淋巴结内FasL蛋白及其mRNA的表达

ＦａｓＬｉｇａｎｄ（ＦａｓＬ）与Ｆａｓ结合诱导细胞凋亡。这一机制对机体免疫反应可能起调节作用。运用ＦａｓＬ多克隆抗体、人工合成ＦａｓＬ寡核苷酸探针分别对淋巴结内ＦａｓＬ蛋白及ｍＲＮＡ进行免疫组织化学检测和原位杂交。结果：正常大鼠淋巴结副皮质区散在分布ＦａｓＬ阳性细胞;非特异性反应性增生的淋巴结内有大量ＦａｓＬ阳性细胞,主要分布在副皮质区,同时输出淋巴管内可见ＦａｓＬ阳性细胞。这为Ｆａｓ系统在参与淋巴细胞凋亡,以维持机体免疫平衡方面的作用提供了证据,同时也提示Ｆａｓ系统可能与某些感染性疾病的发病有关。     

FasL—Fas／APO—1（CD95）系统

Ｆａｓ配体（ＦａｓＬ）与Ｆａｓ受体（Ｆａｓ,即ＡＰＯ－１,又称ＣＤ９５）分别属于肿瘤坏死因子（ＴＮＦ）及ＴＮＦ受体（ＴＮＦＲ）家族,以膜分子或可溶性分子形式存在于哺乳类机体内。ＦａｓＬ或Ｆａｓ单克隆抗体（ＦａｓｍＡｂ）与Ｆａｓ结合可引起Ｆａｓ了性反应细胞的功能增强,继而出现程序性细胞死亡（ＰＣＤ）。ＦａｓＬ与Ｆａｓ相互作用及其所介导的信息途径是目前ＰＣＤ研究的重要内容之一。     

CD40／CD40L介导信号的免疫调节作用

Ｂ细胞的ＣＤ４０分子与活化Ｔ细胞的ＣＤ４０配体（ＣＤ４０Ｌ）结合可调节Ｂ细胞生长分化、克隆转换、Ｉｇ分泌、阻断Ｂ细胞凋亡和导Ｆａｓ表面分隔表达等,直接参与调节体液免疫。ＣＤ４０与ＣＤ４０Ｌ结合也影响细胞免疫功能,诱导Ｔｈ１和Ｔｈ２细胞因子产生《ＣＤ４０和ＣＤ４０Ｌ连接ＴＲＡＦ蛋白多聚化,从而调节基因的转录。ＣＤ４０介导的信号传导过程与蛋白酷氨酸酶、蛋白酪氨酸磷酸化有等有关。     

竞争性PCR检测MLC_2-糜酶融合基因在转基因小鼠中的组织特异性表达

 用竞争性 Ｐ Ｃ Ｒ 方法定量检测了大鼠肌球蛋白轻链 ２ 启动子（ｍ ｙｏｓｉｎ ｌｉｇｈｔ ｃｈａｉｎ ２ ｐｒｏｍ ｏｔ ｅｒ, Ｍ Ｌ Ｃ２） 糜酶（ｃｈｙｍ ａｓｅ）融合基因在转基因小鼠不同组织中的表达情况,发现 Ｍ Ｌ Ｃ２ ｃｈｙｍ ａｓｅ融合基因在转基因小鼠的心脏中有较高水平的表达,在骨骼肌中表达水平较低,在肾脏中有微弱表达;而在肝脏和肺中未检测到．表明 Ｍ Ｌ Ｃ２ ｃｈｙｍ ａｓｅ 融合基因改变了野生型 ｃｈｙｍ ａｓｅ 在生物体内的表达特性,不仅提高了表达效率,而且赋予了一定的心脏组织特异性,为进一步研究 ｃｈｙｍ ａｓｅ 基因在心脏中的功能奠定了基础．     

Fas与FasL的分子及细胞生物学研究进展

Ｆａｓ是Ｉ型膜蛋白,属ＴＮＦ受体家庭成员,许多细胞表达Ｆａｓ。而Ｆａｓ配体（ＦａｓＬ）只在激活Ｔ淋巴细胞表达,是Ⅱ型膜蛋白,属ＴＮＦ家庭成员,当ＦａｓＬ与Ｆａｓ结合时,Ｆａｓ可向细胞传递“死亡信号”,细胞在数小时内凋亡,Ｆａｓ－ＦａｓＬ介导的细胞凋亡在免疫调失及免疫病理中,可能有重要作用。     

Nramp基因家族及其功能

Ｎｒａｍｐ基因家族首先在动物中发现并得以克隆。小鼠 (Ｍｕｓｍｕｓｃｕｌｕｓ)对细胞内病原微生物侵染所具有的抗性或敏感性是受 1号染色体上显性基因Ｂｃｇ、Ｉｔｙ或Ｌｓｈ控制的[1] ,由此将该类基因命名为Ｎｒａｍｐ(ｎａｔｕｒａｌｒｅｓｉｓｔａｎｃｅ ａｓｓｏｃｉａｔｅｄｍａｃｒｏｐｈａｇｅｐｒｏｔｅｉｎ)基因[2 ] 。编码Ｎｒａｍｐ这一膜整合蛋白家族的基因 ,在植物、真菌乃至细菌中也得以克隆。对其功能的分析表明 ,该家族基因可能通过转运金属离子而使生物体产生抵抗病菌侵染的能力。目前 ,国内还未见到有关Ｎｒａｍｐ基因…     

不同刺激对小鼠B细胞表面IgD表达的影响

本文通过细胞流式计（ＦＡＣＳ）技术,利用单克隆抗体抗－ＩｇＤ［Ｉｇ（５ｇ（５ａ）７．２］研究了膜表面ＩｇＤ（ｓＩｇＤ）在新制备的小鼠Ｂ细胞上的表达。并通过用不同的丝裂原刺激脾脏休止Ｂ细胞,研究了不同刺激对Ｂ细胞ｓＩｇＤ表达的调节,发现脾脏Ｂ细胞形成两个主要细胞群,高浮力密度小Ｂ细胞高度表达ｓＩｇＤ的细胞主要是低浮力密度大Ｂ细胞。无论用细菌脂多糖（ＬＰＳ）或抗－ＩｇＭ和白细胞介素－６（ＩＬ－６）刺激     

中华鳖血清中抗体含量的研究

脊椎动物抗体的类型较多 ,如在哺乳类中可分为ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＥ及ＩｇＤ。在血清中的含量也各异 ,如在人血清中ＩｇＧ为 12 5ｍｇ/ｍＬ ,ＩｇＭ为 1 2 5ｍｇ/ｍＬ (杨廷彬和尹学念 ,1994)。低等脊椎动物 (如硬骨鱼 )免疫系统欠发达 ,一般认为其血清中只有 1种抗体即ＩｇＭ ,如大西洋鳕(Ｇａｄｕｓｍｏｒｈｕａ)的抗体浓度达 5 6 2ｍｇ/ｍＬ (Ｌｅｓｌｉｅ＆Ｃｌｅｍ ,1972 )。中华鳖 (Ｔｒｉｏｎｙｘｓｉｎｅｎｓｉｓ)血清中也只含有 1种高分子量的抗体 (简纪常等 ,1998) ,但其血清浓度尚不清楚 ,这有碍于对中华鳖体液免疫…     

口服福氏2a和宋内氏痢疾双价活菌苗FS人群粪抗体检测

本文测试了经三剂（共１×１０１１ｃｆｕ）ＦＳ双价活菌苗口服免疫的１６３名中学生免前及免后１、３和６个月时特异性ｓＩｇＡ粪抗体水平。结果发现,服苗后半年内,分别有７１％和７７．９％的免疫人群特异性抗福氏２ａ和抗宋内氏ｓＩｇＡ有四倍以上升高,且有８２．９％的人群粪便中,两种抗体的应答状况表现一致,说明该菌苗具有良好的免疫原性,并具有双价菌苗的免疫学特性。     

TRAIL及其受体介导的信号转导通路

1 .概述新近克隆的肿瘤坏死因子ＴＮＦ(ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒ,ＴＮＦ)家族新成员———肿瘤坏死因子相关的凋亡诱导配体 (ＴＮＦ ｒｅｌａｔｅｄａｐｏｐ ｔｏｓｉｓ ｉｎｄｕｃｉｎｇｌｉｇａｎｄ ,ＴＲＡＩＬ) [1] 因其结构和功能与ＴＮＦ家族的另一成员ＦａｓＬ/Ａｐｏ 1Ｌ极为相似 ,又称Ａｐｏ 2Ｌ[2 ] 。与其他所有ＴＮＦ家族成员一样 ,ＴＲＡＩＬ属Ⅱ型跨膜糖蛋白 ,Ｃ末端胞外区 ( 1 1 4～ 2 81位氨基酸残基 )含有同源三聚体亚单位 ,在溶液中可形成三聚体或二聚体。可溶性ＴＲＡＩＬ在体外和体内均可诱导多种肿瘤细…     

Induction of Fas and Fas-ligand expression in plasmacytoma cells by a cytotoxic factor secreted by murine macrophages

Induction of tumoricidal activity is one of the major functions of activated macrophages. Our previous study demonstrated that P388D1 murine macrophage-like cells secreted a plasmacytoma cytotoxic factor (PCF) that selectively killed certain tumor cell lines including MOPC-315 plasmacytoma in vitro. Our subsequent studies demonstrated that PCF killed MOPC-315 cells by induction of apoptosis. In this report, the involvement of Fas and Fas ligand (FasL) in PCF-induced apoptosis was investigated. Results suggest that expression of Fas mRNA time-dependently increased in PCF-treated cells and reached an optimal level after 36 h of treatment. The augmented effect of PCF on Fas mRNA expression was significantly reduced by the addition of CB7.C2, an anti-PCF monoclonal antibody. The expression of FasL mRNA was also induced by PCF and reached an optimal level at 24 h, but sharply decreased after 36 h of treatment. Caspase-3 is one of the proteolytic enzymes that can be activated by the Fas-FasL interaction. In our studies, the enzymatic activity of caspase-3 was significantly induced by PCF after 6 h of treatment and reached an optimal level at 12 h. The augmented effect of PCF on caspase activity was significantly reduced by the addition of CB7.C2 and the caspase-3-specific inhibitor, DEVD-fmk. Therefore, PCF-treated plasmacytoma cells might undergo apoptosis via interaction between Fas and FasL.     

人Fas N端融合蛋白的原核表达、纯化及初步分析

目的：对Fas蛋白N端的30～108位肽段进行原核表达,制备可溶性蛋白,并完成二级结构分析。方法：利用BLAST程序进行同源性分析,确定所要构建的FasN端半胱氨酸富集结构域（CRD）,PCR扩增目的基因片段,将其克隆入表达载体pGEX-6P-1,并转化大肠杆菌BL21（DE3）,于16℃用0.2mmol/L的IPTG诱导25h表达GST-Fas融合蛋白;用Glu-tathione Sepharose 4B柱分离GST-Fas融合蛋白,用PreScission蛋白酶切去GST标签,将洗脱的蛋白样品经Superdex 75凝胶预装柱进一步纯化;对所获得的可溶性、高纯度蛋白进行圆二色谱分析。结果与结论：获得重组质粒pGEX-6P-1-fas,并在大肠杆菌中可溶性表达了FasN端CRD功能结构域蛋白;经亲和层析、酶切、分子筛层析后,获得了可溶的、高纯度的FasN端CRD功能结构域蛋白;圆二色谱结果揭示目标蛋白富含β-折叠,为进一步研究Fas的结构和功能奠定了基础。     

宫颈癌SiHa细胞中丛生蛋白、存活素和Fas的表达的研究

目的:Clusterin、Survivin和Fas与肿瘤细胞凋亡密切相关.本研究拟通过检测宫颈癌SiHa细胞中上述这些蛋白的表达水平,以及羟基喜树碱对这些蛋白表达的影响.方法:SiHa细胞分别在普通培养基和舍有羟基喜树碱的培养基中培养,检测细胞死亡,annexin V和PI染色检测细胞凋亡,Western-blotting方法检测Cluste血、Survivin和Fas表迭水平.结果:我们的结果表明,在羟基喜树碱培养的细胞中,在浓度为0.5和3umol/L的培养基培养24小时后凋亡和死亡的肿瘤细胞数量增多,明显高于无化疗药的培养基中的细胞.Western结果显示经过羟基喜树碱处理的细胞Clusterin水平和fas蛋白水平明显高于未经过处理的细胞.在经过0.5和3umol/L的化疗药处理的SiHa细胞Survivin表达下降.结论:与survivin相比,clusterin在对化疗药物抵抗的过程中发挥的作用更大.     

Fas-Fas ligand system as a possible mediator of spermatogenic cell apoptosis in human maturation-arrested testes

To elucidate the mechanism of maturation arrest, known as one of the male infertility, we addressed whether germ cell apoptosis occurs during maturation arrest, and if so, whether Fas and Fas ligand expressions are involved in the apoptosis. By electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), typical apoptotic features were frequently found around the spermatocytic stage in maturation arrest, compared to that in normal testes. When paraffin-embedded sections reacted with anti-Fas antiserum, staining for Fas was found in the plasma membranes of spermatocytes in the maturation-arrested testes, while no positive spermatogenic cells were seen in the normal testes. On the other hand, positive immunostaining for Fas ligand was restricted to Sertoli cells in the maturation-arrested testes as well as in the normal testes, although the intensity of staining for Fas ligand in normal testicular Sertoli cells was much weaker than that of maturation-arrested ones. Thus, these findings demonstrate that "maturation arrest" is characterized by frequent apoptosis of spermatocytes, and that Fas and Fas ligand staining are associated with a high frequency of apoptosis.     

Fas activation in adipocytes impairs insulin-stimulated glucose uptake by reducing Akt

Fas (CD95) belongs to the superfamily of the tumor necrosis factor (TNF) receptors. Besides its key role in apoptosis, Fas contributes to non-apoptotic pathways such as cell proliferation and inflammation. In 3T3-L1 adipocytes, activation of Fas by Fas ligand decreased insulin-stimulated glucose uptake, without affecting cell viability. This decrease in glucose uptake was accompanied by reduced protein expression and diminished phosphorylation of Akt. Similarly, insulin-stimulated glucose incorporation and protein levels of Akt were increased in isolated adipocytes from Fas deficient mice when compared to wild-type mice. In conclusion, Fas activation in adipocytes decreases Akt expression and thereby impairs insulin sensitivity.     

Expression of Inducible Nitric Oxide Synthase and Fas/Fas Ligand Correlates with the Incidence of Apoptotic Cell Death in Atheromatous Plaques of Human Coronary Arteries

It was recently reported that inducible nitric oxide synthase was expressed in advanced atheromatous plaques. So we investigated the effect of NO or peroxynitrite reactive product of NO or O(2)(-) released by iNOS induced in macrophages or T lymphocytes on inflammatory cells in atheromatous plaques of human coronary arteries by immunohistochemistry. We found that iNOS was expressed in T lymphocytes and macrophages in T lymphocytes and macrophages coexisted advanced atheromatous areas. Most of the smooth muscle cells are not coexisted with T lymphocytes. We could not find iNOS in those smooth muscle cells. Only a small number of iNOS-positive smooth muscle cells were found close to T lymphocytes and macrophages. Markers for apoptotic cells induced in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) showed that many apoptotic T lymphocytes and macrophages existed near iNOS induced cells. Fas and Fas ligand were expressed in almost same areas that iNOS was expressed. By double-label immunostaining, Fas was expressed in T lymphocytes but Fas ligand was expressed in macrophages and in some T lymphocytes. These results suggest that NO from iNOS induces Fas and Fas ligand-mediated apoptosis and associates with regression of atherosclerosis. On the other hand, nitrotyrosine was detected wider areas than iNOS. So peroxynitrite from iNOS damages cells and tissues widely and may associate with progression of atherosclerosis. These results suggest an important role of iNOS in mediating both regressive changes and progressive change in atheromatous plaques.     

汉滩病毒诱导小鼠骨髓瘤SP2/0细胞凋亡的初步研究

为研究汉滩病毒对肿瘤细胞的诱导凋亡作用,以一定量病毒悬液感染体外培养的SP2/0细胞,接种后定时间将细胞消化甩片行Gimsa染色观察凋亡细胞核的变化,制细胞悬液以流式细胞仪测细胞周期,并用免疫组化的方法检测凋亡分子Fas和FasL的表达.结果示经病毒诱导后细胞出现生长特性及形态学变化,Giemsa染色观察到典型凋亡细胞;流式细胞仪显示有凋亡峰出现;免疫组化检测出感染后SP2/0细胞中Fas和FasL表达明显升高.该结果表明汉滩病毒可诱导体外培养SP2/0细胞凋亡,其发生可能与凋亡分子Fas和FasL有关.