青蒿琥酯对迟发型超敏反应小鼠脾脏T淋巴细胞的免疫调节作用

背景:青蒿琥酯是青蒿素低毒、高效的衍生物,具有免疫调节功能,但其具体机制仍需研究阐明。目的:初步探讨青蒿琥酯对迟发型超敏反应小鼠脾脏T淋巴细胞的免疫调节作用。方法:建立迟发型超敏反应小鼠模型,40只小鼠随机数字表法均分正常对照组、青蒿琥酯干预组、p-38 MAPK抑制剂组、基质对照组进行实验观察。T淋巴细胞转化实验检测小鼠T淋巴细胞增殖水平;Western blot方法检测p38丝裂酶原激活蛋白激酶(p38 MAPK)蛋白的活性表达。结果与结论:局部给药后青蒿琥酯明显减轻迟发型超敏反应小鼠耳肿胀、降低脾指数、抑制刀豆蛋白A诱导的T淋巴细胞增殖;同时减弱p38MAPK的磷酸化活性表达。提示青蒿琥酯可以有效抑制小鼠迟发型超敏反应,其作用途径可能与抑制p38MAPK信号通路有关。     

Fas及FasL和Caspase-3在青蒿琥酯诱导人胚肺成纤维细胞凋亡中的表达

背景：青蒿琥酯具有减轻肺纤维化的作用,但相关机制的研究罕见报道。目的：探讨青蒿琥酯对人胚肺成纤维细胞凋亡的作用及其与Fas,FasL,Caspase-3表达的关系。方法：用1,10,100mg/L青蒿琥酯分别干预体外培养的人胚肺成纤维细胞。采用CCK-8法检测青蒿琥酯对人胚肺成纤维细胞增殖的影响,流式细胞术测定细胞凋亡率,RT-PCR法测定Fas,FasL,Caspase-3的mRNA的表达。结果与结论：青蒿琥酯呈浓度依赖性抑制人胚肺成纤维细胞增殖,细胞经青蒿琥酯作用后凋亡率明显增加（P〈0.05或P〈0.01）,Fas,FasL,Caspase-3mRNA的表达显著高于对照组（P〈0.05）。结果证实,青蒿琥酯可通过上调Fas,FasL,Caspase-3mRNA的表达抑制人胚肺成纤维细胞增殖、并促进细胞凋亡,发挥抗肺纤维化作用。     

青蒿琥酯对结肠癌HCT116细胞间黏附分子蛋白表达的影响

目的：研究青蒿琥酯对结肠癌细胞锚着不依赖性增殖及其细胞间黏附分子-1（intercellular adhesion molecule-1，ICAM-1）基因蛋白表达的影响。方法：以人结肠癌细胞株HCT116为模型，以软琼脂集落培养试验检测青蒿琥酯对肿瘤细胞的增殖抑制效应；Western blot方法检测青蒿琥酯作用前后细胞ICAM-1蛋白表达的变化。结果：青蒿琥酯能有效地抑制结肠癌细胞HCT116的恶性增殖，且呈剂量依赖性。青蒿琥酯能有效地抑制ICAM-1基因蛋白表达，作用呈时间和浓度依赖性。结论：青蒿琥酯可明显抑制结肠癌HCT116细胞增殖，可能与下调ICAM-1表达有关。     

Fas及FasL和Caspase-3在青蒿琥酯诱导人胚肺成纤维细胞凋亡中的表达

背景:青蒿琥酯具有减轻肺纤维化的作用,但相关机制的研究罕见报道.目的:探讨青蒿琥酯对人胚肺成纤维细胞凋亡的作用及其与Fas,FasL,Caspase-3表达的关系.方法:用1,10,100 mg/L青蒿琥酯分别干预体外培养的人胚肺成纤维细胞.采用CCK-8法检测青蒿琥酯对人胚肺成纤维细胞增殖的影响,流式细胞术测定细胞凋亡率,RT-PCR法测定Fas,FasL,Caspase-3 的mRNA的表达.结果与结论:青蒿琥酯呈浓度依赖性抑制人胚肺成纤维细胞增殖,细胞经青蒿琥酯作用后凋亡率明显增加(P < 0.05或P < 0.01),Fas,FasL,Caspase-3 mRNA的表达显著高于对照组(P < 0.05).结果证实,青蒿琥酯可通过上调Fas,FasL,Caspase-3 mRNA的表达抑制人胚肺成纤维细胞增殖、并促进细胞凋亡,发挥抗肺纤维化作用.     

青蒿琥酯治疗MRL/lpr狼疮鼠肾炎的病理变化及机制

目的研究青蒿琥酯对MRL/lpr狼疮鼠肾炎的疗效,探讨青蒿琥酯治疗狼疮鼠肾炎的病理变化及机制,为临床用于治疗狼疮患者提供依据。方法MRL/lpr鼠随机分为青蒿琥酯治疗组、环磷酰胺(CTX)治疗组和对照组。16周龄时青蒿琥酯组给予青蒿琥酯125 mg/(kg.d)治疗16周,CTX组给予CTX 100 mg/kg×2 d腹腔注射。PAS染色观察病理改变,免疫荧光检测补体C3沉积,运用反转录-聚合酶链反应(RT-PCR)检测小鼠肾脏血管内皮生长因子(VEGF)的mRNA表达水平,用免疫组化法检测肾脏中VEGF的蛋白表达。结果①青蒿琥酯组和CTX组肾脏病理损伤较对照组明显减轻;②青蒿琥酯组和CTX组肾脏内补体C3沉积较对照组明显减少;③青蒿琥酯组肾脏VEGF mRNA表达(0.72±0.11)和CTX组肾脏VEGFmRNA表达(0.66±0.19)均低于对照组(0.92±0.06)(P<0.05);④青蒿琥酯组和CTX组肾脏VEGF表达比对照组明显减少。结论青蒿琥酯治疗MRL/lpr狼疮鼠可以改善肾脏病理损伤。抑制C3的沉积及VEGF的产生是青蒿琥酯治疗MRL/lpr狼疮鼠肾炎的有效的可能机制。     

青蒿琥酯诱导人急性白血病原代细胞凋亡、胀亡的作用机制研究

目的:探讨青蒿琥酯诱导人急性白血病(AL)原代细胞凋亡、胀亡作用的可能机制。方法:提取11例急性白血病患者骨髓液进行原代细胞培养,以对数生长期细胞为干预点,将不同浓度青蒿琥酯作用于细胞,分别采用四甲基偶氮唑盐(MTT)法检测青蒿琥酯对人AL原代细胞增殖的影响;透射电子显微镜观察青蒿琥酯作用后细胞的形态变化;免疫印迹法检测凋亡相关蛋白。结果:与对照组比较,青蒿琥酯对人AL骨髓原代培养细胞有抑制作用;青蒿琥酯具有诱导人AL骨髓原代细胞凋亡和胀亡的作用,青蒿琥酯可下调凋亡抑制蛋白Bcl-2含量,同时促进凋亡蛋白Bax、细胞色素C的释放。结论:青蒿琥酯可诱导人AL原代细胞凋亡和胀亡,线粒体途径可能是青蒿琥酯诱导白血病细胞死亡的主要途径。     

青蒿琥酯通过抑制ICAM-1治疗鼠狼疮性肾炎的研究

目的探索青蒿琥酯对狼疮模型鼠-MRL/lpr鼠狼疮性肾炎(LN)的疗效及作用机制。方法分别以青蒿琥酯50mg/(kg.d)和相同体积生理盐水灌胃治疗MRL/lpr鼠,治疗16周后检测小鼠24 h蛋白尿、血肌酐水平,检测小鼠外周血血清中ICAM-1浓度及淋巴细胞表面CD54阳性率,检测小鼠肾组织中ICAM-1表达。结果青蒿琥酯能够显著减少狼疮鼠的蛋白尿(P0.01)及血肌酐水平(P0.05),显著降低狼疮鼠的血清中可溶性ICAM-1浓度(P0.05)及淋巴细胞表面CD54的阳性率(P0.05),导致肾组织中ICAM-1的表达明显下降。结论青蒿琥酯显著缓解MRL/lpr鼠LN病情,此作用可能与抑制ICAM-1的表达有关。     

青蒿琥酯通过抑制ICAM-1治疗鼠狼疮性肾炎的研究

目的探索青蒿琥酯对狼疮模型鼠-MRL/lpr鼠狼疮性肾炎（LN）的疗效及作用机制。方法分别以青蒿琥酯50mg/（kg.d）和相同体积生理盐水灌胃治疗MRL/lpr鼠,治疗16周后检测小鼠24 h蛋白尿、血肌酐水平,检测小鼠外周血血清中ICAM-1浓度及淋巴细胞表面CD54阳性率,检测小鼠肾组织中ICAM-1表达。结果青蒿琥酯能够显著减少狼疮鼠的蛋白尿（P〈0.01）及血肌酐水平（P〈0.05）,显著降低狼疮鼠的血清中可溶性ICAM-1浓度（P〈0.05）及淋巴细胞表面CD54的阳性率（P〈0.05）,导致肾组织中ICAM-1的表达明显下降。结论青蒿琥酯显著缓解MRL/lpr鼠LN病情,此作用可能与抑制ICAM-1的表达有关。     

青蒿琥酯治疗MRL／lpr狼疮鼠肾炎的病理变化及机制

目的研究青蒿琥酯对MRL／lpr狼疮鼠肾炎的疗效，探讨青蒿琥酯治疗狼疮鼠肾炎的病理变化及机制，为临床用于治疗狼疮患者提供依据。方法MRL／lpr鼠随机分为青蒿琥酯治疗组、环磷酰胺（CTX）治疗组和对照组。16周龄时青蒿琥酯组给予青蒿琥酯125mg／（kg·d）治疗16周，CTX组给予CTX100mg／kg×2d腹腔注射。PAS染色观察病理改变，免疫荧光检测补体G沉积，运用反转录-聚合酶链反应（RT-PCR）检测小鼠肾脏血管内皮生长因子（VEGF）的mRNA表达水平，用免疫组化法检测肾脏中VEGF的蛋白表达。结果①青蒿琥酯组和CTX组肾脏病理损伤较对照组明显减轻；②青蒿琥酯组和CFX组肾脏内补体C3沉积较对照组明显减少；③青蒿琥酯组肾脏VEGFmRNA表达（0．72±0．11）和CFX组肾脏VEGFmRNA表达（066±0．19）均低于对照组（0．92±0．06）（P〈0．05）；④青蒿琥酯组和CTX组肾脏VEGF表达比对照组明显减少。结论青蒿琥酯治疗MRL／lpr狼疮鼠可以改善。肾脏病理损伤。抑制C3的沉积及VEGF的产生是青蒿琥酯治疗MRL／lpr狼疮鼠肾炎的有效的可能机制。     

青蒿琥酯对人肺成纤维细胞TGF-β1/smad通路的影响

目的:研究青蒿琥酯对人肺成纤维细胞(HFL-I)的TGF-β1/smad通路及其对细胞增殖的影响。方法:使用青蒿琥酯和重组人TGF-β1刺激因子的干预下对HFL-I在体外培养,然后Western bloting(WB)检测TGF-β1、Smad3和Smad7蛋白的表达;并使用流式细胞仪检测细胞周期。结果:青蒿琥酯可抑制TGF-β1及Smad3蛋白的表达(P<0.05),刺激Smad7的磷酸化蛋白的表达(P<0.05);对Smad3非磷酸化蛋白无影响;对青蒿琥酯处理的HELF细胞使用重组人TGF-β1刺激因子可使TGF-β1蛋白的表达升高(P<0.05),磷酸化的Smad3蛋白的表达升高(P<0.05)。HELF细胞经过青蒿琥酯处理后细胞主要停滞于G1期,经过重组人TGF-β1刺激因子处理的细胞主要分布于S期。结论:青蒿琥酯可以通过TGF-β1/smad通路对HFL-I有抑制的作用,抑制的细胞停滞在细胞周期的G1期。     

桑色素对二硝基氟苯诱导的迟发型超敏反应的影响

目的研究桑色素（morin）对二硝基氟苯诱导迟发型超敏反应的影响。方法建立二硝基氟苯（dinitroflu-orobenzene,DNFB）诱导迟发型超敏反应（delayed type hypersensitivity,DTH）模型,观察morin对DTH模型小鼠耳组织肿胀、炎性细胞浸润程度和胸腺指数、脾指数的影响,光学显微镜下观察小鼠耳廓组织学变化,噻唑兰（methyl thia-zolyl tetrazolium,MTT）法检测淋巴结细胞和脾脏淋巴细胞增殖情况。结果双耳称重的结果显示,morin（70mg/ml）处理组与DTH组具有显著的差异（P〈0.05）,能明显抑制DNFB引起的炎症反应;耳廓局部组织学检测也表明,morin能够抑制DNFB诱导的DTH,明显减少淋巴细胞的浸润。morin能降低DTH小鼠胸腺指数和脾脏指数,抑制淋巴结淋巴细胞（P〈0.01）和脾脏淋巴细胞的增殖（P〈0.05）。结论 morin可显著抑制DTH小鼠耳组织肿胀和淋巴细胞浸润程度,降低胸腺和脾脏指数,抑制淋巴细胞的增殖可能是其作用机制之一。     

肝星状细胞增殖与p38丝裂原活化蛋白激酶信号传导通路的关系

背景：肝星状细胞的激活、增殖导致肝纤维化,p38丝裂原活化蛋白激酶信号通路可参与调控细胞增殖。目的：探讨SB203580作用于乙醛刺激的大鼠肝星状细胞后p38丝裂原活化蛋白激酶活性变化和细胞增殖变化。方法：体外培养大鼠肝星状细胞株,在乙醛干预的基础上加入不同浓度的p38特异性抑制剂SB203580进行培养,并设置对照。以Westernblot检测磷酸化p38蛋白表达水平变化,MTT比色法检测细胞增殖。结果与结论：乙醛刺激后大鼠肝星状细胞内磷酸化p38水平增强,细胞增殖明显。使用5,10,20μmol/L SB203580能明显抑制乙醛刺激的肝星状细胞增殖（P〈0.05）,加大浓度至30μmol/L时,抑制作用更明显（P〈0.01）,抑制率为43.9%,而磷酸化p38水平也降低（P〈0.05）。结果证实,抑制p38丝裂原活化蛋白激酶活性可能影响肝星状细胞的增殖。     

重组人促红细胞生成素与骨髓间充质干细胞的迁移及黏附☆

背景:促红细胞生成素可促进内皮祖细胞的增殖分化并增强其黏附性。目的:观察重组人促红细胞生成素干预对人骨髓间充质干细胞迁移和黏附能力及相关细胞信号传导通路的影响。方法:体外培养人骨髓间充质干细胞,使用重组人促红细胞生成素干预第6代人骨髓间充质干细胞。分别用PI3K/Akt通路特异抑制剂Ly294002或p38MAPK通路特异激动剂anisomycin或ERK1/2通路特异抑制剂U0126预处理。结果与结论:重组人促红细胞生成素可使人骨髓间充质干细胞的PI3K/Akt通路磷酸化水平升高,抑制p38MAPK通路磷酸化水平,对人骨髓间充质干细胞的ERK通路和总Akt、总p38MAPK水平无明显影响。重组人促红细胞生成素作用组中迁移细胞数目显著高于对照组(P<0.01),黏附细胞数亦明显增加(P<0.01),PI3K/AKT通路特异抑制剂Ly294002预处理后重组人促红细胞生成素对迁移能力的作用消失,p38MAPK通路特异激动剂anisomycin预处理对两种作用影响均不明显。提示重组人促红细胞生成素具有增强人骨髓间充质干细胞迁移和黏附能力的作用,其增强迁移能力的作用与激活人骨髓间充质干细胞的PI3K/Akt通路有关,但是它对黏附能力的作用与PI3K/Akt和p38MAPK通路均无关。     

Virus-replicating T cells in the immune response of mice. I. Virus plaque assay of the lymphocytes reactive to sheep erythrocytes

Virus plaque-forming cell assay with vesicular stomatitis virus (VSV), which had been originally introduced by Bloom and his colleagues as a tool for the enumeration of activated lymphocytes, was first applied to the immune response of mice to a widely used antigen, i.e. sheep red blood cells (SRBC). When spleen cells taken from mice previously primed with SRBC were cultured in the presence of the antigen, lymphocytes capable of replicating VSV (antigen-induced virus plaque-forming cells, Ag-V-PFC) were generated in the culture. They seemed to appear as early as 1 day of culture, and the peak was attained by the 2nd day. Most of Ag-V-PFC belonged to T-cell population, since 80-90% of Ag-V-PFC was killed by the treatment of cultured cells with anti-thymocyte serum plus complement. In vitro generation of Ag-V-PFC seemed to be highly cross-reactive (about 40%) with a related antigen (horse red blood cells). Ag-V-PFC detected in the present experiment may not represent helper T cells, effector T cells, or their precursors because of the following: (a) The generation of Ag-V-PFC was completely suppressed by the addition of anti-SRBC mouse serum in the culture, though the helper activity was apparently augmented by the same treatment. (b) Development of Ag-V-PFC was almost completely suppressed by the pretreatment of mice with cyclophosphamide 2 days before immunization, by which delayed-type hypersensitivity (DTH) was markedly augmented. (c) After the immunization of mice, Ag-V-PFC began to develop just when the level of DTH declined, at which point helper activity of the spleen cells also diminished. A possible role of Ag-V-PFC in the immune response was discussed.     

EphB6-null mutation results in compromised T cell function

So far, there is very limited knowledge about the role of Eph kinases, the largest family of receptor tyrosine kinases, in the immune system. Here, using EphB6(-/-) mice, we demonstrated that in vitro and in vivo T cell responses such as lymphokine secretion, proliferation, and the development of delayed-type skin hypersensitivity and experimental autoimmune encephalitis in EphB6(-/-) mice were compromised. On the other hand, humoral immune responses, such as serum levels of different Ig isotypes and IgG response to tetanus toxoid, were normal in these mice. Mechanistically, we showed that EphB6 migrated to the aggregated TCRs and rafts after TCR activation. Further downstream, in the absence of EphB6, ZAP-70 activation, LAT phosphorylation, the association of PLCgamma1 with SLP-76, and p44/42 MAPK activation were diminished. Thus, we have shown that EphB6 is pivotal in T cell function.     

28 醇对MPTP 致帕金森病模型小鼠行为学的作用及其机制

目的观察28 醇对帕金森病模型小鼠行为学改善作用,并探讨其可能的机制。方法75 只成年雄性C57 小鼠随机分为5 组：对照组,模型组,28 醇低剂量组(25 mg/kg)、中剂量组(50 mg/kg)、高剂量组(100 mg/kg),每组15 只。除对照组外,其他各组间隔2 h 腹腔注射MPTP 16 mg/kg 共4 次造模。28 醇各剂量组按剂量连续灌胃给药2 周,其他组给予等量0.5%缩甲基纤维素钠(CMC)无菌水。行为学检测采用转杆试验和自发运动试验,Nissl 染色组化实验检测纹状体神经元数量及状态,Western blot 实验检测黑质区p-Erk1/2、p-p38 MAPK、p-JNK表达。结果与对照组相比,28 醇高剂量组转杆试验和自发运动试验成绩改善(P<0.05)。28 醇中剂量和高剂量组含有Nissl 小体的神经元显著增多并染色加深(P<0.05),黑质区p-p38 MAPK、p-JNK蛋白表达与模型组相比下降(P<0.05),p-Erk1/2 表达与模型组无显著性差异。结论28 醇可改善帕金森病小鼠行为学指标。可能通过调节MAPKs 家族信号转导因子p-p38 MAPK、p-JNK蛋白表达,减少黑质纹状体通路的损伤发挥其作用。     

新型免疫抑制剂J2对角膜移植小鼠淋巴细胞中白细胞介素10及干扰素γ分泌的影响

背景：小分子化合物J2是以CD4为靶点的新型免疫抑制剂,课题组以往的实验已经验证了J2对正常小鼠及角膜移植后小鼠脾细胞的影响。目的：探讨小分子化合物J2对异基因角膜小鼠的淋巴细胞中白细胞介素10及干扰素γ分泌的影响。方法：BalB/c（H2d）小鼠随机数字表法分为4个组,安慰剂组、环孢素A组和J2组接受C57BL/6-BALB/c同种异体角膜移植后给予相应药物干预,空白对照组不接受角膜移植,观察各组角膜移植片存活时间。取各组大鼠脾细胞给予刀豆蛋白Ⅵ型刺激细胞增生,采用ELISA法测定细胞上清中白细胞介素10及干扰素γ水平,比较各组细胞增生指数及细胞因子含量。结果与结论：J2可能通过抑制CD4＋T淋巴细胞而抑制角膜排斥反应的发生,延长角膜植片存活时间;J2能够明显抑制ConA刺激角膜移植后小鼠脾细胞的增生及Th1细胞因子的产生。这些效应与环孢素A的效果相似。     

Lack of enteral nutrition blunts extracellular-regulated kinase phosphorylation in gut-associated lymphoid tissue

The mitogen-activated protein kinase (MAPK) family (extracellular-regulated kinase [ERK], p38, etc.) of signal transduction proteins includes important intracellular mediators of inflammation, playing critical roles in host defense. Phosphorylations of ERK and p38 are responsible for cell proliferation, cell differentiation, and cell death. We hypothesized that impaired gut-associated lymphoid tissue (GALT) function in the absence of enteral nutrition is associated with reduced MAPK phosphorylation in GALT cells. Fifty-three male Institute of Cancer Research mice were randomized into 3 groups; ad libitum chow, intragastric (i.g.)-TPN, and intravenous (i.v.)-TPN. TPN groups were administered a standard TPN solution. After 5 days of feeding, lymphocytes from Peyer patches (PPs), the lamina propria (LP) cells, and intraepithelial (IE) spaces in the small intestine were isolated. GALT lymphocyte numbers were determined. The lymphocytes were incubated with or without 50 ng/mL of phorbol myristate acetate (PMA) for 15 min, and phosphorylated ERK (p-ERK) and p38 (p-p38) levels were determined using laser scanning cytometry. In PP (GALT inductive site) lymphocytes, p-ERK was increased after PMA in all three groups. However, ERK phosphorylation in GALT effector sites (IE and LP) was enhanced only in the enteral groups. p38 phosphorylation was not increased in any GALT sites, in any of the three groups, in response to PMA. In another set of mice (n = 33), in vitro LP lymphocyte proliferation was assessed with BrdU with or without PMA. Cell proliferation was increased or maintained at high level with PMA in the i.g.-TPN and chow group, but remained low in the i.v.-TPN group. In conclusion, lack of enteral feeding blunts ERK activation and cell proliferation in response to PMA stimulation in GALT effector sites, which may be an important mechanism underlying reduced GALT function. The influence of nutrition on GALT p38 phosphorylation must be assessed with other types and dosages of stimulants.