鸡短且相对滞后,死亡率也较低,表明21日龄雏鸡免疫能力较强,具有一定的抗AIV感染能力.     

A colorimetric assay to evaluate the cytotoxic activity of the intestinal intraepithelial lymphocytes of chickens

After the successful use of 3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) in cell proliferation assays, its use has been established by different workers in cytotoxicity assays and research on leukaemia. In the present study, a colorimetric assay using MTT was adopted to evaluate the cytotoxic activity of chicken intestinal intraepithelial lymphocytes (iIELs), which constitute an important cellular component of the gut-associated lymphoid tissue (GALT). These iIELs are found to exhibit natural killer (NK) cell-like cytotoxic activity, which is spontaneous, non-MHC-restricted, and does not need to be primed. Hitherto, conventional chromium-release assays have been used to evaluate the cytotoxic activity of iIELs, but these assays have disadvantages such as radiation hazards and loss of the cells in washing steps. The mean percentage cytotoxic activity of chicken iIELs evaluated by the colorimetric assay was 90.37±2.53 in a group of 5-week-old chickens and 80.2±3.45 in a group of 8-week-old chickens. These findings established the successful use of a colorimetric assay using MTT for evaluating the cytotoxic activity of chickens iIELs.Abbreviations DMEM Dulbecco's modified Eagle's medium - DMSO dimethyl sulphoxide - E effector cells - GALT gut-associated lymphoid tissue - GM growth medium - iIELs intestinal intraepithelial lymphocytes - MTT 3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide - NK cell natural killer cell - OD optical density - RPMI Rosewell Park Memorial Institute medium - T target cells