旁观者效应与免疫

旁观者效应是自杀基因治疗恶性肿瘤的一大特点,它明显地扩大了自杀基因杀伤肿瘤细胞的能力。动物实验结果显示,体内自杀基因的旁观者效应主要与机体的免疫功能有关。如自杀基因疗法与增强机体免疫功能的其它方法结合起来,有望提高杀伤肿瘤细胞的能力     

旁观者效应

1964年3月,在纽约昆士镇的克尤公园发生了一起谋杀案,这起案件很快成为《纽约时报》的头版新闻,并使全国感到震惊。这件谋杀案受注意的原因与凶手、被害者或其谋杀手段都没有什么关系。吉娣·格罗维斯是一位年轻的酒吧经理,她于早上3点回家途中被温斯顿·莫斯雷刺死。莫斯雷是个事务处理机操作员,根本不认识她,他以前还杀死过另外两名妇女。使这场谋杀成为大新闻的原因是,这次谋杀共用了半个小时的时间(莫斯雷刺中了她,离开,几分钟后又折回来再次刺她,又离开,最后又回过头来再刺她),这期间,她反复尖叫,大声呼救,有38个人从公寓窗口听见和看…     

HSV1—tk／GCV基因治疗系统的旁观者效应观察

探讨逆转录病毒（ｒｅｔｒｏｖｉｒｕｓ,ＲＶ）载体介导的单纯疱疹病毒胸苷激酶（ＨＳＶ－ｔｋ）基因转染,联合抗病毒药物核苷类似物羟甲基无环鸟苷（ｇａｎｃｉｃｌｏｖｉｒ,ＧＣＶ）对人卵巢上皮癌细胞系ＴＹＫ细胞杀伤过程中所产生的旁观者效者。采用脂质体介导法将ＰＬＮＴＫ５质粒转入包装细胞ＰＡ３１７后,以滴度最高的ＰＡ３１７病毒上清液感染ＴＹＫ细胞,遗传霉素Ｇ４１８筛选后,得到带有ＨＳＶ１－ｔｋ基因的ＴＹＫ细     

HSV_1tk/GCV基因治疗系统的旁观者效应观察

探讨逆转录病毒 (retrovirus ,RV )载体介导的单纯疱疹病毒胸苷激酶 (HSV1 tk )基因转染 ,联合抗病毒药物核苷类似物羟甲基无环鸟苷 (ganciclovir,GCV )对人卵巢上皮癌细胞系TYK细胞杀伤过程中所产生的旁观者效应。采用脂质体介导法将PLNTK5质粒转入包装细胞PA317后 ,以滴度最高的PA317病毒上清液感染TYK细胞 ,遗传霉素G418筛选后 ,得到带有HSV1 tk基因的TYK细胞。将细胞按不同比例混合培养后 ,给予 10 μg/mlGCV ,4d后用MTT法计算细胞存活率 ,观察旁观者效应。PLNTK5质粒成功转入PA317细胞 ,病毒滴度最高者为 6× 10 5cfu/ml。用病毒上清液感染TYK细胞 ,成功地得到了表达HSV1 tk的卵巢癌细胞株TYK/tk。混合培养结果显示 ,TYK/tk细胞占混合细胞 10 %时 ,低浓度的GCV就可使一半以上的细胞杀死 ,证明了HSV1 tk/GCV治疗方法存在着旁观者效应。逆转录病毒可介导HSV1 tk基因转入TYK细胞并获稳定表达 ,HSV tk/GCV系统存在旁观者效应。     

连接蛋白介导的GJIC与肿瘤自杀基因治疗时旁观者效应的关系

旁观者效应的存在是自杀基因疗法之所以格外引人注目的重要原因之一,但并非对于所有肿瘤细胞均存有这一效应.研究表明,靶细胞连接蛋白的表达及细胞间缝隙连接状况与这一现象关系密切.本文分连接蛋白及缝隙连接概述,旁观者效应在自杀基因疗法中的重要性及其与缝隙连接细胞间交流的关系,加强缝隙连接细胞间交流以促进旁观者效应的方法和策略等三个方面进行了叙述.     

TRAIL受体在肿瘤细胞系上的表达及意义

目的 检测TNF相关凋亡诱导配体（TRAIL）的受体，在来源于血液系统、肝脏、肺脏和大肠的8个肿瘤细胞系中的表达，并探讨其意义。方法 采用半定量RT－PCR，对TRAIL受体的表达进行半定量检测。结果 TRAIL凋亡通路中，能够诱导凋亡反应的死亡受体DR4和DR5，在所检测的肿瘤细胞系中都有表达，其中DR5在所有肿瘤细胞系中的表达水平均显著高于DR4（P＜0．05）。而能够竞争性与TRAIL诱导的凋亡反应的诱骗受体DcR1和DcR2，在所有的肿瘤细胞中都呈低水平表达或不表达。结论 DR5可能在TRAIL诱导凋亡的通路中发挥最重要的作用。TRAIL死亡受体和诱骗受体在肿瘤细胞系中的表达具有差异性，这种差异性可在一定程度上解释不同细胞对TRAIL诱导凋亡的敏感度。     

自杀基因治疗中的旁观者效应及机制

自杀基因治疗肿瘤的作用很大程度上取决于旁观者效应。目前为止 ,旁观者效应机制尚未有明确结论。本文仅从缝隙连接、细胞凋亡、机体免疫、介质扩散及肿瘤缺血坏死等五个方面就旁观者效应机制作一综述     

自杀基因治疗中的旁观者效应及机制

自杀基因治疗肿瘤的作用很大程度上取决于旁观效应，目前为止，旁观效应机制尚未有明确结论，本仅从缝隙连接，细胞凋亡，机体免疫，介质扩散及肿瘤缺血坏死等五个方面就旁观效应机制作一综述。     

人群中，你为何孤立无援——旁观者效应

“2014年2月28日上午,35岁的梁娅倒在深圳地铁出口的台阶上,并保持这一姿势达50分钟监控录像显示,梁娅倒下后发出过求救的动作。三分钟后,有市民发现并告知地铁工作人员随后,地铁工作人员赶到,民警也在半小时后赶到。11点18分,急救人员到达现场,发现梁娅已经死亡。”     

凋亡诱导配体TRAIL与细胞膜脂筏形成的关系

目的: 探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)与细胞膜微结构域脂筏的关系。方法: 采用间接免疫荧光流式细胞术, 分析K562细胞表面TRAIL分子的表达。用FITC标记的霍乱毒素B亚单位 (CTx)对K562细胞的脂筏进行染色; 用抗FITC单克隆抗体(mAb)交联脂筏后, 以兔抗TRAIL分子抗体及Cy3标记的羊抗兔抗体进行间接免疫荧光染色, 激光共聚焦显微镜分析TRAIL分子与脂筏微结构域的关系。结果: FITC -CTx和抗FITCmAb可使脂筏发生交联。脂筏交联的同时TRAIL分子发生聚集。动态观察表明, 抗FITC抗体作用 20min后, 脂筏发生交联, 作用 30min时交联最明显; 随着脂筏的交联, TRAIL分子发生聚集。抗FITC抗体作用 40min后, 脂筏交联程度减弱, 且TRAIL分子逐渐被排除于脂筏之外。结论: 抗FITC抗体可用于CTx-FITC脂筏染色后脂筏交联的研究。TRAIL分子可能与细胞膜脂筏微结构域有关。     

Expression of TRAIL (TNF-related apoptosis-inducing ligand) and its receptors in normal colonic mucosa,adenomas, and carcinomas

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumour cell lines. Four membrane-bound receptors for TRAIL have been identified, two apoptosis-mediating receptors, DR4 and DR5, and two apoptosis-inhibiting receptors, DcR1 and DcR2. The aim of this study was to examine the role of TRAIL and its receptors in colorectal cancer development. The immunohistochemical expression and localization of TRAIL and its receptors were investigated in normal mucosa (n=10), adenomas (n=19), and carcinomas (n=21). Correlations between the expression of TRAIL and its receptors and the degree of apoptosis (assessed by M30 expression) and histopathological characteristics were explored. TRAIL and its receptors were expressed in normal mucosal epithelium. Expression of the receptors was seen in adenomas and carcinomas. TRAIL expression was lost in a subset of colorectal tumours, more frequently in carcinomas than in adenomas (p<0.05). DR4 and DR5 staining was stronger in neoplastic cells than in normal cells and was accompanied by a higher degree of apoptosis. No differences were found between tumour and normal cells regarding DcR1 and DcR2 expression. No correlations were found between TRAIL or TRAIL receptor expression and histopathological characteristics. In conclusion, marked changes were seen in the course of the adenoma-carcinoma sequence with respect to the expression of TRAIL and TRAIL receptors DR4 and DR5. The stronger expression of DR4 and DR5 in neoplastic cells than in normal cells, together with a higher degree of apoptosis, suggests a possible functional role for these receptors in apoptosis induction in neoplastic colorectal cells.     

人TRAIL在毕氏酵母表达系统中的表达

目的 在甲醇营养型酵母（Ｐｉｃｈｉａ）中,表达人可溶性肿瘤坏死因子相关的凋亡诱导配体（ＴＲＡＩＬ）分子。方法 将可溶性ＴＲＡＩＬ基因片段插入酵母表达载体ｐＩＣ３．５,经氯化锂转化酵母ＧＳ１１５,甲醇诱导表达,５ｄ后用ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ进行分析,并用Ｌ９２９细胞测定其活性。结果 在酵母细胞中表达的ＴＲＡＩＬ,ＳＤＳ－ＰＡＧＥ分析占总蛋白的５０％以上。用抗人ＴＲＡＩＬ多抗检测表     

PEGylated TNF-related apoptosis-inducing ligand (TRAIL) for effective tumor combination therapy

Although PEGylated TNF-related apoptosis-inducing ligand (PEG-TRAIL) has good tumor cell specificity and stability, its therapeutic potential is restricted by the development of tumor cell resistance. The purpose of this study was to develop an effective combination therapy with sustained biological activity based on microspheres. Doxorubicin (DOX), PEG-TRAIL, and DOX plus PEG-TRAIL (dual agent) were microencapsulated into poly (lactic-co-glycolic acid) (PLGA) microspheres using a double-emulsion solvent extraction method. Prepared dual agent microspheres showed the encapsulation efficiency 69.4 ± 2.3 for DOX and 87.7 ± 2.9% for PEG-TRAIL. Potential anti-tumor efficacy of this system was investigated in vitro and in vivo in a human colon cancer (HCT116) and in a human prostate cancer (PC-3). DOX and PEG-TRAIL release from dual agent microspheres were biologically active and significantly inhibited the TRAIL-sensitive HCT116 and resistant PC-3 cells in vitro. Dual agent microspheres simultaneous delivery of DOX and PEG-TRAIL was superior to all other DOX or PEG-TRAIL microspheres in vivo. A single local injection of PLGA microspheres loaded with low amounts of DOX, PEG-TRAIL, or dual agent resulted in 14.8, 30.2, and 63.6% reductions in HCT116 tumor volume and 20.4, 14.2, and 67.7% reductions in PC-3 tumor volume at 35 days. Our findings show that dual agent microspheres offer a promising means of delivering DOX and PEG-TRAIL to tumor sites.     

Hydrogen peroxide upregulates TNF-related apoptosis-inducing ligand (TRAIL) expression in human astroglial cells, and augments apoptosis of T cells

The brain is particularly vulnerable to oxygen free radicals, and these radicals have been implicated in the pathology of several neurological disorders. In this study, the modulation of TNF-related apoptosis-inducing ligand (TRAIL) expression by oxidative stress was shown in LN215 cells, an astroglioma cell line. Hydrogen peroxide (H2O2) treatment increased TRAIL expression in LN215 cells and H2O2-induced TRAIL augmented apoptosis in Peer cells, a cell line sensitive to TRAIL- mediated cell death. Our findings suggest that the upregulation of TRAIL in astroglial cells may abrogate immune cell effector functions.     

TNF-related apoptosis-inducing ligand (TRAIL) regulates inflammatory neutrophil apoptosis and enhances resolution of inflammation

Novel therapeutics targeting neutrophilic inflammation are a major unmet clinical need in acute and chronic inflammation. The timely induction of neutrophil apoptosis is critical for inflammation resolution, and it is thought that acceleration of apoptosis may facilitate resolution at inflammatory sites. We previously demonstrated that a death receptor ligand, TRAIL, accelerates neutrophil apoptosis in vitro. We examined the role of TRAIL in neutrophil-dominant inflammation in WT and TRAIL-deficient mice. TRAIL deficiency did not alter constitutive neutrophil apoptosis, whereas exogenous TRAIL accelerated apoptosis of murine peripheral blood neutrophils. We compared TRAIL-deficient and WT mice in two independent models of neutrophilic inflammation: bacterial LPS-induced acute lung injury and zymosan-induced peritonitis. In both models, TRAIL-deficient mice had an enhanced inflammatory response with increased neutrophil numbers and reduced neutrophil apoptosis. Correction of TRAIL deficiency and supraphysiological TRAIL signaling using exogenous protein enhanced neutrophil apoptosis and reduced neutrophil numbers in both inflammatory models with no evidence of effects on other cell types. These data indicate the potential therapeutic benefit of TRAIL in neutrophilic inflammation.     

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in central nervous system inflammation

In a wide variety of acute and chronic central nervous system (CNS) disorders, inflammatory processes contribute to the damage of brain cells and progression of the disease. Along with other regulatory cytokines, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is involved in the pathology of multiple sclerosis (MS) and murine experimental autoimmune encephalomyelitis (EAE), bacterial meningitis (BM), HIV encephalitis (HIVE), stroke and Alzheimer's disease (AD). In these conditions, TRAIL is released within the brain mainly by activated microglia and leukocytes infiltrating from the blood stream. TRAIL promotes apoptosis of parenchymal cells in MS/EAE, HIVE, AD and stroke through interaction with TRAIL death receptors expressed on these cells. Frequently, cells in the diseased brain display increased susceptibility to apoptosis induction by TRAIL due to upregulation of death receptors and downregulation of decoy receptors. On the other hand, TRAIL inhibits the proliferation of encephalitogenic T cells in EAE, and it is involved in the clearance of infected brain macrophages in HIVE and of activated neutrophils in BM by interaction with their death receptors. Especially in BM, the ability of TRAIL to limit an acute granulocyte-driven inflammation carries significant neuroprotective potential. Given the diversity of beneficial and harmful effects in the immune and nervous system, TRAIL is a double-edged sword in diseases involving CNS inflammation.     

Evidence for a role of TNF-related apoptosis-inducing ligand (TRAIL) in the anemia of myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are characterized by impaired erythropoiesis, possibly caused by proapoptotic cytokines. We focused our study on the cytokine TRAIL (TNF-related apoptosis-inducing ligand), which has been shown to exhibit an anti-differentiation activity on erythroid maturation. Immunocytochemical analysis of bone marrow mononuclear cells (BMMC) showed an increased expression of TRAIL in MDS patients with respect to acute myeloid leukemia (AML) patients and normal BM donors. TRAIL expression was increased predominantly in myeloid precursors of granulocytic lineage and in a subset of monocytes and pro-erythroblasts. Significant levels of soluble TRAIL were released in 21 of 68 BMMC culture supernatants from MDS patients. On the other hand, TRAIL was detected less frequently in the culture supernatants of AML (4 of 33) and normal BMMC (0 of 22). Analysis of peripheral blood parameters revealed significantly lower levels of peripheral red blood cells and hemoglobin in the subset of patients whose BMMC released TRAIL in culture supernatants compared to the subgroup of patients who did not release TRAIL. Moreover, TRAIL-positive BMMC culture supernatants inhibited the differentiation of normal glycophorin A+ erythroblasts generated in serum-free liquid phase. Thus, increased expression and release of TRAIL at the bone marrow level is likely to impair erythropoiesis and to contribute to the degree of anemia, the major clinical feature of MDS.     

TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation from monocyte/macrophage lineage precursor cells

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. Recent evidence indicates that the TNF-related apoptosis-inducing ligand (TRAIL) of the TNF ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated T cells. We show in this work that TRAIL can induce osteoclast formation from human monocytes and murine RAW264.7 macrophages. We demonstrated that both cell models differentiate into osteoclast-like cells in the presence of TRAIL in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. The TRAIL-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. However, TRAIL-induced osteoclastogenesis is dependent on activation of NF-kappaB, ERK, and p38 MAP kinase. Thus, our data demonstrate that TRAIL induces osteoclast differentiation via direct engagement with the TRAIL death receptor through a signaling pathway distinct from apoptosis. Our results indicate that in addition to triggering apoptosis, TRAIL induces osteoclast differentiation. It provides a novel role for TRAIL in regulating osteoclast differentiation and in osteoimmunology.     

Improved biological half-life and anti-tumor activity of TNF-related apoptosis-inducing ligand (TRAIL) using PEG-exposed nanoparticles

TRAIL has received considerable attention as a potential anti-cancer agent due to its specific ability to target tumors. However, recombinant TRAIL has several limitations, such as, its short biological half-life, its inherent instability, and its potential hepatotoxicity. In this study, we developed a sustained release nanoparticle formulation of TRAIL and investigated its therapeutic effects in tumor-bearing mice. TRAIL-loaded nanoparticles (NPs) were prepared by mixing PEGylated heparin (PEG-HE), poly-L-lysine (PLL), and TRAIL. NPs prepared by the ionic interaction between polymer and TRAIL showed uniform spherical structures of diameter 213.3 ± 9.7 nm and a surface charge of 5.33 ± 1.2 mV. An in vitro study of the bioactivity of TRAIL in NPs showed that TRAIL-loaded PEG-HE/PLL NPs (TRAIL-PEG-NPs) were slightly less cytotoxic than TRAIL in vitro. To investigate pharmacokinetic parameters, TRAIL and TRAIL-PEG-NPs were intravenously injected into SD rats. The PEG-NP-based formulation demonstrated a 28.3 fold greater half-life than TRAIL alone. To evaluate the anti-tumor effect, TRAIL, TRAIL-loaded HE/PLL NPs (TRAIL-NPs), and TRAIL-PEG-NPs were intravenously injected into HCT-116 tumor-bearing BALB/c athymic mice. The TRAIL-PEG-NP formulation efficiently suppressed tumor growth (>70%), and histological findings confirmed that NPs induced significant tumor cell apoptosis without inducing liver toxicity. The PEG-exposed NP fabrication method applied in this study could be widely applied to protein and peptide delivery systems.