放射性AnnexinⅤ活体细胞凋亡显像在肿瘤研究中的应用及进展

肿瘤治疗的目的之一是诱导肿瘤细胞凋亡.在细胞凋亡早期,由于磷脂酰丝氨酸暴露在细胞表面,导致与其有高特异性结合的放射性AnnexinⅤ的摄取增加.放射性核素标记AnnexinⅤ体内凋亡显像具有无创、可早期预警和能够定位、定性的特点,将在肿瘤科学研究和临床诊治中发挥重要作用,可监测肿瘤治疗疗效,评价肿瘤患者预后,指导治疗方案;同时在抗肿瘤新药研发领域也具有重要意义.     

放射性AnnexinⅤ活体细胞凋亡显像在肿瘤研究中的应用及进展

肿瘤治疗的目的之一是诱导肿瘤细胞凋亡。在细胞凋亡早期，由于磷脂酰丝氨酸暴露在细胞表面．导致与其有高特异性结合的放射性Annexin V的摄取增加。放射性核素标记Annexin V体内凋亡显像具有无创、可早期预警和能够定位、定性的特点，将在肿瘤科学研究和临床诊治中发挥重要作用．可监测肿瘤治疗疗效，评价肿瘤患者预后．指导治疗方案：同时在抗肿瘤新药研发领域也具有重要意义。     

凋亡分子影像研究进展

诱导细胞凋亡是各种抗肿瘤治疗的主要机制之一,凋亡分子影像能在活体内动态、无创检测抗肿瘤治疗所引起的细胞凋亡,有助于肿瘤治疗疗效的早期评判和预后分析。本文对目前细胞凋亡分子影像学技术最新研究进展进行综述,主要包括放射性核素显像、MRI、光学成像等。     

流式细胞术Annexin V结合PI或7-AAD双染色法进行细胞凋亡分析的原理是什么?

答 :　在凋亡的早期阶段 ,胞浆膜磷脂的不对称性丧失 ,导致膜内侧磷脂酰丝氨酸 (PS)从细胞膜内层暴露于外层 ,从而可被PS特异的Annexin V探针所标记。PS转移到细胞膜外不是细胞凋亡特有的 ,也可发生在细胞坏死中。但在凋亡的早期细胞膜是完整的 ,而细胞坏死时细胞膜的完整性被破坏。由于碘化丙锭 (PI)或 7 AAD对细胞膜完整的活细胞和早期凋亡细胞是拒染的 ,而对膜完整性被破坏的晚期凋亡细胞或坏死细胞可以染色。因此 ,Annexin V结合PI或 7 AAD进行双染色可以用于检测活细胞、凋亡细胞和坏死细胞。正常活细胞不会被染色 ,凋亡细胞可…     

细胞凋亡超声成像早期评估肿瘤治疗反应的研究进展

细胞凋亡在癌症治疗中具有重要作用,是杀死癌细胞的主要途径。监测细胞凋亡情况可以评价药物疗效,评估肿瘤对临床治疗的早期反应。细胞凋亡超声成像对临床了解肿瘤发展变化、指导个体化治疗、判断预后均有重要意义。本文就超声成像在细胞凋亡监测中的研究进展及在早期评估肿瘤治疗反应中的应用进行综述。     

细胞凋亡近红外线荧光显像在评价肿瘤放疗早期疗效中的作用

目的：研究近红外线荧光（NIRF）标记的annexinV是否能显示小鼠体内细胞凋亡的情况。方法：合成了Cy5．5-annexinV，并对其体外与磷脂酰丝氨酸（PS）的结合特性进行研究。建立小鼠乳腺癌（MCA29）动物模型，将其分为放射治疗组与非放射治疗组，于放射治疗后6h静脉注射Cy5．5-annexJnV，24小时后进行显像，比较两组浓聚Cy5．5-annexinV的差异。结果：Cy5．5-annexinV与PS结合的结合力（Kd）为2．51×10^-9，未标记的annexinV与Ps的Kd为1．54×10^-9，两者间无显著差异，表明经Cy5．5标记后并未改变annexinV与PS的Kd。采用流式细胞检测仪及共聚焦激光扫描显微镜研究表明Cy5．5-annexinV与PS结合的特性与FITC—annexinV相似，说明Cy5．5-annexinV在体外具有鉴别凋亡细胞与正常细胞的能力。小鼠MCA29肿瘤细胞动物模型显像表明经放射治疗后的肿瘤细胞浓聚Cy5．5-annexinV高于未治疗的肿瘤细胞。结论：Cy5．5-annexinV可以用于肿瘤放射治疗的早期疗效评价。     

肿瘤治疗反应的早期评估方案:细胞凋亡超声成像研究进展

细胞凋亡在癌症治疗中起着非常重要的作用，是杀死癌细胞的主要途径。凋亡监测可以判断药物疗效，评估肿瘤对临床治疗的早期反应。凋亡成像对临床全面了解肿瘤发展变化，指导个体化治疗、判断预后效果均具有重要意义。本文主要阐述超声成像在凋亡监测的研究进展以及在肿瘤治疗反应早期评估中的应用。     

细胞因子诱导的杀伤细胞在临床常见肿瘤中的应用现状

细胞因子诱导的杀伤(CIK)细胞是由多种细胞因子,如白细胞介素2(IL-2)、γ干扰素(IFN-γ)和CD3单克隆抗体等诱导而成的,对多种肿瘤具有杀伤活性的细胞毒性T细胞,其溶瘤活性具有非MHC限制性,并且能特异地聚集于肿瘤局部发挥抗肿瘤作用[J].CIK细胞是以CD3+ CD8+、CD3+ CD56+为主的一群异质性细胞,具有较强的增殖活性、杀伤活性和广谱抗肿瘤活性.CIK细胞主要通过3条途径杀伤肿瘤细胞:(1)通过表面表达NKG2D受体(natural killergroup 2D receptor)识别肿瘤细胞表面表达的NKG2D配体,直接杀伤肿瘤;(2)进入体内的CIK细胞能分泌多种细胞因子:IL-2、γ-IFN、肿瘤坏死因子(TNF)等,不仅对肿瘤细胞有直接抑制作用,还可以通过调节机体免疫系统间接杀伤肿瘤细胞,同时增强T细胞的抗瘤功能;(3)通过表达Fasl诱导肿瘤细胞凋亡,同时表达抗凋亡基因(Bcl-2、Bcl-xL、survivin)来抵抗Fasl阳性的肿瘤细胞对其的反作用,使CIK细胞可以耐受表达凋亡相关因子配体的肿瘤细胞诱导的凋亡,从而发挥持久的抗肿瘤效应[2].     

靶向survivin在肿瘤治疗中的研究进展

Survivin蛋白是人类凋亡抑制因子家族中的一员,具有抗凋亡及调节细胞有丝分裂的双重功能。大量研究证明,survivin与肿瘤的发生、发展间关系密切,对肿瘤的诊断及预后判断具有深远意义。在该基础上,survivin已成为新的抗肿瘤靶点。一些临床前研究已通过不同的人类肿瘤移植模型,证明运用各种策略来降低肿瘤细胞中survivin的表达水平,削弱其功能,可增加肿瘤细胞的凋亡率及其对化疗、放疗的     

靶向超声造影剂在肿瘤分子显像中的研究进展

靶向超声造影剂可与肿瘤新生血管内皮细胞或肿瘤组织细胞特异性表达的生物分子相结合,实现肿瘤分子显像,从而提高超声诊断的准确性与敏感性,为肿瘤的早期诊断与治疗以及研究肿瘤细胞发生、发展及凋亡的在体活动规律开辟了新领域.     

In vivo fluorescence molecular imaging of the vascular endothelial growth factor in rats with early diabetic retinopathy

Anti-vascular endothelial growth factor (anti-VEGF) therapy is effective for reducing the severity level of diabetic retinopathy (DR). However, it is difficult to determine the in vivo spatial and temporal expression of VEGF in the DR retina at an early stage. Here, we report a quantitatively fluorescence molecular imaging and image analysis method by creating a VEGF targeted fluorescence imaging probe, which can potentially detect and predict anti-VEGF treatment response. Moreover, the ex vivo multiscale fluorescence imaging demonstrated the spatial correlation between VEGF relative expression and vascular abnormalities in two and three dimensions. It revealed that VEGF was mainly abnormally expressed at the bifurcation of the microvessels, which advances the knowledge of the DR progression by molecular fluorescence imaging. Our study has the potential to achieve early detection of DR disease, provide more insight into understanding anti-VEGF treatment, and may help stratify patients based on the molecular imaging of retinal VEGF.     

New TB vaccines: is there a requirement for CD8 T cells?

MHC class I-restricted CD8(+) T cells are necessary to mount an immune response against Mycobacterium tuberculosis. M. tuberculosis antigens can enter MHC class I cross-processing pathways through a number of different mechanisms, including via the uptake of antigen-containing apoptotic vesicles released by infected cells. A study in this issue of the JCI by Hinchey and colleagues shows that M. tuberculosis inhibits host cell apoptosis and thus may interfere with optimal cross-priming and action of CD8(+) T cells (see the related article beginning on page 2279). M. tuberculosis genetically modified to induce apoptosis is shown to be more effective in priming CD8(+) T cells in vivo and therefore may be a more effective vaccine against tuberculosis than the currently utilized M. bovis BCG vaccine.     

Delta(9)-tetrahydrocannabinol-induced apoptosis in the thymus and spleen as a mechanism of immunosuppression in vitro and in vivo

Delta(9)-tetrahydrocannabinol (THC), the main psychoactive component of marijuana has been shown to suppress the immune response. However, the exact mechanism of THC-induced immunosuppression remains unclear. In the current study, we tested the hypothesis that exposure to THC leads to the induction of apoptosis in lymphocyte populations. Splenocytes of C57BL/6 mice cultured in the presence of 10 microM or greater concentrations of THC showed significantly reduced proliferative response to mitogens, including anti-CD3 monoclonal antibodies (mAbs), concanavalin A (Con A), and lipopolysaccharide (LPS) in vitro. Thymocytes and naive and activated splenocytes exposed to 10 microM or 20 microM THC showed significantly increased levels of apoptosis. Treatment with CB2 antagonist inhibited THC-induced apoptosis in thymocytes and activated splenocytes. Administration of 10 mg/kg body weight of THC into C57BL/6 mice led to thymic and splenic atrophy as early as 6 h after treatment. This effect could be partially inhibited by treatment with a caspase inhibitor in vivo. THC exposure led to reductions in the numbers of all subpopulations of splenocytes and thymocytes examined. Functional studies revealed that splenocytes from THC-treated mice had significantly reduced proliferative response to anti-CD3 mAbs, Con A, and LPS in vitro. Finally, thymocytes and splenocytes exposed to THC in vivo exhibited apoptosis upon in vitro culture. Together, these results suggest that in vivo exposure to THC can lead to significant suppression of the immune response by induction of apoptosis.     

Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl

A critical event during programmed cell death (PCD) appears to be the acquisition of plasma membrane (PM) changes that allows phagocytes to recognize and engulf these cells before they rupture. The majority of PCD seen in higher organisms exhibits strikingly similar morphological features, and this form of PCD has been termed apoptosis. The nature of the PM changes that occur on apoptotic cells remains poorly defined. In this study, we have used a phosphatidylserine (PS)-binding protein (annexin V) as a specific probe to detect redistribution of this phospholipid, which is normally confined to the inner PM leaflet, during apoptosis. Here we show that PS externalization is an early and widespread event during apoptosis of a variety of murine and human cell types, regardless of the initiating stimulus, and precedes several other events normally associated with this mode of cell death. We also report that, under conditions in which the morphological features of apoptosis were prevented (macromolecular synthesis inhibition, overexpression of Bcl-2 or Abl), the appearance of PS on the external leaflet of the PM was similarly prevented. These data are compatible with the notion that activation of an inside-outside PS translocase is an early and widespread event during apoptosis.     

体内肿瘤凋亡显像中PET探针的研究进展

细胞凋亡是生命的一种基本生理机制,对恶性肿瘤的发生和发展有重要意义。近年来,对细胞凋亡机制的研究已有重大进展。检测体内肿瘤细胞的凋亡状态及凋亡水平,对预测肿瘤生物学行为、指导个体化治疗具有重要意义。PET在凋亡显像研究中发挥着重要作用,新型分子探针的研制开发是核医学界研究的热点。     

1alpha,25-Dihydroxyvitamin D3 potentiates cisplatin antitumor activity by p73 induction in a squamous cell carcinoma model

1alpha,25-Dihydroxyvitamin D3 (1,25D3) exhibits antitumor activity in a variety of cancers including squamous cell carcinoma (SCC). Intrinsic resistance of SCC cells to cisplatin was observed and led to the investigation into whether 1,25D3 sensitizes SCC cells to cisplatin. Pretreatment with 1,25D3 followed by cisplatin enhanced growth inhibition in SCC cells compared with 1,25D3 alone as assessed by cytotoxicity and in vitro clonogenic assays. In addition, 1,25D3 sensitized SCC cells to cisplatin-mediated apoptosis. Treatment of tumor-bearing C3H mice with 1,25D3 before cisplatin reduced clonogenic survival using in vivo excision clonogenic assay. These results were not observed in a 1,25D3-resistant SCC variant, indicating the critical role of 1,25D3 in sensitizing SCC cells to cisplatin. Further, a marked decrease in fractional tumor volume was observed when SCC tumor-bearing mice were treated with 1,25D3 before cisplatin compared with either agent administered alone. Cisplatin has been shown to modulate p73 protein level in certain cancer cells. Our data showed that p73 level was not affected by cisplatin but increased by 1,25D3 in SCC cells. Knocking down p73 by small interfering RNA protected SCC cells against 1,25D3 and cisplatin-mediated clonogenic cell kill and apoptosis. Increasing p73 protein level by knocking down UFD2a, which mediates p73 degradation, promoted 1,25D3 and cisplatin-mediated clonogenic cell kill. These results suggest that 1,25D3 potentiates cisplatin antitumor activity in vitro and in vivo in a SCC model system possibly through p73 induction and apoptosis. The combination treatment may provide a more effective therapeutic regimen in cancer treatment.     

Splenic B lymphocyte programmed cell death is prevented by nitric oxide release through mechanisms involving sustained Bcl-2 levels.

Incubation of ex vivo cultured mature B cells in the presence of nitric oxide or nitric oxide-donor substances delays programmed cell death as determined by the appearance of DNA laddering in agarose gel electrophoresis or by flow-cytometry analysis of DNA. Nitric oxide also rescues B cells from antigen-induced apoptosis but fails to provide a co-stimulatory signal that converts the signal elicited by the antigen into a proliferative response. The protective effects of nitric oxide against programmed cell death can be reproduced by treatment of the cells with permeant analogues of cyclic GMP. Regarding the mechanisms by which nitric oxide prevents apoptosis in B cells, we have observed that nitric oxide release prevents the drop in the expression of the protooncogene bcl-2, both at the mRNA and protein levels, suggesting the existence of an unknown pathway that links nitric oxide signaling with Bcl-2 expression.     

Applications of imaging biomarkers in the early clinical development of central nervous system therapeutic agents

The early clinical drug development process increasingly utilizes imaging biomarkers to provide key information in response to a sequential series of questions about potential therapeutic agents. We present several examples of how imaging can answer some of these questions pertaining to the central nervous system (CNS) during the early phases of development of drugs to treat diseases involving the CNS. We also present an overview of the challenges and the potential of using and further qualifying imaging biomarkers for clinical trials.     

Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-β1 secretion and the resolution of inflammation

Ingestion of apoptotic cells in vitro by macrophages induces TGF-beta1 secretion, resulting in an anti-inflammatory effect and suppression of proinflammatory mediators. Here, we show in vivo that direct instillation of apoptotic cells enhanced the resolution of acute inflammation. This enhancement appeared to require phosphatidylserine (PS) on the apoptotic cells and local induction of TGF-beta1. Working with thioglycollate-stimulated peritonea or LPS-stimulated lungs, we examined the effect of apoptotic cell uptake on TGF-beta1 induction. Viable or opsonized apoptotic human Jurkat T cells, or apoptotic PLB-985 cells, human monomyelocytes that do not express PS during apoptosis, failed to induce TGF-beta1. PS liposomes, or PS directly transferred onto the PLB-985 surface membranes, restored the TGF-beta1 induction. Apoptotic cell instillation into LPS-stimulated lungs reduced proinflammatory chemokine levels in the bronchoalveolar lavage fluid (BALF). Additionally, total inflammatory cell counts in the BALF were markedly reduced 1-5 days after apoptotic cell instillation, an effect that could be reversed by opsonization or coinstillation of TGF-beta1 neutralizing antibody. This reduction resulted from early decrease in neutrophils and later decreases in lymphocytes and macrophages. In conclusion, apoptotic cell recognition and clearance, via exposure of PS and ligation of its receptor, induce TGF-beta1 secretion, resulting in accelerated resolution of inflammation.