凋亡过程中膜联蛋白V（AnnexinV）的检测

1原理 细胞凋亡具的各种不同的形态学特征,细胞膜的改变是最早的变化之一.在凋亡细胞,位于细胞膜内的磷脂酰丝氨酸( phospholipid phosphatidyl-serine, PS)转位到细胞膜外.膜联蛋白V是一种35～36kDa的钙依赖的.磷脂结合蛋白,其与PS有高度的亲和性,能与表达ps的细胞结合.膜联蛋白可与萤光染料异硫氰酸FITC或生物素(biotin)结合.因此可用于流式细胞术检测凋亡过程中的细胞.     

人膜联蛋白V的重组表达与细胞凋亡显像

移植肿瘤模型的研究表明,化疗和放疗对肿瘤的杀伤效应是通过对细胞凋亡过程的迅速诱导来实现的[1]。膜联蛋白V(Annexin-V)结合细胞膜磷脂酰丝氨酸(phosphatidyl-serine,PS)。PS是组成型的阴离子细胞膜磷脂,在正常情况下,它位于细胞膜脂质双层结构的内侧,而启动程序性细胞死亡的     

慢性肾衰竭病人红细胞表面磷脂酰丝氨酸的表达

目的: 研究血液透析(HD)、腹膜透析(CAPD)、无透析慢性肾衰竭(Non-D)病人及正常人红细胞表面磷脂酰丝氨酸(PS)的表达情况, 以探讨PS的表达对红细胞功能影响的关系。方法: 采用对PS有高度亲和力的磷脂结合蛋白annexin V检测技术, 利用流式细胞仪对细胞进行荧光测定。结果: HD病人红细胞PS的表达较高(2113±1112)%, CAPD组(1135±0184)%和Non-D病人(1139±0170)%相对较低, 但都明显高于正常人对照组(0171±0134)%。结论: 慢性肾衰竭(CRF)病人PS的高表达与尿毒症之间有明显的相关性。     

脓毒症患者外周血红细胞膜磷脂酰丝氨酸的外露

目的研究脓毒症患者外周血红细胞的形态改变及红细胞膜表面磷脂酰丝氨酸（PS）的外露情况。方法采用配对设计随机分组方法,分别对正常对照组（n=30）和脓毒症患者组（n=30）进行静脉采血,行瑞氏染色血涂片观察和测定红细胞聚集指数,并采用对Ps有高度亲和力的磷脂结合蛋白AnnexinV检测技术,利用流式细胞仪对红细胞进行荧光测定。结果显微镜下观察脓毒症患者外周血红细胞形态发生明显异常改变,呈棘形、泪滴形、球形、缗线形等变化,细胞明显聚集;同时红细胞聚集指数测定结果显示脓毒症组较正常对照组也明显升高,其差异有统计学意义（P〈0．05）。脓毒症组Ps外露的红细胞比例明显高于正常对照组,其差异有统计学意义（P〈0．001）。结论脓毒症患者外周血红细胞表面Ps的外露表达明显增加,同时红细胞形态发生明显异常变化。     

膜联蛋白A2与肿瘤进展的相关性北大核心CSCD

膜联蛋白A2（ANXA2）是众所周知的钙离子依赖性磷脂结合蛋白,广泛分布于各种真核细胞的胞核、胞质及细胞外膜。它作为功能多样的蛋白质影响多种细胞和分子功能活动。ANXA2的表达或调节失常涉及一系列疾病,包括自身免疫性疾病、神经系统退行性变、抗磷脂抗体综合征、炎症、糖尿病及各种肿瘤。     

巨噬细胞识别清除衰老红细胞的研究进展

红细胞在体内循环过程中逐渐受损、衰老,其膜表面分子磷脂酰丝氨酸(PS)、CD47和带3(Band 3)蛋白等发生改变,并通过与巨噬细胞膜表面受体相互作用,进而被巨噬细胞识别、吞噬。本文综述了巨噬细胞识别衰老红细胞的主要途径以及识别后吞噬清除红细胞的研究进展。     

依托泊苷通过线粒体信号通路释放细胞色素C诱导Jurkat白血病细胞凋亡

目的：研究在人类Jurkat白血病细胞株中依托泊苷诱导凋亡的分子机制，揭示由依托泊苷启动的凋亡信号通路。 方法：分别用annexin V-FITC和碘化丙啶（PI）染色，通过流式细胞仪测定annexin V阳性和出现亚二倍体DNA的凋亡细胞。以3,3＇-dihexyloxyacarbocyanine iodide ［DiOC6(3)］为染色剂，采用流式细胞术检测细胞线粒体膜电位的变化。采用离心技术分离细胞的胞浆与线粒体。细胞色素c从线粒体转入胞浆，caspase-3的激活，多聚二磷酸腺苷核糖聚合酶（PARP）的切割等蛋白质的表达由免疫印迹技术（Western blotting）检测。 结果：依托泊苷诱导Jurkat白血病细胞凋亡，细胞凋亡与依托泊苷的作用时间呈线性关系。广谱的caspase抑制剂zVAD.fmk可抑制依托泊苷诱导的DNA片段化和磷脂酰丝氨酸外翻。依托泊苷引起的线粒体膜电位下降早于DNA片段化和磷脂酰丝氨酸外翻，形成明显对照的是zVAD.fmk不能阻断依托泊苷诱导的线粒体膜电位的下降。依托泊苷介导细胞色素c从线粒体释放到胞浆，激活caspase-3，caspase-3的底物PARP被切割。 结论：依托泊苷诱导Jurkat白血病细胞株凋亡的机制是降低线粒体膜电位和释放细胞色素c到细胞浆启动线粒体信号转导通路, 最终激活caspase而导致细胞凋亡。     

NO介导的小鼠胸腺细胞中线粒体的变化在细胞凋亡过程中的作用

目的研究一氧化氮(NO)介导的胸腺细胞凋亡中线粒体膜电位(ΔΨm)和心磷脂(CL)含量变化的特点。方法以S-亚硝基-N-乙酰青霉胺(SNAP)作为NO的供体诱导胸腺细胞凋亡,以地塞米松(DEX)作为阳性对照药物;设空白对照组、SNAP组和DEX组3个实验组;经膜联蛋白V(annexinVmAb)和碘化丙啶(PI)染色后,用流式细胞术(FCM)检测细胞磷脂酰丝氨酸(PS)外翻;用3,3’-二已基噁羰花青碘化物[DiOC6(3)]和PE-anti-annexinVmAb检测凋亡中ΔΨm变化;用壬基吖啶橙(NAO)和PE-anti-annexinVmAb检测凋亡中线粒体CL变化。结果SNAP作用后6h,胸腺细胞出现典型的细胞凋亡特征,多数annexinV阳性的细胞出现皱缩。DEX组ΔΨm降低且未凋亡的细胞比例显著高于空白对照组(P<0.01);而SNAP组该群细胞所占比例与空白对照组比较,差异无统计学意义(P>0.05)。各组中约40%~50%的DiOC6(3)阴性细胞同正常细胞的大小。SNAP组CL含量降低的凋亡细胞所占比例显著高于对照组(P<0.01),未见CL含量降低且未凋亡的细胞群。空白对照组和SNAP组中分别有(48.32±3.96)%、(43.64±4.90)%的细胞CL含量降低但大小同正常细胞。结论NO介导的小鼠胸腺细胞的凋亡过程,依次为磷脂酰丝氨酸外翻、线粒体去极化、CL氧化及细胞皱缩。同DEX模型组相比较,NO介导的小鼠胸腺细胞线粒体的变化为凋亡过程中较晚期的变化。     

磷脂信使与细胞凋亡

近年来，磷脂类信号分子如磷脂酸，溶血磷脂酸，神经酰胺，神经鞘氨醇及1-磷酸-鞘氨醇等与其特异性的膜受体结合在调节细胞生长，分化及凋亡中的作用引起了人们的极大关注。根据靶细胞种类，磷脂分子剂量以及相关受体信号的不同其产生的生物效应各不相同，磷脂类信号分子与内皮分化基因（Edg)家族G蛋白耦联受体。MAPK/ERK，PI3K/Akt,JNK/SAPK等信号系统相互作用，从而影响细胞凋亡。磷脂酰丝氨酸暴露于细胞表面是细胞凋亡过程中的重要事件，氨基磷脂易位酶及磷脂scramblase酶与此事件密切相关。暴露于细胞表面的磷脂酰丝氨酸在鞍细胞的识别及凋亡细胞的清华中发挥重要的信号作用。对磷脂类信号分子及其受体信号系统的调节为某些相关疾病的防治提供了有意义的靶点。     

小胶质细胞体外吞噬模型的建立及相关炎症因子的研究

目的: 建立以细胞凋亡信号－磷脂酰丝氨酸(PS)介导的小胶质细胞体外吞噬模型,研究小胶质细胞发挥吞噬功能时炎症因子的表达变化。方法: 用N-乙基马来酰胺(NEM)预处理红细胞,随后整合氧化PS,制备含氧化PS信号的红细胞凋亡模型,并测定小胶质细胞对整合氧化PS信号红细胞吞噬率的变化。同时, real-time PCR检测小胶质细胞中炎症因子白细胞介素1β(IL-1β)和肿瘤坏死因子(TNF-α)的表达情况,研究小胶质细胞吞噬功能与炎症功能之间的关系。结果: 小胶质细胞对整合氧化PS红细胞的吞噬率显著高于对照组(P<0.01),同时炎症因子IL-1β和TNF-α的mRNA表达水平明显减少。结论: 成功制备小胶质细胞体外吞噬凋亡细胞模型,小胶质细胞的吞噬作用可能是自身炎症因子IL-1β和TNF-α在转录水平上降低的诱因。     

Migration of 99mTc-labelled syngeneic lymphocytes in the rat. Biological and theoretical models predict radiation damage and poor scintigraphic detectability

The possibility of obtaining useful scintigrams of secondary lymphoid organs after infusion of syngeneic lymphocytes labelled with technetium-99m (99mTc) was explored in a rat model. Thoracic duct lymphocyte (TDL) accumulation in various organs was measured with both 99mTc and 51Cr labelled cells, the latter processed with a method that has been shown not to damage lymphocytes. 99mTc labelled TDL did not localize properly in the lymph nodes and spleen. We could not visualize lymph nodes in scintigrams, neither could we demonstrate any difference between normal and hyperplastic spleens. Our conclusion is that radiation from the 99mTc label readily influences lymphocyte migration so that useful scintigraphy in rats and other small experimental animals becomes impossible. This was supported by results from culture experiments with 99mTc labelled, radiosensitive mouse haemopoietic progenitor cells. Theoretical considerations, including the calculations of lymphocyte self-irradiation and signal/noise ratios during scintigraphy of rat tissues, supported our conclusion that scintigraphy in small animals, to disclose the physiological migration of lymphocytes, may be impossible with the present sensitivity of gamma cameras.     

Detection of medullary carcinoma of thyroid, with liver metastasis, using 99mTc DMSA(V) scintigraphy

A sixty year old female referred for thyroid and liver scintigraphy had a clinical history of progressive swelling in the neck with hepatomegaly. A large cold area was detected in the right thyroid lobe using 99mTc pertechnetate and in the right lobe of liver using 99mTc phytate. Subsequent whole body scan with 99mTC DMSA(V) showed avid tracer uptake in right lobe of thyroid and liver. Aspiration cytology of thyroid and liver showed medullary carcinoma of thyroid with its metastasis in liver. Histopathology following thyroidectomy confirmed the diagnosis. Thus 99mTc pentavalent DMSA contributes specificity to diagnose medullary carcinoma of thyroid and metastatic lesions.     

Technetium-99m-labeled annexin V imaging for detecting prosthetic joint infection in a rabbit model

Accurate and timely diagnosis of prosthetic joint infection is essential to initiate early treatment and achieve a favorable outcome.In this study,we used a rabbit model to assess the feasibility of technetium-99m-labeled annexin V for detecting prosthetic joint infection.Right knee arthroplasty was performed on 24 New Zealand rabbits.After surgery,methicillin-susceptible Staphylococcus aureus was intra-articularly injected to create a model of prosthetic joint infection(the infected group,n = 12).Rabbits in the control group were injected with sterile saline(n=12).Seven and 21 days after surgery,technetium-99m-labeled annexin V imaging was performed in 6 rabbits of each group.Images were acquired 1 and 4 hours after injection of technetium-99 mlabeled annexin V(150 MBq).The operated-to-normal-knee activity ratios were calculated for quantitative analysis.Seven days after surgery,increased technetium-99m-labeled annexin V uptake was observed in all cases.However,at 21 days a notable decrease was found in the control group,but not in the infected group.The operated-to-normal-knee activity ratios of the infected group were 1.84 ± 0.29 in the early phase and 2.19 ±0.34 in the delay phase,both of which were significantly higher than those of the control group(P=0.03 and P=0.02).The receiver operator characteristic curve analysis showed that the operated-to-normal-knee activity ratios of the delay phase at 21 days was the best indicator,with an accuracy of 80%.In conclusion,technetium-99m-labeled annexin V imaging could effectively distinguish an infected prosthetic joint from an uninfected prosthetic joint in a rabbit model.     

^99mTc—NOEt心肌显像的最佳显像时间的研究

目的:研究~(99m)Tc-NOEt(NOEt:N—乙氧基—N—乙基氨荒酸钠)的最佳负荷显像时间及再分布显像时间,方法:32例CAD患者及8例正常对照者进行~(99m)Tc-NOEt动态心肌断层SPECT显像研究,分析心肌显像质量及心肌显像结果 结果:~(99m)Tc-NOEt心肌显像质量随时间的递增而明显改善,但再分布显像时间过长反而造成部分心肌显像质量下降;~(99m)Tc-NOEt再分布现象可能发生较早.结论:建议负荷心肌显像时间直在15分钟进行,再分布显像在2小时进行,最好不超过4小时.     

Bone extraction and blood clearance of diphosphonate in the dog.

The transcapillary extraction of diphosphonate, as [99mTc]EHDP, a substance used in bone scanning and for management of certain metabolic bone diseases, has been examined. The maximum instantaneous extraction for [99mTc]EHDP was 0.27 +/- 0.05 (mean +/- SD, N = 10) and the net extraction at 5 min was 0.18 +/- 0.05 (N = 10). The permeability ratio of [99mTc]EHDP to the freely diffusible compound, sucrose, using the formula PS = -Fs loge (1 - Emax), was 0.71. This is similar to the ratio of diffusion coefficients of EHDP to sucrose, which is estimated to be 0.78. These results suggest that the mechanism by which [99mTc]EHDP passes through the capillaries in bone is passive diffusion. Tissue level estimations of EHDP confirm a rapid blood clearance associated with an increase in the rate of urinary excretion; the level of [99mTc]EHDP in bone, however, remains constant. The fractional excretion of [99mTc]EHDP was 27.3 +/- 2.0% in control dogs and was unchanged by thyroparathyroidectomy and subsequent infusion of parathyroid hormone.     

Frequent reversible membrane damage in peripheral blood B cells in human T cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP)

Apoptosis in peripheral blood lymphocyte populations in HTLV-I-infected people in vivo was examined, to study the lymphocyte dynamics in HTLV-I infection. Freshly isolated lymphocytes from 10 non-infected healthy people, eight asymptomatic HTLV-I carriers and 15 patients with HAM/TSP were stained with FITC-labelled annexin V to detect phosphatidylserine (PS) residue exposure at the outer plasma membrane leaflet as an early marker of apoptosis. There was no significant difference in annexin V positivity in CD4+ and CD8+ lymphocytes between non-infected subjects, asymptomatic carriers and HAM/TSP patients, but there was a greatly increased exposure of PS on CD19+ lymphocytes (B cells) detected by FITC-annexin V in 12 out of 15 (80%) HAM/TSP patients, while only two out of eight (25%) asymptomatic carriers and none of the non-infected healthy people showed this aberrant PS exposure on B cells. The intensity of annexin V staining of B cells in HAM/TSP was intermediate, as distinct from the high annexin V staining on advanced apoptotic cells. However, annexin V positivity was decreased when the cells were stained after 24 h of culture, suggesting that the intermediate PS exposure on the B cell in HAM/TSP is not a consequence of an apoptotic process, but rather reflects reversible membrane damage. B cells with PS exposure in vivo might provide a site for coagulation and inflammation, and so contribute to the pathogenesis of HAM/TSP and its complications.     

~（99m）Tc标记DTPA-生物素和hIgG在正常小鼠体内分布实验

目的：探讨DTPA-生物素和hIgG的99mTc标记方法,并观察标记物在体内、外的稳定性及其在正常小鼠体内分布。方法：99mTc标记DTPA-生物素采用直接标记法,99mTc标记hIgG采用2-巯基乙醇还原法,标记率及放化纯测定采用纸层析法,并分别观察99mTc-生物素和99mTc-IgG在生理盐水（NS）中的稳定性;取24只正常小鼠,分为两组,分别经尾静脉注入99mTc-生物素和99mTc-hIgG,注入剂量为7.4MBq/100μl,于注入后1h、3h、6h、12h行SPECT显像并处死小鼠分离各脏器,测量放射性计数,计算各脏器每g组织百分注射剂量（%ID/g）。结果：99mTc标记DTPA-生物素的标记率〉80%,99mTc标记hIgG的标记率〉70%,99mTc-hIgG的放化纯〉90%;在NS中温育12h未观察到99mTc-生物素和hIgG放化纯度明显降低;体内生物分布显示99mTc-生物素肾内摄取高,主要经泌尿系统排泄;99mTc-hIgG在体内主要分布于肝、脾、肾,且在血液中存留时间较长。结论：99mTc标记DTPA-生物素和hIgG具有良好的标记率及体外稳定性,小鼠体内分布实验表明,99mTc-生物素和99mTc-hIgG体内分布的明显不同,为下一步基于亲和素-生物素系统预定位技术各组分的应用提供实验依据。     

Performing 18F-FDG PET studies following injections of 99mTc-sestamibi.

When SPECT studies are followed by PET studies on the same day, substantial 99mTc activity may be present in patients during the PET scans. Degraded PET camera performance results unless the low-energy gamma rays are absorbed by lead shields. Spatial resolution, camera count rates, energy spectra, image contrast and noise, and image quality have been measured for phantoms with varying levels of 99mTc activity, and both with and without thin lead shields placed in front of the detectors. In addition examples of the results of twelve 18F-FDG PET cardiac studies performed within 6 h of 99mTc-sestamibi injections are reported. The presence of 99mTc (140 keV gamma rays) causes light pile-up with 511 keV photons resulting in distorted energy spectra, degraded spatial resolution, increased Compton background and reduced count-rate capability. These effects are avoided using thin lead shields. Studies performed with 99mTc activity in patients but using lead shields are of comparable quality to studies performed without 99mTc or shields. Thin lead shields effectively filter low-energy gamma rays during PET studies leading to improved count-rate capability, contrast and image quality.     

The influence of lipid composition and divalent cations on annexin V binding to phospholipid mixtures

Annexin V is a 36-kDa protein which, it has been suggested, is a factor in protecting the vascular endothelium from attack by antibodies to other phospholipid-binding proteins. Competition between annexin V and beta2-glycoprotein I (beta2GPI) for phospholipid surfaces is complicated by empirical observations regarding alterations in binding to anionic phospholipid, primarily phosphatidylserine. In order to elucidate the effect of phospholipid composition and divalent cations (Ca(+2) and Mg(+2)) on annexin V binding to phospholipid, we used biotinylated annexin V and peroxidase-conjugated avidin D to probe the binding of annexin V to phospholipid-coated wells of polystyrene microtiter plates. Binding of annexin V to anionic phospholipid is Ca(+2)-dependent and, in its absence, annexin V was found to bind most avidly to 100% phosphatidylcholine in a saturable manner, followed by decreasing percentages of phosphatidylcholine. Ca(+2) was found to inhibit phosphatidylcholine binding and promote the binding of phospholipid mixtures containing phosphatidylserine. Phosphatidylserine (100%) did not bind annexin V as strongly as mixtures of 50% and 75% phosphatidylserine. The effect with Ca(+2) suggests saturation of Ca(+2)-binding sites on annexin V, reached under our experimental conditions at approximately 1 mM. Under the same conditions, Mg(+2) slightly enhanced the binding of all of the phospholipid compositions studied. Ca(+2)-dependent binding of annexin V was competitively inhibited by Mg(+2); 5 mM Mg(+2) reduced binding significantly (p < 0.0001 by ANOVA, p < 0.05 for post hoc test of 5 mM vs 0 mM). These data suggest that the translocation of membrane phospholipid under the dynamics of ion transport in vascular endothelium may alter annexin V binding.     

Involvement of annexin V in the antiproliferative effect of GnRH agonist on cultured human uterine leiomyoma cells

The objective of this study was to elucidate the role of annexin V, an endogenous inhibitor of protein kinase C (PKC), with regard to the antiproliferative effect of gonadotrophin-releasing hormone (GnRH) agonist (buserelin) on cultured human uterine leiomyoma cells. Uterine leiomyoma tissue was collected from the surgical specimens of patients and cells from 37 specimens (15 cases) were cultured. For up to 96 h after the addition of buserelin to the cultured cells, a time-dependent antiproliferative effect was noted in the group to which 10(-5) mol/l buserelin was added. Both the intracellular concentration of annexin V and the expression of annexin V mRNA increased time-dependently with the addition of buserelin. The intracellular concentration of annexin V increased with the addition of PKC activator (12-O:-tetradecanoylphorbor-13-acetate; TPA) much as it did with the addition of buserelin, and the rise in the concentration caused by the addition of buserelin was completely attenuated by pretreatment with PKC inhibitor (calphostin C). Our findings suggest that buserelin inhibits cell proliferation in cultured human uterine leiomyoma cells accompanied with an increase in the intracellular concentration of annexin V, mediated, at least in part, by the activation of PKC.