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1.
目的:检测慢性移植性肾病(CAN)大鼠脾脏中辅助性T细胞(Th)和B细胞特征性因子表达量的变化,探究这些Th/B细胞免疫状态在CAN病程中的作用。方法:采用Fischer-Lewis左肾原位移植法建立大鼠慢性移植性肾病模型,Lewis-Lewis同种自体移植作为对照组。所有受体大鼠,术后8周处死,取脾脏组织,进行HE染色,拍照后采用双盲法评价脾脏组织病理变化程度及淋巴细胞浸润情况。用Trizol法提取脾脏组织中总RNA,采用实时荧光定量聚合酶链式反应(q RT-PCR)法检测各组脾脏中Th1,Th2,Th17,Treg和B细胞标志性因子的表达情况。结果:与对照组相比,CAN大鼠脾脏出现明显的结构肿胀及淋巴细胞浸润增多,并且Th1细胞特征性因子IFN-γ和T-bet表达量显著增加(P0.001,P0.05);Th2细胞特征性因子GATA-3表达升高(P0.001),但IL-4无变化;IFN-γ/IL-4比例明显上调(P0.001),T-bet/GATA3比例没有显著差异。Th17的特征性因子IL-17未见明显改变,而Treg细胞特征性因子Foxp3表达增加(P0.001),IL-17/Foxp3平衡明显向Treg细胞偏移(P0.05)。B细胞激活相关因子TNFRSF13C和RAG1表达量均显著上调(P0.01,P0.05),而RAG2水平则没有变化。结论:CAN大鼠脾脏中Th1/Th2的活性平衡向Th1偏移,分化平衡未出现显著变化;Th17/Treg的平衡向Treg细胞偏移,B细胞免疫状态也被激活,这些变化在CAN病程的发展中起到了重要作用,并且为临床监测和治疗提供了新的依据。  相似文献   

2.
探讨低分子肝素钙对子痫前期大鼠炎性细胞因子的影响。将Wistar大鼠随机分为A组、B组、C组和D组共4组(n=10),A组和B组大鼠均在妊娠2周后连续5 d注射剂量为200 mg·kg~(-1)·d~(-1)的L-NAME,此后,A组连续5 d注射剂量为50μg·kg~(-1)·d~(-1)的低分子肝素钙注射液,B组连续5 d注射0.5 m L/d生理盐水。C组大鼠为正常妊娠组,妊娠2周后连续10 d注射0.5 m L/d生理盐水。D组大鼠为非妊娠对照组,用药过程中注射0.5 m L/d生理盐水。通过ELISA、免疫组化、荧光定量PCR、Western blotting检测各组大鼠外周血、胎盘、肾脏和脾脏中IFN-γ、IL-2、IL-4和IL-6炎性细胞因子水平的变化。结果显示,B组IFN-γ和IL-2在外周血、胎盘、肾脏和脾脏的表达水平明显高于C组,而IL-4和IL-6的表达水平均明显低于C组(p0.05);A组IFN-γ和IL-2的表达水平均明显低于B组,且IL-4和IL-6表达水平与B组相比,其表达水平明显升高(p0.05)。本研究所建立的动物模型可使Th1/Th2发生Th1方向漂移,可以用来模拟子痫前期的免疫失衡;低分子肝素钙可明显抑制促炎性细胞因子的分泌,促进抗炎细胞因子的分泌,可使Th1/Th2的平衡向Th2方向漂移,从而表现出良好的抗炎作用。  相似文献   

3.
为了探讨TLR2和TLR4对新生儿免疫细胞中Th1/Th2细胞因子的影响,本研究采用LPS和PGN刺激新生儿脐带血单个核细胞、成人外周血单个核细胞和人肥大细胞,并用特异性信号分子抑制剂PD98059处理单核细胞和肥大细胞,测定不同处理组ERK的磷酸化强度、IL-6、IL-12、IL-13和RANTES细胞因子水平以及HMC-1细胞脱颗粒情况。研究显示,经过LPS或PGN刺激后,单核细胞和肥大细胞中ERK的磷酸化强度与对照组相比均显著升高(p0.05);通过LPS和PGN共同刺激后,脐带血单个核细胞中ERK的磷酸化高于单个配体刺激。在添加了ERK特异性抑制剂PD98059后,LPS和PGN共刺激的脐带血单个核细胞中IL-6、IL-12和IL-13浓度显著下降,而RANTES未显示明显抑制。经PGN刺激后HMC-1细胞的β-hexosaminidase释放率高于LPS,并且LPS+PGN共刺激的β-hexosaminidase释放率高于单一配体刺激。本研究表明,TLR2/TLR4与LPS/PGN结合后,通过激活ERK信号通路来调节新生儿免疫细胞中Th1/Th2细胞因子,LPS和PGN共刺激对调节新生儿免疫细胞中IL-6、IL-12和IL-13细胞因子具有协同作用。  相似文献   

4.
目的:观察异源反应性自然杀伤细胞(NK细胞)对骨髓移植小鼠脾脏辅助性T细胞Th1/Th2亚群的影响。方法:以BALB/c和CB6F1小鼠为受体,在γ射线照射后通过尾静脉输入C57BL/6J小鼠的骨髓细胞和脾脏单个核细胞,建立移植物抗宿主病模型;然后输入供体的NK细胞,检测受体小鼠脾脏中Th1/Th2淋巴细胞亚群和外周血中γ干扰素(IFN-γ)、白细胞介素10(IL-10)的变化。结果:与单纯输入骨髓细胞和单个核细胞组小鼠相比,输入异源反应性NK细胞后,BALB/c和CB6F1小鼠脾脏中Th1细胞比例均下降,Th2细胞比例均上升,CB6F1小鼠脾脏中Th2虽有升高但没有统计学意义;外周血中IL-10水平显著升高,IFN-γ的水平显著下降。结论:异源反应性NK细胞可能通过降低脾脏中Th1细胞亚群比例和升高Th2细胞亚群比例减轻移植物抗宿主病。  相似文献   

5.
目的:探讨RAGE及其配体S100B在实验性自身免疫性重症肌无力(EAMG)中对T细胞的作用及其相关机制。方法:选取体重160-180g的Lewis大鼠,并将其随机分为EAMG模型组和CFA对照组。EAMG模型组大鼠通过尾根部注入200μL含有R-ACh R97-116肽段的免疫乳剂,并于初始免疫后的第30天尾根部追加免疫一次;CFA对照组大鼠为免疫乳剂中不含有R-ACh R97-116肽段。采用流式细胞术检测和比较两组大鼠CD4+T细胞上RAGE的表达情况;ELISA方法检测淋巴细胞培养上清中IFN-γ、IL-4、IL-17、TGF-β、IL-6和S100B的表达水平;采用S100B体外干预淋巴细胞,进一步检测S100B对EAMG大鼠T细胞增殖、亚型分布、细胞因子分泌的影响。结果:在疾病发生的晚期时相(初次免疫后第45天),EAMG组淋巴细胞CD4+T细胞上RAGE的表达明显高于CFA组(P0.001),血清中RAGE的配体S100B的表达也高于CFA组(P0.001);体外加入S100B干预能促进T淋巴细胞的增殖,与未干预组相比较差异显著(P0.05);S100B刺激后,Th1细胞和Th17细胞的百分比进一步增高,Th2细胞和Treg细胞百分比下降(PTh10.05,PTh170.05,PTh20.05,PTreg0.05),IFN-γ和IL-17的表达上调(PIFN-γ0.05,PIL-170.05),而IL-4和TGF-β表达下降(PTGF-β0.01,PIL-40.05),Th17细胞的调控因子IL-6表达升高(PIL-60.05)。结论:RAGE及其配体S100B参与EAMG的发病过程,在晚期时相中表现出明显的致病性;RAGE与S100B相互作用可以上调T细胞的致病性作用,加重四种辅助性T细胞之间的网络失衡。  相似文献   

6.
目的:分析侵袭性肺曲霉病患者辅助性T细胞(Th)以及调节性T细胞(Treg)在外周血中单个核细胞中的表达情况及其临床相关性,探讨Th和Treg细胞介导的免疫反应在侵袭性肺曲霉病中的作用。方法分离21例侵袭性肺曲霉病患者及19例健康人外周血的单个核细胞,采用流式细胞术分析Th1、Th2、Th17、Treg细胞群的表达情况,Real-timePCR方法检测相关转录因子T-bet、GATA-3、RORγt以及Foxp3的表达,ELISA法检测血清中相关细胞因子IFN-γ、IL-4、IL-17以及TGF-β的表达。结果与健康人对照组相比,侵袭性肺曲霉病患者Th1细胞以及Treg细胞占CD4+T细胞的比例较之对照组明显降低;Th1、Th17、Treg细胞相关转录因子T-bet、RORγt、Foxp3以及相关细胞因子IFN-γ、IL-17A、TGF-β与对照组相比表达明显降低。结论IPA患者的Th1、Th17以及Treg细胞所介导的免疫反应受抑制。  相似文献   

7.
目的:研究妊娠期肝内胆汁淤积症患者外周血中维生素D受体的表达与Th1/Th2型细胞因子干扰素-γ/白细胞介素-4(IFN-γ/IL-4)的变化关系,探讨ICP发病机制。方法:选取ICP患者31例(ICP组),孕周相匹配的正常孕妇31例(正常对照组)。采用酶联免疫吸附试验(ELISA法),检测两组孕妇血清中Th1型细胞因子(IFN-γ)和Th2型细胞因子(IL-4)的水平;采用实时荧光定量逆转录-多聚酶链反应(qRT-PCR),检测两组孕妇外周血单个核细胞维生素D受体(VDR)mRNA的表达水平,采用3-磷酸甘油醛脱氢酶(GAPDH)为内参,根据相对定量公式:2-△△CT分析VDR mRNA的表达水平。结果:(1)ICP组外周血清中IFN-γ的浓度[(230.93±36.04)pg/ml]明显高于正常对照组[(138.37±25.08)pg/ml],差异有统计学意义(P<0.01)。ICP组血清中IL-4浓度[(9.99±3.19)pg/ml]和正常对照组[(8.58±2.43)pg/ml]比较,差异无统计学意义(P>0.05)。ICP组IFN-γ/IL-4比值(24.56±6.91)高于正常对照组(17.13±4.84),差异有统计学意义(P<0.05)。(2)ICP组外周血单个核细胞维生素D受体mRNA的表达明显低于正常对照组(P<0.01),正常对照组VDR的表达定义为1.0,ICP组的表达量为0.4。(3)ICP组外周血中VDR的表达水平与IFN-γ浓度呈明显负相关(r=-0.833,P<0.01),与IL-4浓度无明显相关(r=-0.109,P>0.05),与IFN-γ/IL-4比值呈负相关,但相关性不强(r=-0.356,P=0.049<0.05)。结论:ICP患者外周血Th1/Th2型细胞因子平衡由Th2向Th1偏移,可能与ICP孕妇外周血单个核细胞VDR的表达减少有关。  相似文献   

8.
白细胞介素18   总被引:7,自引:1,他引:6  
白细胞介素-18(IL-18)是新发现的细胞因子,具有多种生物学功能.IL-18能促进外周血单个核细胞产生干扰素-γ(IFN-γ)、IL-2、粒细胞巨噬细胞集落刺激因子(GM-CSF)等细胞因子,增强天然杀伤细胞(NK细胞)的细胞毒作用.IL-18结构上与IL-1相似,而功能更接近IL-12,IL-18与IL-12均能诱导Th1细胞产生IFN-γ,存在协同效应,但它们的作用途径不同.IL-18在抗感染抗肿瘤等方面有着潜在的应用前景,并与自身免疫性疾病的发病密切相关.  相似文献   

9.
目的:研究口腔扁平苔藓(OLP1)患者Th1型和Th2型细胞因子的表达及临床意义。方法:选取2013年1月至2014年3月于我院就诊的21例充血糜烂型及14例光滑型OLP患者为研究对象,18例正常人为对照组,采用密度梯度离心法对各组外周血单个核细胞进行分离,酶联免疫吸附剂测定(ELISA)法对各组外周血单个核细胞中的IL-4和IFN-gamma的表达进行检测,逆转录-聚合酶链反应法对各组血清中IL-4 m RNA和IFN-gamma m RNA的表达进行检测。结果:与正常对照组相比,OLP患者IL-4m RNA及蛋白的表达均增高,而IFN-gamma m RNA及蛋白的表达则降低,差异均有显著统计学意义(均P0.01)。充血糜烂型及光滑型OLP患者组间比较发现,IL-4 m RNA和IFN-gamma m RNA及蛋白的表达差异无统计学意义(P0.05)。结论:OLP发病机制与Th1与Th2的表达失衡有关,为临床治疗提供参考。  相似文献   

10.
本文目的是为了探讨过继转输的父系抗原耐受T细胞对受体孕鼠反应性T细胞的影响。以(?)CBA/J×(?)BALB/c为正常妊娠模型,(?)CBA/J×(?)DBA/2为自然流产模型,将自然流产模型CBA/ J孕鼠于孕第4天(着床期)分别腹腔注射大鼠抗小鼠CD80和CD86 mAb或大鼠同型IgG。于孕第9天,应用免疫磁珠阴性分选孕鼠的脾脏T细胞,将纯化的T细胞先进行CFSE体外荧光标记,再分别过继转输至孕第4天的CBA/J×DBA/2孕鼠。于受体孕鼠孕第9天,在父系抗原刺激下,采用单向混合淋巴细胞反应分析受体孕鼠脾脏免疫细胞对父系抗原的增殖能力,并用流式细胞术分析过继转输的T细胞及受体T细胞内细胞因子IL-2、IL-4、IL-10和IFN-γ及细胞表面协同刺激分子CTLA-4的表达。本文结果显示,与过继转输父系抗原非耐受性T细胞相比,转输父系抗原耐受性T细胞和转输正常妊娠模型孕鼠T细胞均使受体孕鼠脾脏免疫细胞对父系抗原的增殖能力显著下降(P<0.05);同时使受体孕鼠T细胞内细胞因子IL-10及细胞表面CTLA-4的表达显著增加,而细胞因子IL-2和IFN-γ的表达则明显下降(P<0.05),细胞因子IL-4的表达不变(P>0.05)。总之,过继转输的父系抗原耐受T细胞具有抑制性免疫调节功能,诱导受体孕鼠反应性T细胞对父系抗原亦产生免疫耐受,两者协同作用则抑制母体对胚胎的免疫排斥,明显改善了自然流产模型孕鼠的妊娠预后。  相似文献   

11.
Ozenci V  Kouwenhoven M  Press R  Link H  Huang YM 《Cytokine》2000,12(8):1218-1224
The cytokine IL-12 promotes Th(1)type immune responses and plays a key role in immune regulation. The complex nature of IL-12 hampered its detection without use of stimulants that might give less relevant information. To detect circulating IL-12 p40, we developed enzyme-linked immunospot (ELISPOT) assays that allow enumeration of IL-12 p40 secreting cells without prior in vitro stimulation of the cells. In parallel, intracellular staining of IL-12 p40 by flow cytometry was performed to compare the two methods. IL-12 p40 secreting cells were detected in healthy subjects at a mean number of 103+/-155 per 10(5)blood mononuclear cells (MNC). Numbers of IL-12 p40 secreting blood MNC correlated with IL-12 p40 positive blood MNC detected by flow cytometry. Bacterial endotoxins and the inflammatory cytokines TNF-alpha and IFN-gamma control IL-12 production by antigen presenting cells. Utilizing IL-12 p40 ELISPOT assays, we could confirm occurrence of elevated numbers of IL-12 p40 secreting blood MNC after stimulation with TNF-alpha, IFN-gamma, LPS, LPS+TNF-alpha or LPS+IFN-gamma, compared to cultures without stimulant. Due to its central role in inflammation and autoimmunity, IL-12 is an attractive target for immunotherapy. IL-12 p40 ELISPOT assays represent a sensitive, specific and reliable tool for investigating the role of IL-12 in both health and disease.  相似文献   

12.
目的:研究大剂量HBsAg对HBV转基因小鼠其T细胞免疫效果的影响。方法:用大剂量血源性HBsAg免疫HBV转基因小鼠,采用ELISA方法观察转基因小鼠所诱生的HBsAg特异性Th1类细胞因子的水平,ELISPOT方法检测不同免疫方案对小鼠HBsAg特异性分泌IFN-γT细胞数量的影响,同时检测对小鼠淋巴细胞增殖的影响。结果:HBsAg组免疫后脾细胞产生的Th1类细胞因子(IFN-γ、IL-2)、HBsAg特异性分泌IFN-γT细胞及T细胞增殖水平较对照组显著增加(P〈0.05)。结论:大剂量的HBs-Ag可以诱导乙肝转基因小鼠产生高水平Th1类细胞因子并打破免疫耐受。  相似文献   

13.
We have recently reported that in patients with chronic immune thrombocytopenic purpura (IMTP), circulating T and B cells that are responsive to gpIIb-IIIa can induce anti-platelet autoantibody production. In this study, the frequencies and activation status of gpIIb-IIIa-reactive T and B cells were evaluated in the peripheral blood and spleen obtained from nine IMTP patients undergoing splenectomy. There was no difference in gpIIb-IIIa-reactive T cell frequencies between peripheral blood and spleen (6.4 +/- 2.6 vs 5.2 +/- 2.4 per 10(5) T cells), as determined by limiting dilution analysis, but activated T cells responsive to gpIIb-IIIa showing accelerated proliferation kinetics and those expressing CD154 were more frequent in spleen than in peripheral blood. The frequencies of anti-gpIIb-IIIa Ab-producing B cells, as determined by ELISPOT assay, were also similar in peripheral blood and spleen (61.2 +/- 24.0 vs. 77.7 +/- 45.3 per 10(5) B cells); however, an anti-gpIIb-IIIa Ab was spontaneously produced by splenocytes in vitro, but scarcely secreted by PBMCs. CD19(-)/surface Ig(-)/CD38(+)/CD138(+) plasma cells secreting anti-gpIIb-IIIa Ab were exclusively detected in the spleen. In serial analysis, the frequencies of circulating gpIIb-IIIa-reactive T and B cells were markedly decreased after splenectomy in patients with a complete response, but were unchanged in nonresponders. These findings indicate that an interaction between gpIIb-IIIa-reactive T and B cells inducing anti-platelet Ab production in IMTP patients occurs primarily in the spleen and that the significant number of gpIIb-IIIa-reactive T and B cells activated in the spleen are released into the circulation as memory cells.  相似文献   

14.
15.
Most macrophages in the peripheral tissues present Ag optimally to a variety of functionally distinct Th cells. Although thymic macrophages have been implicated in deleting autoreactive thymocytes, their role in influencing the functional capacities of mature T cells is not clear. We have established a normal untransformed macrophage cell line, named TMC, from the mouse thymus. The TMC line presents protein Ag to an IL-4-producing Th2 type Th clone after IFN-gamma treatment as evidence by T cell proliferation and the release of IL-3 and IL-4. However, these thymic macrophages are inefficient at stimulating a well characterized cytochrome C-specific IL-2-producing Th1 clone, A.E7. Ag presentation by TMC results in the production of IL-3 but not IL-2 production or proliferation of A.E7 cells. This selective Ag presentation defect to Th1 cells is corrected by the addition of live but not fixed allogeneic irradiated spleen cells, suggesting that the thymic macrophages lack the expression of costimulatory activity required for Th1 activation. This is further demonstrated by the failure of live thymic macrophages to provide costimulatory activity to A.E7 cells stimulated with fixed spleen cells plus the antigenic peptide 81-104. Exposure of A.E7 cells to paraformaldehyde-treated TMC in the presence of 81-104 peptide induces specific hyporesponsiveness, anergy. These data demonstrate that thymic macrophages can have a profound influence on the response of selected T cells to Ag. Furthermore, the nature of the T cell stimulus is also critical because Th1 and Th2 cells responded equally well to the T cell mitogen, Con A, and a bacterial superantigen presented by the thymic macrophages.  相似文献   

16.
Variable lymphocyte responses in rats after space flight.   总被引:2,自引:0,他引:2  
Most studies of human blood lymphocyte function following space flight have indicated that microgravity suppresses T cell proliferation. However, several other postflight experiments with animals have shown no decrease in proliferation of lymphocytes from peripheral lymphatic tissues, suggesting that different tissues may be variably affected by microgravity. Therefore, we examined the proliferation of lymphocytes from both spleen and lymph nodes of rats following a 4-day flight aboard the Space Shuttle. The experiments were designed to investigate tissue variability as well as potential mechanisms involved in suppressing proliferation. We found that proliferation of lymph node lymphocytes (LNL) from flight (FLT) animals stimulated with the antigen receptor-dependent T cell mitogen concanavalin A was depressed and could not be restored by supplementing cultures with interleukin 1 or interleukin 2 (IL-2). Response to another receptor-dependent mitogen, phytohemagglutinin, was not decreased. However, proliferation of FLT LNL following stimulation with the receptor-independent, mitogenic combination of phorbol ester and ionomycin was depressed. LNL IL-2 activity, cell surface marker expression, and B cell responses to mitogen were normal. Thus, deficits in antigen receptor/ligand interactions, cell surface marker expression, or IL-2 did not account for the suppressed lymphocyte proliferation observed postflight. In contrast to LNL, FLT splenocyte proliferation was not depressed. Assayable IL-2, IL-2 receptor expression, and cell surface marker expression likewise were unaffected by space flight. The differences between lymph node and splenic responses demonstrate the tissue-specific nature of microgravity effects on individual lymphatic tissues.  相似文献   

17.
Interleukin (IL)-27, a heterodimeric cytokine, has been reported to be involved in the pathogenesis of autoimmune diseases through mediating differentiation of Th1 or Th17 cells and immune cell activity or survival. However, the origin and effects of IL-27 in joints of rheumatoid arthritis (RA) remain unclear. In this study, we investigated the distribution and anti-inflammatory roles of IL-27 in RA synovium. The IL-27 levels in plasma of RA patients, osteoarthritis (OA) patients, or healthy volunteers (n=15 per group) were equivalent and were at most 1 ng/ml, but the IL-27 level in synovial fluid of RA patients (n=15, mean 0.13 ng/ml; range 0.017-0.37 ng/ml) was significantly higher than that in synovial fluid of OA patients (n=15, mean 0.003 ng/ml; range 0-0.033 ng/ml) and potentially lower than in plasma. We analyzed the protein level of IL-27 produced by RA fibroblast-like synoviocytes (FLSs) or mononuclear cells (MNCs) from RA or OA synovial fluid or peripheral blood and showed that IL-27 in RA joints was derived from MNCs but not from FLSs. We also found by flow cytometry that IL-27-producing MNCs were CD14(+), and that these CD14(+)IL-27(+) cells were clearly detected in RA synovium but rarely in OA synovium by immunohistochemistry. Furthermore, we demonstrated that a relatively physiological concentration of IL-27 below 10 ng/ml suppressed the production of IL-6 and CCL20 from RA FLSs induced by proinflammatory cytokines through the IL-27/IL-27R axis. In the synovial fluid of RA, the IL-27 level interestingly had positive correlation with the IFN-γ level (r=0.56, p=0.03), but weak negative correlation with the IL-17A level (r=-0.30, p=0.27), implying that IL-27 in inflammatory joints of RA induces Th1 differentiation and suppresses the development or the migration of Th17 cells. These findings indicate that circulating IL-27-producing CD14(+) cells significantly infiltrate into inflamed regions such as RA synovium and have anti-inflammatory effects in several ways: both directly through the reduction of IL-6 production, and possibly through the induction of Th1 development and the suppression of Th17 development; and indirectly by regulation of recruitment of CCR6(+) cells, such as Th17 cells, through the suppression of CCL20 production. Our results suggest that such a serial negative feedback system could be applied to RA therapy.  相似文献   

18.
IL-7 is critically involved in regulating peripheral T cell homeostasis. To investigate the role of IL-7 on lymphopenia-induced proliferation of polyclonal lymphocytes, we have transferred CFSE-labeled cells into a novel T-lymphopenic, IL-7-transgenic mouse line. Results obtained indicate that T and B cells do not respond in the same way to IL-7-homeostatic signals. Overexpression of IL-7 enhances proliferation of both CD4(+) and CD8(+) T cells but with distinctly temporal effects. Expansion of naturally arising CD4(+)-regulatory T cells was like that of conventional CD4(+) T cells. IL-7 had no effect on B cell proliferation. By immunohistology, transferred T cells homed to T cell areas of spleen lymphoid follicles. Increasing IL-7 availability enhanced T cell recovery by promoting cell proliferation and reducing apoptosis during early stages of lymphopenia-induced proliferation. Taken together, these results provide new insights into the pleiotropic effects of IL-7 on lymphopenia-induced T cell proliferation.  相似文献   

19.
Production of monoclonal antibodies (mAb) using genetic immunisation is a potential alternative when purified antigen is difficult to obtain, or when induction of an antibody response to a limited part of an antigen is wanted. DNA immunisation using only the constant parts of trout immunoglobulin light chains coding regions was attempted here, because mAbs against the variable (V) part of immunoglobulins do not recognise the whole repertoire of the isotype. After positive results with the light chains and establishing of a proper screening system (ELISA), generation of monoclonal antibodies against trout T cell receptor was also performed.The DNA constructs were used both for immunisation of mice and for protein expression in EBNA 293 cells. Mice were immunised with the constructs 3-5 times by intramuscular injection, with or without adjuvants during 1-3 months. Spleens of positive mice were fused with myeloma Sp2/0 cells and clones were screened by ELISA using double-screening (recombinant protein/trout cells).MAbs 46E5 (anti-IgL2C), 4F2 (anti-TCRalpha), 18B3 (anti-TCRalphaC) and 4E5 (anti-TCRalphaC) show specific binding to its antigen in Western blot, mAb 18B3 and 7H7(anti-TCRalpha) shows specific staining of trout splenocytes in flow cytometry and mAb 7H7 induces proliferation of trout peripheral blood leucocytes (PBL) in vitro.  相似文献   

20.
An in vitro immunization (IVI) protocol enables antigen specific antibody production from L-Leucyl-L-Leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBL) upon antigen stimulation in the presence of IL-2, IL-4, and muramyl dipeptide. In the course of our studies, we have found that IL-10 added at the antigen sensitization significantly augmented antibody production level from the LLME-treated PBL. In the present study, we tried to demonstrate the role of IL-10 in the augmentation of antibody production in an IVI protocol by clarifying the cytokine expression profiles in CD4(+) and CD8(+) T cells. The results showed that IL-10 skewed the Th1/Th2 balance to Th2-type responses by suppressing Th1-type cytokine production and augmenting Th2-type cytokine production in CD4(+) and CD8(+) T cells, as well as in CD19(+) B cells. Furthermore, IL-10 augmented the expression of CD38, an antigen marker of plasma cells, on B cells, which clearly indicates that IL-10 promoted differentiation and maturation of B cells in an IVI protocol. These results indicate that IL-10 plays an important role in setting the cellular milieu to produce antibodies in an IVI protocol.  相似文献   

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