The Potential of the Tumor Microenvironment to Influence Apo2L/TRAIL Induced Apoptosis

Apo2L/TRAIL ligation of specific cell surface receptors (DR4 and DR5) induces apoptosis of many malignant cells with little effect on normal cells. This anti-tumor capability has been demonstrated using cell lines of many tumor types, both in vitro and in vivo when the cells are grown as xenografts. We have extended these studies to investigate the efficacy of Apo2L/TRAIL against patient tumor xenografts in SCID mice and found that the growth of many tumors, both of primary and metastatic origin, can be inhibited by Apo2L/TRAIL. The basis of resistance to Apo2L/TRAIL induced apoptosis in malignant cells and normal cells is not completely understood, but it is known that a variety of factors including hypoxia, MMPs and cytokines present in the tumor microenvironment can influence the response of malignant cells to Apo2L/TRAIL. Currently, the clinical potential of several molecules targeting the Apo2L/TRAIL receptors DR4 and DR5 is being investigated. Our goal in this review is to provide a brief overview of a number of factors that have potential to influence the response of patient tumors to Apo2L/TRAIL.     

Species-dependent serum interference in a sandwich ELISA for Apo2L/TRAIL

To support pre-clinical studies of Apo2L/TRAIL in rodents and non-human primates, a sandwich ELISA was developed using two mouse monoclonal anti-Apo2L/TRAIL antibodies. Mouse, rat, cynomolgus monkey, and chimpanzee serum at concentrations of > or =1% were found to interfere with accurate quantitation of Apo2L/TRAIL. Moreover, the characteristics of the serum interference for each species were different. In order to resolve the observed serum effect, studies were performed in which salts, detergents, and blocking proteins were added to the sample diluent, and optimized sample diluents that eliminated serum interference were developed for mouse, cynomolgus monkey, and chimpanzee serum. These buffers consisted of a base assay diluent (PBS/0.5% BSA/0.05% Tween-20/10 ppm ProClin 300) supplemented with: NaCl (mouse serum); NaCl, EDTA, CHAPS, bovine gamma globulin (BGG), and human IgG (cynomolgus monkey serum); and NaCl and EDTA (chimpanzee serum). Full characterization studies were performed for the "buffer" ELISA run in base assay diluent (intended for non-serum samples) as well as the assays optimized for mouse serum and cynomolgus monkey serum. Precision, accuracy, linearity, and specificity were found to be satisfactory. With the availability of a rabbit polyclonal antibody against Apo2L/TRAIL, a new pAb/mAb ELISA was developed. This assay was not only more sensitive by > or =6-fold, but it was also much less subject to serum interference.     

Influence of the corticospinal tract on the cutaneous silent period: a study in patients with pyramidal syndrome

Celecoxib is a cyclooxygenase 2-selective nonsteroidal anti-inflammatory drug (NSAID) that exhibited therapeutic activity in cancer. In this study three malignant glioma, U87-MG, U251 and A172, were treated with celecoxib, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Single treatment with celecoxib (25-100muM) for 24h resulted in a concentration-dependant decrease of cellular viability in U87-MG, U251 and A172. Combining subtoxic concentrations of celecoxib with TRAIL strongly increased cell death in human malignant glioma cells. After 8h treatment with celecoxib we found down-regulation of the inhibitor of apoptosis protein survivin that was mediated by proteasomal degradation. In addition, over-expression of survivin not only attenuated celecoxib-induced cytotoxicity but also cytotoxicity induced by the combination of celecoxib and TRAIL. Taken together, in malignant glioma survivin is a key regulator in celecoxib- and TRAIL-celecoxib-mediated cell death.     

The potential of the tumor microenvironment to influence Apo2L/TRAIL induced apoptosis

Apo2L/TRAIL ligation of specific cell surface receptors (DR4 and DR5) induces apoptosis of many malignant cells with little effect on normal cells. This anti-tumor capability has been demonstrated using cell lines of many tumor types, both in vitro and in vivo when the cells are grown as xenografts. We have extended these studies to investigate the efficacy of Apo2L/TRAIL against patient tumor xenografts in SCID mice and found that the growth of many tumors, both of primary and metastatic origin, can be inhibited by Apo2L/TRAIL. The basis of resistance to Apo2L/TRAIL induced apoptosis in malignant cells and normal cells is not completely understood, but it is known that a variety of factors including hypoxia, MMPs and cytokines present in the tumor microenvironment can influence the response of malignant cells to Apo2L/TRAIL. Currently, the clinical potential of several molecules targeting the Apo2L/TRAIL receptors DR4 and DR5 is being investigated. Our goal in this review is to provide a brief overview of a number of factors that have potential to influence the response of patient tumors to Apo2L/TRAIL.     

WtCosmc质粒转染对Tn~+肿瘤细胞恶性行为的影响

目的:以Tn~+肿瘤细胞为靶标,研究野生型Cosmc(WtCosmc)质粒转染对其恶性行为的影响,以期为肿瘤的诊断及治疗提供新思路。方法:流式细胞术检测肿瘤细胞表面Tn抗原阳性率,免疫磁珠分选Tn~+与Tn-肿瘤细胞;分选后的Tn~+肿瘤细胞经Fugene 6转染WtCosmc质粒,并用免疫荧光检测细胞表面Tn抗原表达状况。分别采用荧光法、CCK-8和Transwell检测转染前后Tn~+肿瘤细胞的T-synthase活性、细胞增殖、迁移能力及对Apo2L/TRAIL的敏感性。结果:与Tn~-细胞相比,Tn~+肿瘤细胞T-synthase活性及对Apo2L/TRAIL诱导凋亡的敏感性较低;细胞增殖、迁移能力较强。经WtCosmc质粒转染后,Tn细胞T-synthase活性及其对Apo2L/TRAIL诱导凋亡的敏感性明显增高;Tn抗原表达受抑;细胞增殖及迁移能力明显下降。结论:转染WtCosmc可恢复Tn~+细胞T-synthse活性,抑制Tn抗原的表达,进而增强其对Apo2L/TRAL诱导凋亡的敏感性,降低细胞增殖、迁移能力,有效抑制Tn~+肿瘤细胞的恶性行为。     

Involvement of APO2 ligand/TRAIL in activation-induced death of Jurkat and human peripheral blood T cells

The interaction of Fas with Fas ligand (FasL) mediates activation-induced cell death (AICD) of T hybridomas and of mature T lymphocytes. The TNF/TNF receptor system also plays a significant role in AICD of mature T cells and in the maintenance of peripheral tolerance. We previously demonstrated that in human Jurkat leukemia cells, AICD is triggered mainly by the rapid release of preformed FasL upon TCR stimulation. In the present work, we show that the cytotoxic cytokine APO2 ligand (APO2L; also known as TRAIL) is constitutively expressed as an intracytoplasmic protein in Jurkat T cells and derived sublines. APO2L is also detected in fresh human peripheral blood mononuclear cells (PBMC) from a significant number of donors, and the amount of both FasL and APO2L substantially increases upon blast generation. A neutralizing anti-APO2L monoclonal antibody (mAb) partially suppresses the cytotoxicity induced by supernatants of phytohemagglutinin (PHA)-prestimulated Jurkat or human PBMC on non-activated Jurkat cells, indicating that APO2L is released by these cells and contributes to AICD. A combination of neutralizing anti-APO2L and anti-Fas mAb blocks around 60 % of the toxicity associated with supernatants from PHA-activated human PBMC. These results show that FasL and APO2L account for the majority of cytotoxic activity released during AICD, and suggest that additional uncharacterized factors may also contribute to this process.     

缺氧复氧诱导大鼠大脑皮层神经细胞凋亡的形态学研究

目的　　应用原代分离培养的Ｗｉｓｔａｒ胎鼠大脑皮层神经细胞缺氧复氧模型,探讨缺氧复氧诱导神经细胞凋亡的形态学变化。方法　　采用光镜、透射电镜观察并用原位末端标记法（ＴＵＮＥＬ）检测ＤＮＡ断裂。结果　　缺氧复氧可使胎鼠大脑皮层神经细胞发生凋亡,凋亡的细胞皱缩,细胞核染色质凝集、边聚呈半月形等多种形式形成核碎块,内质网扩张,线粒体肿长,其它细胞器未见明显变化。结论　　凋亡参与了缺氧复氧诱导大脑皮层神经细胞死亡过程。     

TNF-α下调bcl-2mRNA表达与HL-60细胞凋亡的关系

以不同浓度TNF-α刺激HL-60细胞,采用DNA凝胶电泳、流式细胞术、RT-PCR等技术,于6h,12h,24h,48h,72h对细胞进行凋亡鉴定.发现一定剂量的TNF-α可诱导HL-60细胞凋亡,HL-60细胞bcl-2mRNA表达明显降低,且与药物呈时间、剂量上的依赖性;IL-2的同时应用,可减轻TNF-α诱导的DNA降解,但bcl-2mRNA的表达无明显改变.结果提示:在粒细胞发生过程中,一定量的TNF-α可直接诱导其产生凋亡,凋亡信号的传导与抑凋基因bcl-2基因的表达有密切关系.     

乙型肝炎病毒I97L变异核壳蛋白对诱导HepG2细胞凋亡的影响

目的研究197L变异核壳蛋白对诱导HepG2细胞凋亡的影响。方法构建EGFP-野毒株核壳蛋白、197L变异核壳蛋白融合表达载体（pEGFP—WT和pEGFP—L97），酶切和测序鉴定；将pEGFP—WT、pEGFP—L97和对照质粒分别转染HepG2细胞．筛选阳性细胞株；荧光显微镜观察阳性克隆细胞荧光蛋白表达，Western—blot检测核壳蛋白表达：以TNF-α、Act-D诱导HepG2细胞株凋亡，0、16、32和48h采用流式细胞术检测细胞凋亡，32h时采用共聚焦检测细胞凋亡比例。结果成功建立了融合表达蛋白表达载体；荧光显微镜显示各细胞克隆有较好的荧光蛋白表达，Western-blot检测各细胞系核壳蛋白表达没有差异；16、32、48h时，pEGFP-WT、pEGFP-L97表达细胞株凋亡率均明显低于pEGFP-C1细胞株（P〈0．05）；32h和48h时，pEGFP-L97细胞株凋亡率明显高于pEGFP-WT细胞株（P〈0．05）；32h时激光共聚焦检测结果与流式细胞术检测结果一致。结论乙型肝炎病毒197L变异核壳蛋白对诱导HepG2细胞凋亡的影响与野毒株核壳蛋白不同，与野毒株核壳蛋白相比，197L变异核壳蛋白对凋亡因素可能更敏感。     

The activity of the Nodal antagonist Cerl-2 in the mouse node is required for correct L/R body axis

Correct establishment of the left/right (L/R) body asymmetry in the mouse embryo requires asymmetric activation of the evolutionarily conserved Nodal signaling cascade in the left lateral plate mesoderm (L-LPM). Furthermore, the presence of Nodal in the node is essential for its own expression in the L-LPM. Here, we have characterized the function of cerl-2, a novel Nodal antagonist, which displays a unique asymmetric expression on the right side of the mouse node. cerl-2 knockout mice display multiple laterality defects including randomization of the L/R axis. These defects can be partially rescued by removing one nodal allele. Our results demonstrate that Cerl-2 plays a key role in restricting the Nodal signaling pathway toward the left side of the mouse embryo by preventing its activity in the right side.     

mCD99L2基因沉默对小鼠B淋巴瘤细胞A20生物学特性的影响

的探究mCD99L2基因沉默对小鼠B淋巴瘤细胞系A20细胞生物学特性的影响。方法采用MTT法和流式细胞仪检测mCD99L2基因沉默前后干扰组A20-LV—mCD99L2和未干扰组A20细胞的增殖率和细胞周期;Transwell法测定两组细胞的体外侵袭能力;采用裸鼠与BALB／c鼠皮下成瘤实验观察比较两组细胞在体内的成瘤时间和成瘤率。结果MTT法显示干扰组A20．LV—mCD99L2增殖率为（0．61±0．12）．低于未干扰组增殖率（0．75±0．20）,差异有统计学意义（P=0．000）;干扰组A20-LV-mCD99L2处于G2期的细胞比例为（10．58±4．97）,高于未干扰组（3．33±1．31）,差异有统计学意义（P=0．009）;Transwell法显示干扰组A20-LV-mCD99L2运动能力较未干扰组下降。裸鼠皮下成瘤时间干扰组（13．33±4．63）d长于未干扰组（9．50±2．90）d。成瘤率均为100％;BALB／c鼠皮下成瘤时间干扰组（10d）长于未干扰组（7．0±0．82）d,成瘤率干扰组为14．3％。显著低于未干扰组（100％）,差异有统计学意义（P=0．000）。结论mCD99L2基因沉默可抑制小鼠B淋巴瘤细胞系A20细胞的生物学行为,降低其在体外的增殖能力和运动能力,显著降低其在BALB／c鼠体内的成瘤率。     

mCD99L2基因沉默对小鼠B淋巴瘤细胞系A20细胞转化为 H/RS样细胞的影响

目的探究mCD99L2基因沉默对小鼠B淋巴瘤细胞系A20细胞转化为H/RS样细胞的影响。方法重组SiRNA表达质粒LV-mCD99L2,体外转染内源性mCD99L2表达阳性的A20细胞,筛选出稳定表达LV质粒的细胞株并扩增培养;采用免疫荧光技术和流式细胞仪检测转化前后两组细胞鼠源CD30表达;透射电镜观察转化后细胞超微结构的形态特点;细胞计数方法动态观测培养细胞干扰组A20-LV-mCD99L2和未经干扰组A20细胞的H/RS样细胞(直径≥25μm)转型率,以人霍奇金淋巴瘤细胞系L428作为对照;采用流式细胞仪检测细胞周期变化。结果获得了稳定表达LV质粒的单克隆细胞株A20-LV-mCD99L2;免疫荧光标记显示转化细胞CD30( );流式细胞仪检测A20-LV-mCD99L2细胞CD30阳性率为54.4%;透射电镜观察转化后细胞核增大,可见单核、双核及多核,核仁明显的H/RS样细胞;干扰组H/RS样细胞的转型率明显高于未经干扰组(P<0.01)。两组处于S期的细胞无明显差异,两组细胞均未见凋亡峰。结论mCD99L2基因沉默可诱导小鼠B淋巴瘤细胞系A20细胞转化为H/RS样细胞。     

经动脉化疗栓塞兔VX2肝癌后早期肿瘤细胞凋亡

目的　研究介入治疗兔VX2肝癌肿瘤细胞凋亡的早期动态改变 .方法　建立 2 7只新西兰大白兔VX2肝癌模型 ,随机分成肝动脉化疗栓塞组 (Transarterialchemoembolization ,TACE)、灌注化疗组 (Transarterialinfusion ,TAI)、灌注肝素生理盐水组 (对照组 ,Control) ,每组各 9只 .介入治疗后每组再随机分成 3个亚组 ,每亚组 3只 .在治疗后第2 4、72、12 0小时分别处死 3组中一亚组 ,取材肿瘤生长活跃的外带组织用流式细胞仪AnnexinV和PI双标记法定量检测细胞凋亡 ,组织病理切片苏木精 -伊红染色 (HE染色 )和甲基绿 -派洛宁染色 (MG -P染色 )光镜下形态学方法观察不同时间肿瘤细胞的凋亡变化 .结果　TACE、TAI、Control组在治疗后 2 4、72、12 0小时流式细胞仪法定量检测肿瘤细胞凋亡比率分别为 11.4 4± 2 .15、10 .99± 1.74、6 .0 0± 0 .5 8,5 .84± 0 .6 8、4 .6 5± 0 .11、2 .88± 1.2 3和 2 .80± 0 .15、2 .19± 1.6 9、2 .5 1± 2 .13.TACE、TAI、Control组凋亡在各时间点均有显著差异 (p <0 .0 1) ,TACE组 2 4、72小时的凋亡明显高于 12 0小时 (p <0 .0 5 ) .TACE组诱导的肿瘤细胞凋亡比率明显高于TAI、Control组 ;TACE组诱导的肿瘤细胞早期凋亡比率随时间呈下降趋势 ,在 12 0小时凋亡仍明显高于其     

生育年龄妇女早期卵泡细胞凋亡和Bcl-2/BAX蛋白表达

目的:研究生育年龄妇女早期卵泡的细胞凋亡和Bcl-2/BAX蛋白表达。方法:12例卵巢组织标本来自进行妇科手术的生育年龄妇女(年龄23-38岁),并经过组织病理学检查证实无明显形态异常。利用末端标记法(TUNEL)和免疫组织化学方法研究早期卵泡(包括始基卵泡、间期和初级卵泡)细胞凋亡和Bcl-2/BAX蛋白表达。结果:早期卵泡内18.75%卵细胞表现TUNEL阳性,但是卵泡内未见TUNEL阳性颗粒细胞。BAX在早期卵泡的卵细胞内表达,阳性表达率76.07%;相反未见Bcl-2在卵细胞表达。另外,早期卵泡内颗粒细胞也没有表达Bcl-2和BAX。结论:生育年龄妇女早期卵泡的卵细胞发生凋亡,促凋亡蛋白BAX在卵细胞凋亡过程中发挥调节作用,由此提示BAX介导的卵细胞凋亡可能是生育年龄妇女早期卵泡闭锁的机制。     

2D/3D刚性图像配准在肿瘤放疗计划中的应用

目的:在肿瘤放疗中,2D/3D刚性图像配准技术是精确定位病人重要保证。方法:数字重建放射影像技术是2D/3D配准中最为关键的部分,一定程度上影响着配准效率。在对数字重建放射影像进行重点研究的基础上,实现了一种基于Bresenham方法的快速数字影像重建算法,并利用腹部CT体数据进行了2D/3D配准实验。实验以互信息、模式强度和梯度差分为相似性测度并采用了Powell-Brent优化算法。结果:在数字重建放射图像算法实验中,Bresemham方法相比于另外两种光线跟踪算法,时间仅需0.467 s。在2D/3D配准实验中,对三种相似性测度的实验结果进行了比较,互信息和梯度差分有较好的配准结果,模式强度仍存在不少问题。结论:实验结果表明,利用Bresenham方法产生的数字重建影像能使得配准具有较高精度,但配准时间仍然较长。     

麻黄碱介导TSLP/OX40L通路调节变应性鼻炎大鼠Th2型免疫反应的作用研究

目的:探讨麻黄碱对卵白蛋白(OVA)诱导的变应性鼻炎大鼠Th2型免疫反应的抑制作用及可能机制.方法:将雌性SD大鼠随机分为4组,对照组、模型组、麻黄碱组(10 mg/kg)和氯雷他定组(2 mg/kg),每组10只.通过OVA诱导变应性鼻炎大鼠模型,并使用药物连续治疗7 d.治疗后,对大鼠进行鼻部症状评分,ELISA法...