Fas信号传导研究

Ｆａｓ与抗Ｆａｓ抗体或Ｆａｓ配体结合可以诱导Ｆａｓ阳性细胞凋亡，凋亡信号的传导受到正反两个方面的调控。Ｆａｓ胞内死亡区自聚和／或交联启动死亡信号的传导，涉及Ｆａｓ配体、蛋白激酶、蛋白酪氨酸磷酸酯酶、蛋白酶、神经酰胺、Ｃａ２＋、癌基因等。     

心肌细胞凋亡信号传导通路与心肌损伤

在心肌损伤中,心肌细胞凋亡信号通路包括了依赖于CASPASE的信号途径和不依赖于CASPASE的信号途径,以及炎症、氧化及自噬等信号机制的交联和激活信号途径,心肌细胞的凋亡信号通路决定着心肌的生存与死亡,对心肌损伤的发生和发展及预后产生重要影响。     

整合素激活FAK介导的信号传导途径研究进展

整合素量类重要的细胞表面受体家族，主要介导细胞与细胞上基质的粘附。整合素在胞内外信号传导中起重要作用。ＦＡＫ是整合素信号传导途径的关键酪氨酸激酶，整合素、ＦＡＫ及细胞骨架蛋白共聚于焦点粘附物上，使ＦＡＫ自身磷酸化而激活，ＦＡＫ激活后，其磷酸化的Ｔｙｒ３９７与Ｓｒｃ家族激酶结合，通过形成ＦＡＫ／Ｓｒｃ复合物而引起Ｐａｘｉｌｉｎ和Ｃａｓ磷酸化，后二者通过接头蛋白Ｃｒｋ和Ｇｒｂ２激活Ｒａｓ途径的下游激酶     

信号传导的负性调节因子家族SOCS

细胞因子和相应受体结合 ,引发细胞内信号分子级联反应 ,而SOCS蛋白负性调节细胞因子的JAK STATs信号传导途径 ,且SOCS蛋白作用的直接靶点不同。另一方面STATs可以和SOCS基因的调控序列结合 ,调节SOCS基因的表达。小鼠SOCS基因敲除实验显示 ,该信号负反馈途径有助于调节细胞适度应答。结构上 ,SOCS中间为SH2结构域 ,C 末端是保守的SOCS盒 ,因N 末端差异较大而将SOCS家族分为 5组     

TNF-α与TNF受体超家族介导的信号传导

肿瘤坏死因子α(TNF-α)与其相应的受体结合后通过介导复杂的信号传导而表现生物学活性。目前资料显示主要从以下途径:(1)以神经酰胺(CM)作为第二信使,进而激活Raf途径;(2)以甘油二酯(DAG)作为第二信使,然后激活蛋白激酶C(PKC)和核因子κB(NF-κB);(3)也可能还存在Racl途径。同时,近年还发现在TNFR1与Fas的胞内部分存在一个凋亡结构域。并且发现3种蛋白质能与该结构域发生反应,从而介导凋亡及NF-κB的激活。     

MAPK/JNK信号传导通路研究进展

JNK信号途径参与如胚胎发育、免疫反应、细胞分化等许多正常的生理过程。近年来研究表明 ,JNK信号途径也参与许多病理过程 :JNK介导心脏肥大反应 ,与 型糖尿病发病有关 ,介导胰腺 b细胞凋亡 ;JNK信号途径的异常活化与多种人类肿瘤的发生发展密切相关 ,因此 JNK是一个潜在的治疗分子靶 ,已引起人们的关注。对其功能及作用的分子机制的深入研究 ,有助于我们对 JNK相关疾病的了解和寻找可能的干预和治疗途径     

整合素激活FAK介导的信号传导途径研究进展

整合素是一类重要的细胞表面受体家族,主要介导细胞与细胞外基质的粘附。整合素在胞内外信号传导中起重要作用。FAK是整合素信号传导途径中的关键酪氨酸激酶,整合素、FAK及细胞骨架蛋白共聚于焦点粘附物上,使FAK自身磷酸化而激活,FAK激活后,其磷酸化的Tyr397与Src家族激酶结合,通过形成FAK/Src复合物而引起Paxillin和Cas磷酸化,后二者通过接头蛋白Crk和Grb2激活Ras途径的下游激酶MAPK,通过MAPK改变细胞行为。本文综述了FAK在整合素信号传导中的生物学作用,即介导细胞在ECM上的粘附和迁移,调节细胞增殖和存活等。     

粘附蛋白介导粘附信号传导的机理探讨

为了探讨粘附蛋白介导粘附信号传导的机制 ,我们进行了一系列的实验。首先观察了 CD4 4,Vn在单核细胞粘附于内皮表面过程的作用 ,然后观察了细胞因子 IL- 1、IL- 6、TNF和 IFN对 CD4 4和 Vn表达的调节作用 ,进一步观察了细胞色素 B对 CD4 4、Vn表达和单核细胞粘附的抑制作用 ,最后观察了细胞粘附对单核细胞增殖特性的影响。结果表明 CD4 4和 Vn可介导单核细胞粘附于内皮表面 ,细胞因子可上调 CD4 4和 Vn的表达并促进单核细胞的粘附 ,细胞骨架参与单核细胞的粘附过程 ,细胞粘附过程中单核细胞内 DNA含量和 PCNA表达增加。因此 ,我们推测粘附信号可通过细胞因子作用于粘附蛋白 ,继而通过细胞骨架引起细胞内生物学功能的变化     

蛋白激酶B与信号传导

蛋白激酶B(PKB)是1991年首次被鉴定的一种丝氨酸/苏氨酸类蛋白激酶,它通过依赖或不依赖PI3-K信号途径被多种不同的生长因子激活。PKB在细胞凋亡、糖代谢、蛋白合成中起着十分重要的作用。     

Local Fas/APO-1 (CD95) ligand-mediated tumor cell killing in vivo

Fas/APO-1 (CD95) is a cell surface receptor which mediates apoptosis when ligated by specific antibodies or by its recently cloned natural ligand, FasL. We have studied the cytotoxic potential of FasL in vivo using Fas/APO-1-expressing Yac-1 cells as targets. Supernatant harvested from Neuro-2a cells transfected with the murine FasL cDNA contains FasL and transduces a potent apoptotic signal to Yac-1 cells in vitro. Specificity of FasL-mediated cytotoxicity was confirmed by competition assays using soluble Fas or anti-Fas/APO-1 F(ab')₂ fragments which specifically interfere with FasL-Fas/APO-1 interactions. Intraperitoneal injection of FasL-containing supernatant efficiently killed Yac-1 target cells which had been implanted in capsules into the peritoneal cavity of mice. Analysis of the target cells revealed DNA fragmentation and nuclear changes typical of apoptosis. As previously shown, intraperitoneal injection of anti-Fas/APO-1 antibodies caused liver failure (Ogasawara, J., Watanabe, F. R., Adachi, M., Matsuzawa, A., Kasugai, T., Kitamura, Y., Itoh, N., Suda, T. and Nagata, S., Nature 1993. 364: 806) and was observed at doses which did not reduce Yac-1 cell viability. In contrast, FasL did not induce histopathology in the liver when applied intraperitoneally at doses cytotoxic for Yac-1 cells. However, intravenous administration of FasL induced lethal liver hemorrhages and hepatocyte apoptosis. Thus, locally applied FasL kills tumor cells very efficiently without systemic toxicity and may therefore represent a candidate for local tumor treatment.     

Regulation of cell surface APO-1/Fas (CD95) ligand expression by metalloproteases

APO-1/Fas (CD95) ligand (APO-1L) induces apoptosis in sensitive target cells. Activation-induced T cell death and Ca²⁺-independent cytotoxicity in perforin knockout mice are mediated by APO-1L. To define whether APO-1L is expressed on the surface of activated T cells and to investigate the mechanisms leading to the release of a soluble form, we developed rabbit anti-APO-1L antibodies (Ab). The purified rabbit Ab detected the mature forms of the human and mouse APO-1L of approximately 42 and 40kDa. In addition, the Ab recognized the non-glycosylated form of APO-1L of approximately 32-33 kDa. In activated human T cells, the soluble form of APO-1L was detectable with a moleculas mass of 26 kDa. Immunofluorescence of three human T lymphoblastoid cell lines showed that activation of these cells by phorbol 12-myristate 13-acetate/ionomycin induced a significant increase in cell surface APO-1L only in the presence of metalloprotease inhibitors. Zn²⁺, but not Ca²⁺, prevented the increase in surface APO-1L observed in the presence of 1,10-phenanthroline. Blocking of other classes of proteases (serine- and acid-proteases, chymotrypsin) had no effect. Increased expression of surface APO-1L by metalloprotease inhibitors was not dependent on T cell activation, as the metalloprotease inhibitors also modulated the low level of constitutive APO-1L expression. These results suggest that cell surface expression of human APO-1L is regulated by Zn²⁺-dependent metalloproteases. Cleavage of surface APO-1L may act as a regulatory mechanism to prevent accumulation of the membrane-bound form and may cause systemic effects of the APO-1L.     

松果体及其褪黑素对大鼠胸腺细胞死亡信号通路的影响

目的探讨松果体及其褪黑素对胸腺细胞死亡信号通路的影响。方法选用清洁级SD大鼠,分为正常对照组、假手术对照组、松果体摘除组、松果体摘除 褪黑素腹腔注射7.5 mg/(kg.d)组和松果体摘除 褪黑素腹腔注射15 mg/(kg.d)组。术后4、8周取材。运用免疫细胞化学ABC法染胸腺Fas/FasL、caspase_8、caspase_12阳性细胞,计算机图像分析测量阳性细胞面积及其染色强度。以RT-PCR法检测褪黑素干预原代培养胸腺细胞TNF-β的表达。结果松果体摘除后胸腺细胞Fas染色显示阳性细胞面积显著增大,术后4周皮质阳性细胞面积增加,有统计学意义,术后8周其差异继续扩大。FasL染色结果与Fas类似。松果体摘除后胸腺细胞caspase_8阳性细胞面积术后8周无论皮质还是髓质均显著增加,对caspase_12影响不明显。补充褪黑素后,松果体摘除造成的各种参数的影响逐渐减弱。褪黑素处理后大鼠培养胸腺细胞TNF-β的表达明显减弱。结论松果体能通过影响胸腺细胞死亡信号通路从而调控大鼠胸腺细胞的凋亡,补充褪黑素能缓解相关影响。     

Segregation of APO-1/Fas antigen- and tumor necrosis factor receptor-mediated apoptosis

The cytokine tumor necrosis factor (TNF) and the APO-1/Fas ligand represent typical inducers of apoptosis, i.e. programmed cell death. A limited sequence homology between the TNF receptor TR60 (p55) and the APO-1/Fas (CD95) antigen in their intracellular domains suggests overlapping signaling pathways. The TNF-sensitive cell line KYM-1, which expresses high numbers of both TNF receptors and the APO-1/Fas antigen, is highly sensitive towards triggering of apoptosis by each of the TNF receptors, but resistant to APO-1/Fas stimulation. The opposite response pattern was obtained using the B lymphoid line SKW 6.4, which also co-expresses all three cell surface molecules. Furthermore, no co-modulation of APO-1/Fas by down-regulation of cell surface expressed TR60 and/or TR80 receptors, and vice versa, was found. These data argue against a physical interaction of TNF receptors with APO-1/Fas and, in addition, demonstrate cell specific control of induction of apoptosis and usage of distinct signaling pathways by these receptor molecules.     

RCS大鼠感光细胞凋亡与 Fas蛋白表达

为了探讨遗传性视网膜变性时感光细胞凋亡及其基因调控机制 ,本研究对出生后 9、15、2 0、2 5、3 0、3 5、40、60 d的 RCS大鼠及同龄 SD大鼠各 4只的视网膜进行了 TU NEL 凋亡检测及 Fas蛋白免疫组织化学反应。结果表明 ,出生后 2 5～ 40 d,RCS大鼠视网膜外核层可见 TUNEL阳性的感光细胞核 ,TUNEL阳性细胞数到 3 5 d达高峰 ( P<0 .0 5 )。Fas蛋白免疫组织化学检测发现 ,RCS大鼠视网膜内核层在 15～ 40 d可见 Fas免疫阳性细胞 ,阳性细胞数以 2 5 d为最多 ( P<0 .0 5 ) ;外核层在 2 5 d也可见Fas蛋白免疫阳性反应 ,一直持续到 40 d;节细胞层在 15～ 40 d可见 Fas蛋白表达。到 60 d时则各层又都不见明显的 Fas蛋白阳性反应。本研究结果提示 ,在 RCS大鼠视网膜变性过程中 ,感光细胞发生凋亡 ,Fas蛋白高表达可能与感光细胞的凋亡有关     

Mapping of the linear site on the Fas/APO-1 molecule targeted by the prototypic anti-Fas mAb

Fas/APO-1 is a member of the nerve growth factor/tumor necrosisfactor (TNF) family of receptors and has been shown to mediateapoptotic cell death upon binding of specific mAb. We reporthere that the prototypic anti-human Fas mAb (clone CH-11) inducesapoptosis by binding to a linear epitope present on the extracellulardomain of the Fas/APO-1 protein. Synthetic peptides correspondingto this epitope blocked the apoptotic effect of the antibodyin a susceptible Jurkat T cell line. Based upon the similaritybetween the Fas/APO-1 protein and the recently crystallizedsoluble TNF receptor type I, we generated a molecular modelof Fas/APO-1. Our computer modeling indicates that the antibodybinding region forms a hairpin loop on the surface of the Fas/APO-1protein. These findings further our understanding of the Fas/APO-1-mediatedapoptotic signal and may provide a useful tool in future investigationsof programmed cell death.     

APO-1(CD95)-dependent and -independent antigen receptor-induced apoptosis in human T and B cell lines

Certain B and T cell lines respond to activation signals, e.g.through the antigen receptor, by undergoing apoptotlc cell death.In T cells it has been recently shown that TCR-mediated apoptosisinvolves APO-1/Fas (CD95) receptor-ligand interaction. To investigatewhether the TCR-CD3 complex can trigger alternative apoptosispathways we generated subclones of the T cell line Jurkat whichwere completely resistant towards APO-1-mediated apoptosis.These Jurkat^R cells differed phenotypically from sensitive parentalJurkat^S cells only by the lack of APO-1 protein expression.Although Jurkat^R cells responded normally to anti-CD3 stimulationby expression of APO-1 ligand they failed to undergo anti-CD3-inducedapoptosis. Thus, in Jurkat cells APO-1 -mediated apoptosis wasthe main, and might be the only, mechanism for anti-CD3-inducedcell death. However, BL-60 B cells, highly sensitive to anti-IgM-inducedapoptosis, did not use the APO-1 receptor-ligand system becausethey failed to express APO-1 ligand mRNA. Taken together, ourresults suggest that malignant T and B cell lines may use APO-1receptor-ligand-dependent and -independent antigen receptor-inducedapoptosis pathways respectively. Similarly, differential pathwaysmay be used by T and B cell subsets.