髓鞘碱性蛋白致敏小鼠及其免疫机制的研究

采用有机溶剂、稀盐酸抽提及CM 5 2离子交换提取人髓鞘碱性蛋白 (MBP )抗原 ,并用以致敏KM小鼠 ,观察小鼠实验性变态尖性脑脊髓炎 (EAE)发病情况并检测其免疫功能 ,包括MBP特异性淋巴细胞转化、NK功能、IL 2及MBP抗体。实验结果显示 :按四级打分法 ,6 0只KM小鼠有 41只小鼠出现 1～ 2分临床症状 ,发病率为 6 8 3% ;发病组MBP特异性淋巴细胞转化指数 (SI ) ,NK杀伤功能及IL 2水平明显增高 (P <0 0 5 ) ;MBP抗体与发病程度呈负相关 (r= 0 986 )。实验证明 :自制的人MBP具有生物活性 ,可诱导KM小鼠为EAE模型。     

凋亡相关分子的表达上调在免疫性肝损伤中的作用探讨

检测刀豆蛋白A(concanavalin A,ConA)诱导的小鼠急性肝损伤模型中凋亡相关分子的表达,并探讨其意义。以小鼠尾静脉注射15 mg/kg ConA建立急性肝损伤模型;AnnexinⅤ染色检测肝细胞的凋亡率;流式细胞术检测不同时间点肝细胞表面凋亡受体Fas、DR5的表达,以及肝浸润淋巴细胞表面凋亡配体FasL、TRAIL的表达。结果显示,与正常小鼠肝细胞凋亡率3.36%±0.69%相比,ConA诱导小鼠急性肝损伤后肝细胞的凋亡率显著上升,12 h为13.52%±0.68%,24 h达20.92%±0.66%。同时,肝细胞表面Fas、DR5的表达明显上升,肝浸润淋巴细胞表面FasL、TRAIL亦呈显著上调表达。结果表明,在ConA诱导的急性肝损伤发生发展过程中,肝细胞表面凋亡相关分子Fas、DR5与肝浸润淋巴细胞表面凋亡配体FasL、TRAIL的表达上调以及相互作用是导致肝损伤的重要因素。     

骨髓间充质干细胞Fas／FasL介导的免疫调节在结肠炎治疗中的作用北大核心CSCD

目的探索卵巢摘除术(OVX)所致骨质疏松症小鼠骨髓间充质干细胞(BMSC)与假手术组(sham)BMSC治疗结肠炎效果差异,明确Fas/FasL表达水平改变情况及其下游T淋巴细胞趋化及凋亡程度的差异。方法建立实验性结肠炎模型,OVX小鼠骨质疏松及假手术模型,对比注射OVX组与sham组小鼠BMSC治疗结肠炎的效果,检测2组BMSC对T淋巴细胞趋化及凋亡的差异,采用RT-PCR和Western blot法对BMSC的凋亡相关基因Fas/FasL的表达进行对比。结果注射雌激素缺乏所致骨质疏松小鼠的BMSC与注射假手术组的BMSC相比,表达Fas/FasL降低,从而使T淋巴细胞趋化及凋亡能力减弱,导致用OVX-BMSC治疗结肠炎效果较差。结论 OVX-BMSC通过降低自身Fas/FasL表达减弱肠炎小鼠T淋巴细胞的趋化和凋亡。     

饮水中二溴乙酸对小鼠免疫功能影响机制的研究

目的 探讨二溴乙酸对小鼠免疫功能影响的机制.方法 清洁级BALB/c小鼠40只,分为四组:即二溴乙酸5、20和50 mg/kg剂量组以及阴性对照.连续灌胃28 d后,检测脾和胸腺细胞因子白细胞介素(IL)-2、IL-4分泌水平,脾和胸腺内细胞凋亡率,以及凋亡相关基因(Fas,Bcl-2,TRAF-2和bax)和蛋白(Fas/FasL)的表达.结果 与阴性对照组相比,二溴乙酸染毒组脾细胞的IL-2分泌水平明显增加(P＜0.01)而胸腺细胞的IL-2分泌水平降低(50 mg/kg剂量组有统计学意义,P＜0.05),脾细胞和胸腺细胞的IL-4的分泌水平明显降低(P＜0.01).脾和胸腺细胞凋亡率明显增加.胸腺和脾内的Fas和TRAF2基因表达显著增加,Fas/FasL的蛋白表达显著增加,具有统计学意义(P均为＜0.05).结论 饮水中二溴乙酸对小鼠免疫器官的损伤是通过Fas/FasL途径诱导细胞凋亡.     

血必净对活化诱导T细胞凋亡的调节

目的 观察活化诱导对脾脏T淋巴细胞凋亡、凋亡相关基因mRNA表达及caspase3活性的影响,以及活血化瘀中药的调节作用.方法 提取BALB/c小鼠脾脏T淋巴细胞并培养,以Con A+IL-2诱导T细胞活化凋亡,MTT法检测细胞增殖活性,流式细胞仪检测细胞凋亡率,RT-PCR检测Fas、FasL、Bcl-2、Bax、IL-2 mRNA表达水平,分光光度法测定caspase3酶活性,并观察活血化瘀中药对上述各项指标的影响.结果 活化T淋巴细胞于诱导18h后凋亡率明显增加,于诱导6h时未见FasL、Bax mRNA表达,Fas、Bcl-2 mRNA表达无明显变化;于诱导18 h后Fas、FasL、Bax mRNA表达升高,Bel-2 mRNA表达下降,caspase3活性增高.活血化瘀中药可降低T细胞凋亡,并可分别降低Fas、FasL、Bax mRNA表达,提高Bcl-2 mRNA表达,减轻easpase3酶活性.在活化诱导早期(6 h)促进T淋巴细胞内IL-2 mRNA表达,在晚期(18 h)减少IL-2 mRNA表达.结论 过度活化是脾脏T淋巴细胞异常凋亡的诱发因素,而凋亡的发生与Fas、FasL、Bax、Bcl-2 mRNA表达的改变有关.活血化瘀中药可通过调节IL-2及凋亡相关基因mRNA表达而减轻脾脏T淋巴细胞凋亡,同时可以促进T淋巴细胞的增殖活性.     

Fas,FasL与Ⅰ型糖尿病

Fas和FasL是细胞表面的两种跨膜蛋白.当一个细胞的Fas与另一个细胞的FasL结合时,可诱导表达Fas的细胞凋亡;胰岛中浸润的T淋巴细胞表达FasL可以使表达Fas的胰岛β细胞发生凋亡.Fas诱导的胰岛β细胞凋亡可能在1型糖尿病的发生发展中起重要作用.     

Fas,FasL与1型糖尿病

Fas和FasL是细胞表面的两种跨膜蛋白.当一个细胞的Fas与另一个细胞的FasL结合时,可诱导表达Fas的细胞凋亡;胰岛中浸润的T淋巴细胞表达FasL可以使表达Fas的胰岛β细胞发生凋亡.Fas诱导的胰岛β细胞凋亡可能在1型糖尿病的发生发展中起重要作用.     

CpG-ODN下调HeLa细胞功能性Fas配体的表达

为观察CpG-ODN对宫颈癌细胞系HeLa细胞Fas配体(FasL)表达水平的影响,探讨其对由HeLa细胞Fas-FasL途径诱导的淋巴细胞凋亡作用。采用实时荧光RT-PCR方法检测HeLa细胞、正常宫颈上皮细胞中FasL和Jurkat T淋巴细胞中Fas的表达水平,应用HeLa细胞与Jurkat细胞共培养的方法体外研究HeLa细胞FasL诱导T淋巴细胞凋亡作用。结果显示:①HeLa细胞、正常宫颈上皮细胞中FasL表达阳性,其表达水平分别是(0.99±0.05)、(0.68±0.03),差别具有统计学意义(P=0.0007);Jurkat细胞Fas表达呈阳性;②HeLa细胞与Jurkat细胞共培养后Jurkat细胞的凋亡率为(38.23%±4.98%),应用抗体NOK-2中和HeLa细胞的FasL后,Jurkat细胞凋亡率减少为(3.54%±1.61%),两者相比,差别有显著性意义(P=0.0001);③HeLa细胞用CpG-ODN处理前后FasL的表达水平分别是(0.99±0.05)、(0.79±0.04),差别有统计学意义(P=0.005);CpG-ODN预处理的HeLa细胞与Jurkat细胞共培养后Jurkat细胞凋亡率为(6.41%±2.81%),而没有用CpG-ODN预处理的HeLa细胞与Jurkat细胞共培养Jurkat细胞凋亡率为(29.23±6.85)%,二者的差别有统计学意义(t=13.39,P=0.006)。HeLa细胞可能通过表达FasL主动诱导T淋巴细胞凋亡从而在肿瘤的免疫逃逸中发挥作用,CpG-ODN可通过下调FasL的表达而减少肿瘤细胞主动诱导的T淋巴细胞凋亡。     

自然流产模型小鼠蜕膜细胞凋亡及相关基因的表达

目的 通过比较正常妊娠模型小鼠及自然流产模型小鼠蜕膜细胞凋亡及Bcl-2、Bax、Fas、FasL蛋白的表达,从细胞及分子水平探讨自然流产的发病机制.方法建立正常妊娠模型CBAXBALB/c和自然流产模型CBAXDBA/2.用免疫组化SABC法测定两组模型孕13 d蜕膜细胞Bcl-2、Bax、Fas、FasL蛋白的表达,并通过MIAS-2000医用彩色病理图像免疫组化测量系统对其表达进行半定量分析,其结果用平均灰度值表示;同时应用DNA缺口原位末端标记技术(TUNEL)测定两组模型孕13天蜕膜细胞凋亡情况.结果与正常妊娠模型相比,自然流产模型蜕膜细胞Bcl-2蛋白的表达降低(P＜0.01);Bax蛋白的表达明显升高(P＜0.01);FasL的表达明显升高(P＜0.01);Fas的表达两组比较无明显差异(P＞0.05).蜕膜细胞凋亡指数(AI),自然流产模型明显高于正常妊娠模型(P＜0.01).结论 早孕期蜕膜组织细胞凋亡异常是自然流产的机制之一,Bcl-2/Bax,Fas/FasL途径可能是诱导早孕期蜕膜细胞凋亡的重要因素.     

凋亡相关因子参与自身免疫性甲状腺疾病发病的研究

目的:了解细胞凋亡相关蛋白Fas,FasL和Bcl-2表达在自身免疫性甲状腺疾病发病机制及病理变化中的作用及意义。方法:采用免疫组织化学方法,检测20例桥本甲状腺炎,20例Graves病以及20例甲状腺腺瘤(作为对照组)患者甲状腺标本中Fas、FasL和Bcl-2表达及分布。结果:Fas在所有的标本中表达,主要分布于甲状腺滤泡细胞表面和细胞质上。除3例甲状腺瘤标本外,其余均表达FasL。Bcl-2表达于15例桥本甲状腺炎、19例Graves病以及17例甲状腺瘤滤泡细胞上。在甲状腺瘤滤泡细胞上表达中等强度Fas,很少或是没有表达FasL。在桥本甲状腺炎中Fas和FasL免疫染色强阳性甲状腺滤泡细胞多分布于浸润淋巴滤泡附近,浸润淋巴细胞中Fas、FasL免疫染色相对较弱。在Graves病中,Fas表达强度与桥本甲状腺炎类似,但FasL表达却更弱。在Graves病和甲状腺瘤组织中,Bcl-2表达两者类似。但在桥本甲状腺炎组织中,分布于浸润淋巴细胞附近的甲状腺滤泡细胞以及生发中心的淋巴细胞上,Bcl-2表达很弱。结论:Fas、FasL和Bcl-2表达在桥本甲状腺炎和Graves病中相似。FasL高表达和Bcl-2低表达可能引起桥本甲状腺炎滤泡细胞凋亡。进一步证明3种凋亡相关因子在自身免疫性甲状腺疾病发病机制中的作用。在桥本甲状腺炎中,滤泡细胞凋亡并非由浸润淋巴细胞其FasL发挥作用直接杀伤,但是它们能分泌细胞因子促进滤泡细胞自身Fas、FasL表达,从而导致滤泡细胞凋亡。     

Analysis of the expression of Fas, FasL and Bcl-2 in the pathogenesis of autoimmune thyroid disorders

To investigate the expression of apoptosis-related protein (Fas, FasL, and Bcl-2) in the pathogenesis of autoimmune thyroid disorders (ATDs), immunohistochemical staining was performed on 20 Hashimoto‘s thyroiditis (HT), 20 Graves‘ disease (GD), and 20 thyroid follicular adenoma (TFA, as control). All the cases expressed Fas, mainly on the cell surface and cytoplasm. FasL was found in 17 cases of the TFA. Bcl-2 was detected in 15 cases of HT, 19 of GD and 17 of TFA. In T FA, a moderate Fas expression and a minimal or no FasL expression was detected on follicular cells. In HT, the follicles adjacent to infiltrating lymphocytes showed increased levels of Fas and FasL expression. A weaker staining of Fas and FasL was exhibited on infiltrating lymphocytes than on thyrocytes. In a comparison of GD with HT, thyrocytes and lymphocytes showed similar Fas staining, but for FasL the staining was rather weaker in HT. The expression of Bcl-2 was nearly identical in GD and TFA, but much weaker on the follicular cells in vicinity of lymphocytes and on the lymphocytes located in germinal centers of HT tissues. The expression of Fas, FasL, Bcl-2 in Hashimoto‘s thyroiditis and Graves‘ disease were almost same. FasL strong expression and Bcl-2 weak expression on the follicles in HT may induce apoptosis. These results provided evidence for expression of Fas, FasL and Bcl-2 in the pathogenesis of autoimmune thyroid disease. The lymphocytes seem not to be directly engaged in the process v/a their own FasL, but they may provide some cytokines that, in turn, upregulate Fas and/or FasL expression to induce apoptosis.     

Microglia are more susceptible than macrophages to apoptosis in the central nervous system in experimental autoimmune encephalomyelitis through a mechanism not involving Fas (CD95)

Morphological studies have shown that macrophages and microglia undergo apoptosis in the central nervous system (CNS) in acute experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. To assess the relative levels of macrophage and microglial apoptosis, and the molecular mechanisms involved in this process, we used three-colour flow cytometry to identify CD45lowCD11b/c+ microglial cells and CD45highCD11b/c+ macrophages in the inflammatory cells isolated from the spinal cords of Lewis rats 13 days after immunization with myelin basic protein (MBP) and complete Freund's adjuvant. Simultaneously, we analyzed the DNA content of these cell populations to assess the proportions of cells undergoing apoptosis and in different stages of the cell cycle or examined their expression of three apoptosis- regulating proteins, i.e. Fas (CD95), Fas ligand (FasL) and Bcl-2. Microglia were highly vulnerable to apoptosis and were over-represented in the apoptotic population. Macrophages were less susceptible to apoptosis than microglia and underwent mitosis more frequently than microglia. The different susceptibilities of microglia and macrophages to apoptosis did not appear to be due to variations in Fas, FasL or Bcl- 2 expression, as the proportions of microglia and macrophages expressing these proteins were similar, and were relatively high. Furthermore, in contrast to T cell apoptosis, apoptosis of microglia/macrophages did not occur more frequently in cells expressing Fas or FasL, or less frequently in cells expressing Bcl-2. These results indicate that the apoptosis of microglia and CNS macrophages in EAE is not mediated through the Fas pathway, and that Bcl-2 expression does not protect them from apoptosis. Expression of FasL by macrophages and microglia may contribute to the pathogenesis and immunoregulation of EAE through interactions with Fas+ oligodendrocytes and Fas+ T cells. The high level of microglial apoptosis in EAE indicates that microglial apoptosis may be an important homeostatic mechanism for controlling the number of microglia in the CNS following microglial activation and proliferation.      

转染反义Fas阻断T细胞凋亡及对肿瘤的治疗意义

目的 通过阻断Ｔ细胞的Ｆａｓ信号传递途径,探讨消除肿瘤对Ｔ细胞的攻击及其对肿瘤的治疗意义。方法 流式细胞术、ＲＴ－ＰＣＲ方法检测卵巢癌细胞表达Ｆａｓ和ＦａｓＬ。构建ｐｃＤＮＡ３－反义Ｆａｓ真核表达载体,经脂质体转染Ｊｕｒｋａｔ细胞,流式细胞仪检测Ｆａｓ表达变化。以Ａｎｎｅｘｉｎ－Ｖ和ＭＴＴ法检测转染反义Ｆａｓ基因对Ｊｕｒｋａｔ细胞凋亡的影响。采用ＭＴＴ体外杀伤实验观察３ＡＯ对Ｊｕｒｋａｔ细胞杀伤变化。结果 ６种卵巢癌细胞均表达Ｆａｓ和ＦａｓＬ。ｐｃＤＮＡ３－反义Ｆａｓ基因可以使Ｊｕｒｋａｔ细胞表达Ｆａｓ量下降并部分阻断Ｆａｓ单抗诱导的Ｊｕｒｋａｔ细胞凋亡,３ＡＯ对其杀伤减弱。结论 卵巢癌细胞表达ＦａｓＬ可能是其逃逸免疫监视并产生对淋巴细胞攻击的原因之一;应用反义技术阻断Ｆａｓ表达,可部分阻断Ｆａｓ单抗诱导Ｊｕｒ     

Overexpression of Fas and FasL Is Associated with Infectious Complications and Severity of Experimental Severe Acute Pancreatitis by Promoting Apoptosis of Lymphocytes

This study investigated the relationship of Fas and Fas ligand (FasL) expression and apoptosis of lymphocytes in relation to the pathogenic immune response and infectious complications observed in experimental severe acute pancreatitis in mice. Forty male Balb/c mice were randomly divided into control, mild (MAP), and severe acute pancreatitis (SAP) groups. Overexpression of Fas/FasL messenger ribonucleic acid (mRNA) and protein was observed in spleen-derived lymphocytes in SAP (p?<?0.01). Apoptosis of these resulted in a depletion of circulating lymphocytes in this group (p?<?0.05). A further significant change in the SAP group with infectious complications was observed. A positive relationship was found between the Fas/FasL expression and lymphocyte apoptosis, and negative relationships were observed between Fas/FasL expression and CD4⁺ and CD19⁺ lymphocytes and the CD4⁺/CD8⁺ ratio in SAP mice (p?<?0.01). The results suggest that the overexpression of Fas/FasL is associated with infectious complications and severity of experimental severe acute pancreatitis by promoting apoptosis of lymphocytes.     

Polarization and apoptosis of T cell subsets in idiopathic thrombocytopenic purpura

It is well-known that idiopathic thrombocytopenic purpura （ITP） is an acquired organ-specific autoimmune hemorrhagic disease and dysfunctional cellular immunity is considered important in the pathophysiology of ITP. However, polarization patterns and apoptosis profiles of T lymphocytes remain unclear. In this study, we investigated the polarization of T cell subsets, the expressions of apoptotic proteins Fas/FasL on the subsets and the level of anti-apoptotic gene bcl-2 and bax mRNA. It was demonstrated that the ratios of Thl/Th2 and Tcl/Tc2 in ITP children were increased obviously and that the average percentages were increased clearly for Thl and Th2, but not for Tcl and Tc2. In ITP children, the enhancing expressions were detected for FasL on Thl and Tcl and for Fas on Th2 and Tc2. With increasing level of bcl-2 mRNA and decreasing expression of bax mRNA in ITP children, the ratio of bcl-2/bax mRNA was improved obviously, which was positive correlated with the ratio of Thl/Th2. Taken together, our findings indicate that ITP is a Thl predominant disease. This polarization pattern of T cell subsets might be related to the high ratio of bcl-2/bax mRNA and the abnormal expressions of Fas and FasL on T cell subsets.     

Fas/FasL mediated apoptosis of thyrocytes in Graves' disease.

We examined in the present study the possible involvement of Fas and its ligand (FasL) in the process of Graves' disease. Immunohistochemical analysis showed that few normal thyrocytes expressed Fas but many thyrocytes in Graves' disease expressed this molecule. The percentage of FasL-positive thyrocytes in Graves' thyroids was, however, less than in normal thyroids. Several apoptotic thyrocytes and infiltrating mononuclear cells (MNCs) were detected scattered throughout Graves' thyroid tissues and abundant proliferating cell nuclear antigen (PCNA)-positive thyrocytes were present. Apoptotic cells, as well as PCNA-positive cells, were scarcely detectable in normal thyroid glands, however. In vitro treatment of thyrocytes by IL-1beta a cytokine found to be expressed in Graves' thyroid glands, increased Fas but reduced FasL expression. IL-1beta-stimulated thyrocytes became sensitive to apoptosis by anti-Fas IgM monoclonal antibody (mAb). Activated T cells, which strongly expressed FasL, showed cytotoxic activity toward IL-1beta-stimulated thyrocytes but not toward unstimulated thyrocytes. This cytotoxic activity involved the Fas/FasL pathway. Importantly, unstimulated thyrocytes could kill activated, but not resting, T cells. IL-1beta-stimulated thyrocytes, with down-regulated FasL expression, could not efficiently kill activated T cells. The cytotoxic activity of unstimulated thyrocytes toward activated T cells was inhibited by anti-FasL mAb. Interestingly, unstimulated thyrocytes induced apoptosis in IL-1beta-stimulated thyrocytes but not in unstimulated thyrocytes. These interactions were also blocked by anti-FasL mAb. Our results suggest that the apoptotic cell death of both thyrocytes and infiltrating MNCs found in Graves' thyroid glands is regulated by IL-1beta through Fas/FasL interactions.     

雷公藤内酯醇对致敏大鼠淋巴细胞凋亡的影响

目的 探讨雷公藤内酯醇（TP）体内外对致敏大鼠淋巴细胞淋巴细胞凋亡的影响。方法 采用卵蛋白（OVA）致敏并反复刺激建立过敏性气道炎症模型,24只SD大鼠随机分为正常组、阳性对照组和TP处理组,每组8只。用TUNEL原位末端标记法,DNA电泳及电镜等方法,观察体内外TP对致敏大鼠淋巴细胞凋亡的影响及机制。结果 致敏大鼠BALF中嗜酸性粒细胞（Eos)、淋巴细胞均较正常组明显增高（P＜0．05）。体内应用TP可减少致敏大鼠BALF中Eos、淋巴细胞数目,同时可增加其BALF中淋巴细胞凋亡百分率。体外实际显示,不同剂量TP呈剂量依赖性（10^-7-10^5g/ml）的促进OVA抗原刺激的脾淋巴细胞凋亡,该效应随作用时间凋亡,可能是其抗炎机制之一,并可能是通过Fas/FasL途径发挥作用;同时TP可明显增加DXM的促淋细胞凋亡作用。为阐明TP对抗哮喘气道炎症的作用机制和探讨激素依赖性哮喘治疗的新途径提供了有意义的实验资料。