FasL诱导EAE小鼠淋巴细胞凋亡的实验研究

为了解FasL在诱导EAE淋巴细胞凋亡中发挥的作用 ,我们用髓鞘碱性蛋白 (MBP )致敏小鼠 ,建立动物模型 实验性自身免疫性脑脊髓膜炎 (EAE )。分析了EAE动物淋巴细胞膜上Fas及FasL分子的表达水平 ,检测了分泌的细胞因子 ;用FasL分子诱导了EAE淋巴细胞凋亡。结果表明 ,应用MBP致敏KM小鼠 ,成功地建立了EAE模型 ,KM小鼠的发病率为6 8 3% ,发病程度为 1～ 2分 ;小鼠特异性淋转比对照组高 ,并与发病评分成正比 ;细胞因子分泌水平提示以Th2为主 ;EAE淋巴细胞Fas和FasL的表达及凋亡显著高于对照组 ;FasL诱导淋巴细胞凋亡剂量依赖曲线表明 ,在一个较小的浓度范围内呈现正相关 ,若加入单抗可部分阻断FasL诱导的凋亡。结果提示Fas FasL在EAE动物淋巴细胞凋亡中起了重要作用。     

CTLA-4-FasL融合分子的构建及其生物学特性的鉴定

文章通过特异的引物分别扩增出CTLA 4和FasL胞外区的cDNA ,将它们拼接后 ,克隆入真核表达载体pcDNA3 1( + )中 ,进行表达、纯化 ,获得CTLA 4 FasL融合蛋白。Westernblot分析显示了该融合蛋白具有CTLA4 胞外区和FasL胞外区的抗原性。体外试验表明 ,该融合蛋白可以结合Jurkat细胞表面的Fas受体和Raji细胞表面的B7分子 ,表明了该分子双特异性的特点。该融合蛋白能够直接诱导Jurkat细胞发生凋亡 ,且此凋亡效应伴随Raji细胞的参与而增强 ,初步证实了该分子的免疫抑制效应 ,从而为进一步研究该融合蛋白特性及应用奠定了基础。     

反义RNA阻断Hep G2细胞FasL的表达及功能研究

应用分子克隆技术将人FasLcDNA片段反向插入逆转录病毒载体pLXSN ,构建重组质粒pL (hFasL AS )SN ,转染包装细胞PA317后获得FasL反义RNA重组逆转录病毒表达载体假病毒上清 ,经NIH3T3细胞检测其感染滴度后转染肝癌细胞株HepG2细胞并筛选建系 ,命名为HepG2 hFasL AS。半定量RT PCR检测显示HepG2 FasL AS细胞FasL的mRNA明显少于正常HepG2细胞 ;FACS检测显示HepG2 FasL AS细胞FasL的表达与HepG2细胞相比显著下降 ;同时 ,HepG2 FasL AS细胞导致HL 6 0细胞凋亡能力有所下降。表明人FasL反义RNA重组逆转录病毒表达载体能抑制转染细胞FasL的表达并下调其致凋亡功能     

癫痫大鼠海马神经元Fas、FasL和Caspase-3的表达

目的观察Fas、FasL和Caspase-3在癫痫大鼠海马神经元中的表达,探讨Fas、FasL和Caspase-3表达与癫痫的关系,为癫痫的生物治疗提供实验依据。方法通过腹腔注射戊四氮建立癫痫大鼠模型,用免疫组织化学方法和图像分析技术检测Fas、FasL和Caspase-3在癫痫大鼠海马神经元中的表达。结果 Fas、FasL和Caspase-3在癫痫大鼠海马神经元中的表达水平和阳性细胞数量与正常对照组相比均明显增多(<0.05)。结论 Fas、FasL和Caspase-3在癫痫大鼠海马过表达。     

可溶性HLA-G1上调活化的同种反应性T细胞表达FasL促进其凋亡

目的探讨可溶性HLAG1对活化的同种反应性T细胞FasL表达及凋亡的影响。方法借助基因工程技术构建表达可溶性HLAG1的真核表达质粒;把重组质粒转染入宿主细胞,表达可溶性HLAG1并借助免疫亲和层析技术纯化可溶性HLAG1蛋白;用EB病毒转化的同种异体B淋巴细胞作为刺激细胞,通过长期混合淋巴细胞培养,激活同种反应性T细胞。活化T细胞经不同浓度可溶性HLAG1处理12h后,用Westernblot法检测其FasL表达情况;处理24h后,用FACS检测其凋亡情况。结果可溶性HLAG1能够上调活化的同种反应性T细胞表达FasL;能够促进活化的T细胞发生凋亡,且上述作用具有剂量依赖性。结论可溶性HLAG1分子能够使活化的同种反应性T细胞表达FasL升高,进而促进其凋亡。     

人Fas配体蛋白在大肠杆菌中的融合表达与应用

目的：在大肠杆菌中融合表达人Fas配体蛋白。方法：应用RT-PCR技术，从激活的人外周血淋巴细胞中提取总RNA，扩增Fas配体cDNA，克隆入PCR2.1载体，测序验证后，克隆入带有组氨酸盒的表达载体pQE-31，在大肠杆菌中表达，经亲和层析柱纯化后，用SDS-PAGE和Western blot鉴定表达产物。结果：表达的融合蛋白为人Fas配体，其相对分子质量（Mr)为40000。；经透析复性后，具有诱导Jurkat细胞凋亡的作用，用该蛋白分子免疫BALB/c小鼠制备抗血清，以间接ELISA检测了部分自身免疫病与肿瘤患者血清中可溶性的F 苛的含量。结果与进口试剂盒的灵敏性相似。结论：获得FasL单克隆抗体，深入研究FasL的应用提供了材料。     

着丝粒相关蛋白E的抗体制备

目的 制备着丝粒相关蛋白E(CENP-E)的兔源特异性多克隆抗体.方法 应用分子克隆技术构建原核细胞表达质粒pHis-CENPEC410,并将其转化至表达菌株BL-21 (DE3)感受态细胞中;然后用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达His-CENPEC410融合蛋白,再用Ni-NTA镍离子金属螯合树脂柱亲和层析法进行纯化;最后将纯化后的蛋白作为抗原免疫新西兰大白兔,制备特异性CENP-E多克隆抗体,分别用免疫印迹法和免疫共沉淀法对免疫抗体血清进行检测,用免疫荧光染色法检测纯化的抗体.结果 制备的抗体血清可较好地应用于免疫印迹和免疫共沉淀实验,纯化的抗体可用于免疫荧光染色.结论 获得了具有较高特异性和灵敏度的CENP-E多克隆抗体,为后续CENP-E蛋白的研究奠定了基础.     

人CD1d分子胞外区的原核表达及其多克隆抗体的制备

目的:原核细胞表达人CD1d分子胞外区及制备其多克隆抗体.方法:用RT-PCR法扩增人CD1d分子胞外区基因,将其克隆入原核表达载体pET28中,转化大肠杆菌BL21(DE3),用IPTG诱导重组蛋白的表达,用亲和层析法纯化重组蛋白,以之为免疫原免疫小鼠制备多克隆抗体,并以ELISA、Western blot及免疫组织化学法检测抗体.结果:在原核细胞中高效表达和纯化了人CD1d分子胞外区蛋白,用其免疫小鼠,获得了效价高、特异性较好的多克隆抗体,免疫组织化学检测显示该抗体可识别人小肠组织中的天然CD1d分子.结论:成功制备了人CD1d分子胞外区重组蛋白及鼠抗人CD1d分子胞外区抗体,为进一步建立人CD1d分子的免疫学检测方法及其生物学功能的深入研究奠定了基础.     

CTLA4-FasL双功能融合蛋白分子抑制混合淋巴细胞反应及促进淋巴细胞凋亡

目的 构建人CTLA4-FasL融合蛋白真核表达载体，表达CTLA4-FasL融合蛋白，通过体外实验初步研究其生物学特性。方法 通过特异引物分别扩增出CLTA4和FasL胞外区的cDNA，将它们拼接后，克隆入真核表达载体pcDNA3．1( )中，体外表达纯化。Western blot分析CTLA4-FasL融合蛋白的抗原性。体外细胞结合试验研究其结合特异性配体作用。混合淋巴细胞反应研究其抑制免疫应答的效应。结果 测序证实所扩增的PCR产物分别是CLTA4和FasL胞外区的cDNA，其序列与文献报道相符。成功构建了pcDNA3．1-CTLA4-FasL真核表达载体。Western blot分析结果显示，表达获得的蛋白具有CTLA4和FasL的抗原性。体外细胞结合试验显示，CTLA4-FasL融合蛋白可以分别与Jurkat细胞表面的Fas受体和Raji细胞表面的町分子结合。混合淋巴细胞反应结果显示，该融合蛋白可以有效抑制异基因淋巴细胞的刺激作用及诱导淋巴细胞凋亡，并显示了显著的协同效应。结论 成功构建了CTLA4-FasL融合蛋白真核表达载体，体外表达并纯化了CTLA4-FasL融合蛋白，体外实验证实CTLA4-FasL融合蛋白是一个可以有效抑制免疫应答的双功能分子。     

戊型肝炎病毒Ⅰ型ORF3蛋白的表达、纯化及抗原性分析

目的　表达戊型肝炎病毒 (HEV)Ⅰ型ORF3全长基因片段 ,纯化表达产物并进行抗原性分析。方法　将Ⅰ型HEVORF3基因连接到融合表达载体pThioHisB ,IPTG诱导表达 ;Westernblot分析表达产物的抗原活性 ;用经高效液相色谱纯化的表达产物作为抗原 ,制备血清抗 HEVIgG及抗 HEVIgM诊断试剂 ,用国家参考品进行考核 ;用所得抗原检测临床血清中抗 HEV抗体 ,比较其与Genelabs试剂盒检测结果的差异。结果　含有Ⅰ型pThioHisB/ORF3质粒的菌株表达了相对分子质量(Mr)为 2 6× 10 3 的融合蛋白 ,免疫印迹表明其能与戊型肝炎患者恢复期血清发生特异性反应。国家参考品考核结果显示 ,用表达产物制备的抗HEVIgG诊断试剂阳性符合率为 10 0 % (10 / 10 ) ,阴性符合率为 96 .7% (2 9/ 30 ) ,总符合率为 97.5 % (39/ 4 0 ) ;制备的抗 HEVIgM诊断试剂阳性符合率为 83.3%(10 / 12 ) ,阴性符合率为 10 0 % (2 0 / 2 0 ) ,总符合率为 93.8% (30 / 32 )。与Genelabs公司试剂盒检测结果比较 ,抗 HEVIgG和抗 HEVIgMELISA总符合率分别为 94 .5 %和 87.0 %。结论　本实验所得全长ORF3基因工程表达产物具有良好的抗原性 ,可用于制备抗 HEV诊断试剂。     

Fas Ligand-Expressing B-1a Lymphocytes Mediate CD4+-T-Cell Apoptosis during Schistosomal Infection: Induction by Interleukin 4 (IL-4) and IL-10

A previous study of the murine model of Schistosoma mansoni infection has implicated splenic CD19(+) B lymphocytes as Fas ligand (FasL)-bearing mediators of CD4(+) T-lymphocyte apoptosis. The present study shows that B-cell deficiency leads to decreased CD4(+) T-cell apoptosis during infection and compares FasL expression and killer function of B-1a- and CD5(-) B-lymphocyte subsets. B-1a cells from uninfected mice displayed constitutive expression of FasL compared with that of CD5(-) B cells. FasL expression was enhanced following worm egg deposition and antigenic stimulation on both subsets of B cells. Purified B-1a cells from uninfected mice were potent effectors of CD4(+) T-cell apoptosis, and the killing effect was enhanced during schistosome infection. FasL expression by splenic B cells required CD4(+)-T-cell help that was replaced by addition of culture supernatants from antigen-stimulated splenocytes of infected mice. The culture-supernatant-stimulated FasL expression was inhibited by anti-interleukin 10 (IL-10) and anti-IL-4 antibodies. Culture of purified B cells with recombinant IL-4 (rIL-4), rIL-10, and soluble egg antigens (SEA) led to increased expression of FasL on B-1a cells. These results suggest that FasL-expressing, splenic B-1a cells are important mediators of SEA-stimulated CD4(+)-T-cell apoptosis and that maximal FasL expression on B-1a cells is dependent on antigenic stimulation and the presence of IL-4 and IL-10.     

B lymphocytes mediate Fas-dependent cytotoxicity in MRL/lpr mice

The Fas/Fas ligand (FasL) pathway is one of the two major effector mechanisms of T cell-mediated cytotoxicity. To prevent nonspecific killing by lymphoid cells, FasL expression on the cell surface of immune effector cells is strictly regulated. However, MRL/lpr autoimmune-prone mice massively overexpress FasL on their T lymphocytes, which render them able to kill Fas+ targets in vitro and in vivo. It is surprising that we show in the present work that B lymphocytes purified from MRL/lpr spleen cells express FasL to the same extent as T cells at the mRNA and protein level. These B cells are potent cytotoxic effectors against Fas+ but not Fas- targets. The B lymphocyte effectors were used ex vivo without any in vitro activation by B cell stimuli. Furthermore, we found that MRL/lpr B lymphocytes have the same cytotoxic potential as natural killer cells, which have been characterized as potent, Fas-mediated, cytotoxic effectors. The level of membrane-anchored FasL increases with the size of the B cell and cell-surface activation marker CD69 expression, indicating that the expression of FasL is up-regulated in parallel with the activation state of the B cell. The activated B cell population contained the major cytotoxic activity, and a minor part was associated with CD138/Syndecan-1+ plasma cells.     

Soluble egg antigen-stimulated T helper lymphocyte apoptosis and evidence for cell death mediated by FasL(+) T and B cells during murine Schistosoma mansoni infection

Granuloma formation around schistosomal eggs is induced by soluble egg antigens (SEA) and mediated by the activity of CD4(+) Th lymphocytes and their cytokines. Regulation of the inflammatory Th cell response during infection is still insufficiently understood. The hypothesis of this study was that activation-induced cell death (AICD) of CD4(+) T cells is involved in the immune inflammatory response. This study investigated the dynamics of splenic and granuloma CD4(+) Th cell apoptosis and Fas ligand (FasL) expression during the acute and chronic stages of murine schistosomal infection. Enhanced apoptosis of freshly isolated CD4(+) Th lymphocytes commenced after egg deposition and persisted during the peak and modulated phases of granuloma formation. After oviposition, CD4(+), CD8(+), and CD19(+) splenocytes and granuloma cells expressed elevated levels of FasL but FasL expression declined during the downmodulated stage of infection. In culture, SEA induced splenic and granuloma CD4(+) T-cell apoptosis and stimulated expression of FasL on splenic but not granuloma CD4(+) T cells, CD8(+) T cells, and CD19(+) B cells. SEA-stimulated splenocytes and granuloma cells preferentially lysed a Fas-transfected target cell line. Depletion of B cells from SEA-stimulated splenic cultures decreased CD4(+) T cell apoptosis. Coculture of purified splenic B cells with CD4(+) T cells and adoptive transfer of purified B cells indicated that antigen-stimulated B cells can kill CD4(+) Th cells. However, CD4(+) T cells were the dominant mediators of apoptosis in the granuloma. This study indicates that AICD is involved in the apoptosis of CD4(+) T cells during schistosomal infection.     

CD4+ T cell-mediated cytotoxicity toward thyrocytes: the importance of Fas/Fas ligand interaction inducing apoptosis of thyrocytes and the inhibitory effect of thyroid-stimulating hormone

The accumulation of activated CD4+ T cells and antigen (Ag)-dependent cellular interactions between thyrocytes and CD4+ T cells have been determined in thyroid gland from patients with Graves' disease. The Fas/Fas ligand (FasL) interaction between antigen-presenting cells and T cells regulates the apoptosis of the former cells triggered by the latter cells. The inhibition of Fas-mediated apoptosis in thyrocytes could be a underlying mechanism of hyperplasia of thyrocytes in patients with Graves' disease. We investigated the potential role of Fas/FasL interaction between thyrocytes and CD4+ T cells in the induction of Fas-mediated apoptosis of the former cells induced by the latter cells. The presence of only a few specific T cells responsive to a putative autoantigen has hampered the investigation of specific T cell activation toward antigen-presenting cells (APCs). Therefore, we used a superantigen, staphylococcal enterotoxin B (SEB), to examine specific T cell activation toward thyrocytes in vitro since it stimulates a large proportion of T cells with particular Vbeta elements. Spontaneous apoptosis of thyrocytes in culture was not found even in the presence of various kinds of cytokines. In contrast, a clear induction of Fas-mediated apoptosis by anti-Fas IgM was determined in interferon-gamma (IFN-gamma)-stimulated thyrocytes. In addition, a significant cytotoxicity of purified CD4+ T cells toward IFN-gamma-stimulated thyrocytes in the presence of SEB was induced, and the addition of anti-HLA-DR and -DQ monoclonal antibodies (mAbs) or blockade of the Fas/FasL interaction reduced this cytotoxicity. FasL expression of CD4+ T cells cocultured with IFN-gamma-stimulated thyrocytes in the presence of SEB was clearly induced. Furthermore, the addition of mAbs against CD54 and CD58 inhibited both cytotoxicity and FasL expression of CD4+ T cells. The cytotoxicity of CD4+ T cells toward IFN-gamma-stimulated, SEB-pulsed thyrocytes was markedly inhibited when we used thyrocytes cultured with IFN-gamma in the presence of thyroid-stimulating hormone (TSH) as target cells. Our results suggest that 1) CD4+ T cells were activated by thyrocytes expressing MHC class II molecules in an SEB-dependent manner and then expressed FasL. 2) These activated FasL+ CD4+ T cells killed thyrocytes by interacting with Fas on thyrocytes and FasL on activated CD4+ T cells. The presence of costimulating molecules such as CD54 and CD58 on thyrocytes was also necessary to generate activated FasL+ CD4+ T cells. 3) Since the actions of thyroid stimulating antibody (TSAb) toward thyrocytes are similar to those of TSH, one goitrogenic activity of TSAb may, in part, be due to the inhibitory effect on Fas-mediated apoptosis of thyrocytes triggered by activated CD4+ T cells.     

FasL on human nucleus pulposus cells prevents angiogenesis in the disc by inducing Fas-mediated apoptosis of vascular endothelial cells

The intervertebral disc is the largest avascular organ in the human body. However, with the progress of intervertebral disc degeneration (IDD), the disc tends to be vascularized increasingly via angiogenesis. It is well established that both human nucleus pulposus (NP) cells and vascular endothelial cells express FasL and Fas. However, the issue remains open as to whether there are certain active mechanisms preventing angiogenesis in the disc via the FasL-Fas machinery. Here, we established a co-culture system of human NP cells and vascular endothelial (HMEC-1) cells. We found that normal NP cells were more capable of inducing apoptosis in HMEC-1 cells (14.2±3.4%) than degenerate NP cells (6.7±1.9%), p<0.05. By up-regulating the FasL expression in degenerate NP cells, we found that FasL played an essential role in the mediation of HMEC-1 cell apoptosis with the activation of downstream FADD and caspase-3. Furthermore, we found an increased Fas expression in HMEC-1 cells following co-cultured with NP cells, which might be closely linked with FasL produced by NP cells and enhance their interaction. Collectively, this is the first study showing FasL-Fas network might plays an important role in the molecular mechanisms of angiogenesis avoidance of human disc. Consequently, our findings might shed light on the pathogenesis in human IDD and provide a novel target for the treatment strategies for IDD.     

Single base oligodeoxyguanosine upregulates Fas ligand release by nonspecific cytotoxic cells

Nonspecific cytotoxic cells (NCC) are a type of teleost NK-like cell. In the present study a novel stimulus secretion model is described for catfish NCC utilizing single base oligodeoxyguanosine. Binding of guanosine 20-mers (dG20) to NCC up-regulated expression of cytosolic FasL detected by an anti-human FasL monoclonal antibody (mab). In vitro treatment of purified NCC with dG20 produced a 7-fold increase in expression of soluble Fas ligand (sFasL) after 3 h. Antibody binding to NCC was saturable and approximately 30-35% of total NCC were positive for sFasL expression. The teleost FasL equivalent produced programmed cell death of appropriate FasR positive targets. Supernatants from dG20 activated NCC produced hypoploidy and annexin-V binding by FasR bearing HL-60 cells. Treatment of activated supernatants with immobilized anti-FasL mab neutralized these activities. These studies demonstrated that an NK like cell (NCC) produces and secretes sFasL following binding by single base oligodeoxyguanosine.     

Suppression of FasL expression in tumor cells and preventing tumor necrosis factor-induced apoptosis by adenovirus 14.7K is an effective escape mechanism for immune cells

To elucidate if the Fas/FasL signal pathway participates in the immune escape of tumor cells, and if contemporary Fas/FasL and tumor necrosis factor (TNF))-induced apoptosis is better for immune cell survival than just blocking Fas/FasL-induced apoptotic signal. FasL expression in mouse H22 hepatocellular cancer cells was suppressed by the siRNA technique. The wild-type Ad5 14.7K gene was amplified by polymerase chain reaction and transduced into Jurkat T-cells. Apoptosis of target Jurkat cells was detected by flow cytometry. TNF-alpha in the culture supernatant of H22 cells by ELISA was seen. FasL and 14.7K gene expression in stably transfected or transduced clones were determined by Western blotting. As a result, FasL expression in H22 cells was down-regulated after stable transfection with a plasmid encoding antisense FasL cDNA. Down-regulation of FasL expression in H22 cells had no effect on tumor growth in vitro. There was an apparent decrease in the number of apoptotic Jurkat T-cells after coculture with transfected H22 cells, relative to coculture with FasL-expressing untransfected cells. Compared with untransduced Jurkat cells, apoptotic rates in 14.7K-transduced Jurkat cells were significantly reduced in three different E/T ratios (P < 0.01), respectively. We conclude that Fas/FasL signal pathway participates in the immune escape of tumor cells by inducing immune cells apoptosis. Reducing the expression of FasL in tumor cells can decrease the apoptotic rate of immune cells, further blocking the apoptotic signal pathway of immune cells by preventing TNF-induced apoptosis can increase the survival of immune cells.     

The Regulation of FasL Expression—A Distinguishing Feature Between Monocytes and T Lymphocytes/NK Cells with Possible Implications for SLE

Monocytes and lymphocytes from patients with systemic lupus erythematosus (SLE) had a higher cell surface expression of FasL than the corresponding cells from healthy individuals. Inhibitors of metalloproteases upregulated the surface expression of FasL in peripheral blood lymphocytes (PBL), indicating that a metalloprotease is responsible for the cleavage of FasL. The level of sFasL in serum was slightly increased in the patient group compared to the controls. Therefore, the possible contribution of various mononuclear cell types to the release of FasL was analyzed. Isolated NK cells and T lymphocytes released FasL into the medium and the release was prevented by inhibitors of metalloproteases. In contrast, isolated monocytes did not release FasL. FasR expression was elevated in patients with inverted CD4/CD8 ratio, while FasL expression showed no relationship to CD4/CD8 ratio. The absence of FasL release by isolated cells and a high level of surface expression of FasL distinguish monocytes and T lymphocytes/NK cells.