小鼠脾淋巴细胞可T能存在5HT3受体

本文用3H-TdR掺人法和MTT法观察5HT3受体激动剂1-phenylbiguanide(PBG)、5HT3受体拮抗剂格拉司琼(granisetron)和托烷司琼(tropisetron)对体外培养ICR小鼠脾淋巴细胞ConA,LPS刺激的增殖和NK细胞活性的影响.结果表明PBG(10-6～10-4mol/L)抑制ConA刺激的脾细胞增殖反应和脾细胞IL-2的生成(P＜0.05);增强LPS剌激的脾细胞增殖反应(P＜0 05)格拉司琼和托烷司琼双相影响,即在低浓度(10～～10-6mol/L)促进、在较高浓度(10-5～10-4mol/L)抑制ConA刺激的增殖反应(P＜0.05);二药均浓度依赖抑制LPS刺激的脾细胞增殖效应.PBG对脾淋巴细胞增殖的影响被同时加入格拉司琼或托烷司琼拮抗,格拉司琼和托烷司琼减弱PBG 10-5mol/L对脾细胞增殖反应的影响.本实验5HT3受体激动剂或拮抗剂浓度不明显影响脾NK细胞活性和无丝裂原刺激的增殖反应.结果提示小鼠脾T细胞和B细胞表面可能存在对淋巴细胞增殖有不同影响的5HT3受体.     

大鼠海马内5-HT3受体参与神经免疫调制

目的 :探讨大鼠海马不同部位 5 HT3受体对免疫的调制作用。方法 :通过小鼠腹腔和大鼠侧脑室和海马不同结构内注射 5 HT3受体激动剂 1 phenylbiguanide(PBG)后不同时间 ,用3H TdR掺入法观察对ConA、LPS刺激的脾T和B淋巴细胞增殖效应的影响 ,并观察选择性 5 HT3受体拮抗剂托烷司琼 (tropisetron ,Trop)对PBG作用的影响。结果 :小鼠腹腔注射、大鼠侧脑室和双侧腹侧海马注射 5 HT3受体激动剂PBG后可增强ConA、LPS刺激的脾T和B淋巴细胞增殖效应 ;双侧背侧海马内注射PBG后抑制ConA刺激的脾T淋巴细胞增殖效应 ,而不影响LPS刺激的B淋巴细胞增殖效应 ;5 HT3受体激动剂对脾细胞增殖效应的影响可被同时给予的托烷司琼阻断。结论 :提示大鼠海马 5 HT3受体参与了免疫的调制并有明显的部位差异     

西洋参茎叶总皂甙在小鼠体内外的免疫效应

本文研究了西洋参茎叶总皂甙(PNS)对小鼠免疫功能的影响,发现 PNS 能在体内外协同ConA 促进小鼠脾细胞增殖;协同 ConA 增强小鼠脾 T 细胞产生淋巴因子的能力;明显增强小鼠脾 NKC 活性;PNS 在体内还能增强 LPS 刺激小鼠脾细胞的增殖反应;增强小鼠对胸腺依赖性抗原(SRBC)的初次抗体应答能力.     

铜绿假单胞菌外毒素A的抗原性及其与辅助细胞的关系

目的　研究铜绿假单胞菌外毒素A(PE)的某些抗原性及其与辅助细胞的关系。方法用MTT法观察PE在有无辅助细胞参与条件下对鼠脾淋巴细胞的刺激作用 ,并用鼠细胞毒性T细胞株CTLL进行证实。结果　PE在 0 0 1～ 10 0ng/ml浓度范围能刺激鼠脾淋巴细胞增殖 ,PE的丝裂原作用无需粘附细胞的辅助 ,但粘附细胞及其粘附细胞的PE刺激上清能协同PE对鼠脾淋巴细胞的刺激作用。PE在丝裂原作用范围内能刺激CTLL细胞生长 ,并与IL 2有协同作用。PE在体内能使小鼠脾淋巴细胞自发淋转率增高 ,对ConA刺激的增殖反应增强 ,但对淋巴细胞自发分泌IL 2和ConA诱导IL 2的产生水平无影响。结论　PE在无辅助细胞参与条件下也能刺激淋巴细胞增殖     

耐受性CD8~+ NKT细胞体外增殖能力的鉴定

目的:利用CFSE标记细胞,流式细胞术(FCM)检测法,解析超抗原SEB活化的耐受性CD8+ NKT细胞在体外增殖的情况。方法:利用CFSE标记新鲜分离的C57BL/J鼠脾细胞,分别与ConA和LPS共同培养3d,收集细胞进行荧光染色并用FCM解析细胞表面CD69分子的表达率和增殖能力。CFSE标记的鼠脾细胞与SEB共培养5d和10d后,荧光染色并用FCM解析细胞表面CD69的百分数和增殖能力。SEB活化的第10天细胞经CFSE标记后在IL-2的协同作用下继续培养10d,荧光染色,FCM解析这群细胞的增殖能力、活性分子CD69的表达率和NKT细胞亚群的变化情况。结果:ConA、LPS和SEB三者均可以刺激小鼠脾细胞增殖。ConA和LPS在3d内可以使细胞增殖3代,且CD69的表达率为74.19%和41.56%;SEB在5d和10d内分别可以使细胞增殖5代和7代,细胞表面CD69的表达率为32.09%和48.66%。SEB活化的10d细胞可以在IL-2的协同下继续传代培养10d,可以增殖7代;这群细胞中CD8+ NKT细胞亚群,由原始的0.36%增加到38.58%;细胞表面CD69分子由正常值的0.11%提高到83.74%。结论:超抗原SEB活化的CD8+ NKT细胞可以在体外进行增殖培养,且这些细胞是活性化的细胞。利用CFSE标记细胞,FCM可以检测耐受性CD8+ NKT细胞在体外的增殖水平。     

心房肽对自发性高血压大鼠免疫细胞增殖活性的影响

本文探讨了心房肽对免疫细胞增殖活性的影响。结果表明,心房肽可以直接促进大鼠胸腺细胞的增殖反应,亦可协同ConA促进脾细胞及T细胞的增殖反应,协同IL-2促进经ConA诱导的淋巴母细胞增殖;心房肽还可促进ConA诱导的大鼠脾细胞产生IL-2。但心房肽并不影响B细胞的增殖反应,也不影响巨噬细胞产生IL-1。同时发现,自发性高血压大鼠脾细胞和胸腺细胞对心房肽与ConA协同刺激的增殖反应性低下。     

漆黄素对巨噬细胞和T淋巴细胞的免疫调节作用

目的探讨漆黄素(Fisetin,FIS)对小鼠巨噬细胞吞噬功能、NO的释放及对T淋巴细胞体外活化、增殖的影响。方法无菌制备小鼠巨噬细胞悬液及淋巴细胞悬液;荧光微球结合流式细胞术(FCM)分析FIS对巨噬细胞吞噬作用的影响;Griess试剂盒检测巨噬细胞NO的释放;双色荧光抗体染色结合FCM,检测CD3+T细胞CD69的表达水平;CFSE标记技术检测T细胞增殖的情况。结果 2.5μmol/L,5μmol/L,10μmol/LFIS均能明显抑制巨噬细胞的吞噬微球的能力;FIS能抑制LPS和IFN-γ刺激的巨噬细胞的NO的产生(P<0.05);FIS对ConA刺激的T细胞表达CD69有抑制作用,并能有效抑制ConA诱导的T细胞增殖(P<0.01),且均呈剂量依赖性。结论 FIS能显著抑制小鼠腹腔巨噬细胞的吞噬能力和分泌NO的能力,并能够抑制T细胞的活化和增殖,有望发展成为一种新的免疫抑制药物。     

淫羊藿次苷Ⅱ对小鼠脾淋巴细胞体外增殖的影响及其机制研究

目的:研究淫羊藿次苷Ⅱ对正常小鼠脾淋巴细胞体外增殖的影响,并初步探讨其机制。方法:不同浓度的淫羊藿次苷Ⅱ与刀豆素A(Concanavalin A,ConA)体外处理小鼠脾淋巴细胞,观察淫羊藿次苷Ⅱ对细胞增殖的影响,初步确定其发挥作用的最佳浓度;然后以最佳浓度淫羊藿次苷Ⅱ在不同时间点与ConA协同处理细胞,采用细胞增殖-毒性检测试剂盒检测增殖能力;运用流式细胞仪检测细胞周期分布;用荧光定量分析仪检测细胞内游离Ca2+浓度的变化。结果:淫羊藿次苷Ⅱ在10-12～10-9mol/L的浓度范围作用48小时,对细胞增殖有一定的促进作用,呈浓度依赖性。在10-7～10-4mol/L范围出现抑制作用。以10-9mol/L的淫羊藿次苷Ⅱ处理细胞,发现其与ConA在不同时间点促进细胞增殖作用显著优于ConA单独应用;并且促进细胞由G0/G1期进入S与G2/M期,并以第24小时效果更明显;促进细胞内Ca2+浓度升高,效果以第12小时最明显。结论:不同浓度的淫羊藿次苷Ⅱ对小鼠脾淋巴细胞增殖具有促进作用,有浓度、时间依赖性,这种促增殖作用的机制可能与升高细胞内Ca2+浓度,进而加快细胞由G0/G1期进入S与G2/M期有关。     

霉酚酸衍生物对小鼠T细胞免疫功能的抑制作用

目的研究2种新型霉酚酸衍生物(代号JU95-07B、JU100-07E)在体外对小鼠T细胞免疫功能的影响。方法小鼠脾细胞用抗CD3单克隆抗体(mAb)刺激,同时加入不同浓度(10-4、10-5、10-6、10-7、10-8mol/L)及在不同时间点(刺激细胞后第0、6、12 h)加入同一浓度(10-5mol/L)的霉酚酸衍生物后,用酶联免疫吸附法(ELISA)检测培养上清中IFN-γ和IL-2的水平。同时利用流式细胞术(flow cytometry,FCM)检测霉酚酸衍生物对T细胞IFN-γ和IL-2的分泌、表面分子CD25的表达以及增殖的影响。结果 2种霉酚酸衍生物对抗CD3刺激条件下诱导的小鼠脾细胞产生的IFN-γ和IL-2均具有浓度依赖性抑制作用,并且在抗CD3刺激的不同时间点分别加入2种霉酚酸衍生物均能不同程度地抑制小鼠脾细胞产生IFN-γ和IL-2;同时,与单独使用抗CD3刺激相比,分别加入10-5mol/L浓度的2种霉酚酸衍生物后,均能抑制小鼠T细胞表面分子CD25的表达以及增殖。结论体外实验表明2种霉酚酸衍生物均对小鼠T细胞免疫功能具有明显抑制作用,该研究提示这2种霉酚酸衍生物可以抑制自身免疫性疾病和移植排斥反应。     

细胞外信号调节激酶1/2的阻断剂PD98059对小鼠淋巴细胞增殖的影响

目的探讨细胞外信号调节激酶1/2(ERK1/2)信号通路的特异性阻断剂PD98059对小鼠淋巴细胞体外增殖的影响。方法分离小鼠淋巴结细胞,藉羧基荧光素乙酰乙酸(CFDA-SE)染色,用多克隆刺激剂刀豆A蛋白(ConA)或者佛波醇酯(PDB)加离子霉素(Ion)刺激,以流式细胞术分析PD98059对淋巴细胞增殖的影响;利用碘化丙锭(PI)染色分析PD98059对细胞周期的影响。结果CFDA-SE染色分析显示,当浓度为5、10、20、30、40μmol/L时,PD98059对ConA诱导的淋巴细胞增殖具有明显的抑制作用,并呈现明显的剂量依赖关系(r=0.985,P<0.01)。选择最佳浓度30μmol/L的PD98059,观察其对不同时间淋巴细胞增殖的影响,结果发现,随着时间的延长,PD98059对淋巴细胞增殖的抑制作用明显降低(P<0.01),但抑制率随时间的延长而明显升高。细胞周期分析进一步表明,PD98059可阻止ConA刺激的淋巴细胞进入S期和G2/M期,而亚二倍体峰变化并不明显。PD98059对PDB Ion刺激的淋巴细胞细胞周期的影响与ConA刺激相似,不同的是S期和G2/M期的变化较ConA的作用更明显。结论PD98059对小鼠淋巴细胞的增殖有明显地抑制作用,并可阻止其进入S期和G2/M期,提示ERK1/2信号通路的活化在淋巴细胞增殖中起重要作用。     

褪黑素对系统性红斑狼疮样小鼠的影响

为了观察褪黑素 (MT )对CJ S13 1所致的SLE样小鼠改变的影响并探讨其作用机制。采用空肠弯曲菌CJ S13 1和CFA混合免疫动物 ,诱导SLE样小鼠改变。结果表明MT 0 0 1、 0 10、 1 0mg/kg/d× 2 8d ,ig能部分或完全拮抗血清抗ssDNA和组蛋白IgG型自身抗体水平的升高 ,抑制ConA及LPS诱导的淋巴细胞增殖反应的增强 ,降低动物尿蛋白水平 ,减轻肾组织病变程度。提示一定剂量的MT对SLE样小鼠改变具有一定的保护作用。     

Immunomodulation of polypeptides fromChlamys farreri in vitro

Polypeptides fromChlamys Farreri(PCF) ,a wa-ter soluble octopeptide ,wasisolatedfromChlamysFarreribyenzyme engineering.The previous stud-iesin our laboratory showed that PCF was capableof protectingimmunocytesfromthe damages of Ul-traviolet or60Co,andthat PCFcouldresist the de-pression of lymphoproliferation caused by E2[1 ,2] .Based onthe earlier studies ,wefurtherinves-tigatedthe effects of PCF on immunityin vitrosoasto provide experimental basisforthe exploitationof PCF.MATERIAL…     

超抗原SEA活化淋巴细胞抗肿瘤活性的实验研究

纯化SEA在 10 -12 ～ 10 -5g/ml浓度范围对体外培养的BALB/c鼠脾细胞表现了细胞增殖诱导能力 ,并呈剂量依赖关系 ,其中 10 -7～ 10 -5g/mlSEA的作用强于最适量 ( 2 5 μg/ml)PHA。在E/T为 5∶1～ 2 0∶1条件下 ,10 -5g/mlSEA活化 48h的BALB/c鼠脾细胞对YAC 1细胞的杀伤活性高于NK细胞 ,但SEA未能增强BALB/c鼠脾细胞对B16细胞的杀伤活性。 5 μg和5 0 μgSEA体内用药对L615白血病无治疗作用 ,但转输经SEA活化的同系小鼠脾细胞对L615白血病有一定疗效。实验结果提示 ,SEA活化淋巴细胞对NK敏感的淋巴瘤、白血病有杀伤及治疗作用 ,这种作用依赖于较大数量级SEA活化淋巴细胞的存在 ,并且与SEA活化T细胞分泌的细胞因子有关     

Roles of gamma interferon and other cytokines in suppression of the spleen cell proliferative response to concanavalin A and toxoplasma antigen during acute toxoplasmosis.

Suppressed splenocyte proliferation in response to mitogen and toxoplasma lysate antigen (TLA) is observed in mice acutely infected with Toxoplasma gondii. Recently, we reported that NG-monomethyl-L-arginine (NMMA), an inhibitor of reactive nitrogen intermediate (RNI) production, partially restored proliferative responses of splenocytes from infected mice. In the present study we have examined the effect of NMMA on production of cytokines by splenocytes from mice acutely infected with T. gondii and assessed the role of gamma interferon (IFN-gamma) and interleukin-10 (IL-10) in the RNI-mediated suppression. Stimulation with concanavalin A (ConA) or TLA of splenocytes from CBA/Ca mice infected for 7 days resulted in increased production of IFN-gamma, IL-4, and IL-10 but reduced levels of IL-2 when compared with cultures of splenocytes from uninfected mice. Whereas addition of NMMA did not alter levels of cytokines produced by splenocytes from uninfected mice, splenocytes from infected mice stimulated with ConA produced significantly higher levels of IL-10 and reduced levels of IL-2 and IL-4. Addition of anti-IFN-gamma monoclonal antibodies to cultures of spleen cells from mice infected for 7 or 14 days remarkably decreased the levels of nitrite and resulted in a 47- and 4-fold increase in proliferation induced by stimulation with ConA or TLA, respectively. Anti-IL-10 did not reduce levels of nitrite produced in culture but did result in a fourfold increase in the proliferative response of splenocytes from mice infected for 14 days. In vivo administration of anti-IFN-gamma or anti-IL-10 monoclonal antibodies to infected mice partially restored ex vivo spleen cell proliferative responses by approximately 40 and 15%, respectively. Our data indicate that IFN-gamma is important in inducing the RNI-mediated immunosuppression, which, in turn, affects production of cytokines by splenocytes. Our data also demonstrate that IL-10 is involved in the suppression observed but that this activity is independent of RNI.     

IL-3、IL-6联合转基因的基质细胞促进异基因小鼠骨髓移植后的免疫重建

探讨转染IL 3和IL 6基因的骨髓基质细胞系QXMSC1对异基因骨髓移植 (BMT )小鼠免疫功能重建的促进作用。用逆转录病毒载体将小鼠IL 3和IL 6基因转染到骨髓基质细胞系QXMSC1(H 2 d) ,分别建立基质细胞系QXMSC1IL 3、QXMSC1IL 6及QXMSC1IL 3/IL 6 ,供体BALB/c(H 2 d)小鼠骨髓去除T细胞 ,致死量照射 (9 0Gy)受体小鼠C5 7BL/ 6 (H 2 b)后 ,输入供体骨髓细胞 (1× 10 5/只 )的同时输入基质细胞QXMSC1IL 3/IL 6 (5× 10 5/只 )。在骨髓移植后 30d、 6 0d ,检测BMT小鼠脾细胞对LPS、ConA的反应强度、脾细胞中辅助性T淋巴细胞前体频数 (HTLp )、杀伤性T淋巴细胞前体频数 (CTLp )、抗体生成细胞 (PFC )的能力及对迟发型超敏反应 (DTH )的能力来评价BMT后免疫功能的恢复情况。异基因BMT并输入基质细胞系QXMSC1IL 3/IL 6可明显增强BMT后淋巴细胞对LPS、ConA反应强度 ,小鼠对异基因小鼠脾细胞DTH反应增强 ,脾淋巴细胞HTLp、CTLp及PFC数明显增加。基质细胞QXMSC1可作为有效的基因载体明显促进异基因骨髓移植小鼠免疫功能重建。联合转入IL 3和IL 6对BMT后免疫功能的恢复有协同作用     

Luminal hypotonicity increases duodenal mucosal permeability by a mechanism involving 5-hydroxytryptamine

Aim: To investigate whether 5‐hydroxytryptamine (5‐HT) participates in the mediation of the hypotonicity‐induced increase in duodenal mucosal permeability. Methods: Proximal duodenum in anaesthetized rats was perfused in situ with a hypotonic NaCl solution and effects on duodenal motility, net fluid flux, mucosal permeability [blood‐to‐lumen clearance of ⁵¹Cr‐ethylenediaminetetraacetic acid (EDTA)] and the release of 5‐HT into the luminal solution studied in the presence of the cyclooxygenase inhibitor indomethacin. Results: Perfusion of the duodenum with 50 mm NaCl increased mucosal permeability eightfold, increased the luminal output of 5‐HT twofold and induced net fluid absorption. This rise in permeability was enhanced 25% by 5‐HT (3 × 10^?3 m ), reduced by the 5‐HT₃‐receptor antagonists granisetron (10^?4–3 × 10^?4 m ) or ondansetron (10^?5–10^?4 m ) or by the 5‐HT₄ receptor antagonist SB 203186 (10^?4 m ). The 5‐HT_3/4 receptor antagonist tropisetron, at 10^?4 m , did not affect while 3 × 10^?4 and 3 × 10^?3 m augmented the hypotonicity‐induced increase in mucosal permeability. Lidocaine (1.1 × 10^?3 m ) similarly potentiated while tetrodotoxin (TTX) (5 × 10^?5 m ) inhibited the hypotonicity‐induced increase in mucosal permeability. Compared with animals treated with indomethacin alone ondansetron and granisetron augmented (by 30–40%) while tropisetron and lidocaine reduced (by 60–70%) the hypotonicity‐induced net fluid absorption. Tetrodotoxin and all 5‐HT receptor antagonists, except tropisetron, depressed duodenal motility. Conclusions: Luminal hypotonicity increases duodenal mucosal permeability by a neural mechanism involving 5‐HT acting on 5‐HT₃ and 5‐HT₄ receptors. 5‐HT also appears to participate in the regulation of the hypotonicity‐induced fluid flux.     

商陆皂甙辛对小鼠脾脏细胞产生IL-3和IL-6的影响

利用MTT法测定了商陆皂甙辛 (EH )对ConA诱导的小鼠脾淋巴细胞IL 3和IL 6活性的影响 ,采用地高辛标记斑点杂交法测定了IL 3mRNA和IL 6mRNA的表达水平。结果显示EH在 1 0～ 1 0 0 μg/ml浓度范围内能增强ConA诱导的小鼠脾淋巴细胞IL 3和IL 6活性 ,伴随mRNA水平的上升。提示EH通过提高IL 3和IL 6基因转录水平 ,从而使其产物活性增强 ,是其免疫调节和提高造血功能的分子机制之一     

Deficit of interleukin 2 production associated with impaired T-cell proliferative responses in Mycobacterium lepraemurium infection.

C57BL/6 and BALB/c mice were infected intravenously with 10(7) Mycobacterium lepraemurium (MLM). At various times after infection, spleen cells were tested for their capacity to proliferate in vitro in response to concanavalin A (ConA) and to allogeneic cells. The generation of alloreactive cytotoxic T lymphocytes was also studied. The mitogen- and allogeneic-cell-induced blastogenesis of splenocytes from MLM-infected C57BL/6 and BALB/c mice was shown to be depressed during infection. The maximal decrease occurred 6 months after infection. Conversely, no reduction in the ability to generate alloreactive cytotoxic T lymphocytes was observed even after 6 months of infection. At the same time, interleukin 2 (IL2) activity generated by ConA stimulation of infected splenocytes was measured in both strains. IL2 activity in the ConA-stimulated culture supernatants was decreased as early as 1 month after MLM inoculation as compared with supernatants from age-matched control mice. Thus, IL2 production by infected-mouse spleen cells was shown to decline earlier than their proliferative responses to ConA and to allogeneic cells. ConA-induced T-cell blasts from infected mice showed a reduced ability to proliferate when incubated with an IL2-containing reference supernatant from ConA-stimulated normal spleen cells. These data suggest that a defect in IL2 production and utilization might contribute to the impairment of T cell-mediated immunity observed in MLM-infected mice.     

Immune deficiency of the cts mouse. I. Deficiency of in vitro t cell-mediated immune response

The cataract Shionogi (CTS) mouse characterized by cataracts and microphthalmia is a sister strain of the NOD mouse. We have made the immunological characterization of the CTS mouse by means of in vitro assays. Splenocytes of the CTS mouse were very low in the responsiveness to T cell mitogens such as Con A and PHA but not to a B cell mitogen, LPS. The production of IL 2 and expression of IL 2-receptor of spleen cells after in vitro stimulation with Con A decreased in the CTS mouse, when compared with those in the NOD and the other reference strains. In mixed lymphocyte culture, CTS splenocytes did not proliferate and did not generate cytotoxic T lymphocytes when cocultured with splenocytes of the C3H/He mouse. The NK activity against YAC-1 target cells was lower in the CTS mouse than in the C3H/He mouse, an NK high responder, but higher than in the NOD mouse, a low responder. These results suggest that the CTS mouse is deficient in T cells. Subset analysis of splenic lymphocytes of the CTS mouse using flow cytometry revealed that the percentage of T cells in the CTS mouse was significantly lower than those in the reference strains, which was consistent with the reduced responsiveness to T cell mitogens in the CTS mouse. The deficiency in the Ly-2⁺ T cell subset was particularly striking. However, the response to PHA of the splenocytes of the CTS mouse was normalized when T cells were enriched by nylon wool-passing and cell-sorting. Therefore, it seems that decreased T cell activity is due to a decrease in T cell number and not to dysfunction of individual T cells.     

海马内注射5-HT3受体激动剂后c-fos在不同脑区的表达

目的：探讨海马5-HT3受体神经免疫调节的神经功能通路及可能的途径。方法：采用SABC免疫组化染色法，测定不同脑区c-fos的表达。结果：给海马核团内注射5-HT3受体激动1-phenylbiguanide(1-PBG)后1h，海马及大脑皮层中有大量的c-fos表达，随时间的推移表达量渐减少。下丘脑在注射1-PBG后8h，中脑导水管周围灰质在16h出现有意义的表达。1-PB诱导的c-fos的表达可被其相应受体拮抗剂tropisetron(TROP)所阻断。结论：海马-大脑皮层-下丘脑-中脑导水管周围灰质形成一个神经网络，这个神经网络可能与免疫调节有关。