生物材料诱导细胞凋亡的研究进展

针对近些年生物材料诱导细胞凋亡的现象和机制的研究情况，从凋亡信号传导角度分类综述了生物材料诱导细胞凋亡的途径，包括传统信号传导途径、死亡受体途径、以及近年发现的线粒体途径，另外也论述了生物材料诱导活性氧产生而发生的凋亡和影响细胞黏附导致的细胞凋亡等途径，发现生物材料诱导的细胞凋亡产生的途径具有明显的多样性、交叉性和多途径同时作用的特点。本综述对于生物材料诱导细胞凋亡机制的深入研究特别是生物材料的研制具有重要的指导意义。此外也为生物材料广泛应用于人类疾病特别是应用于与细胞凋亡紧密相关的癌症治疗提供了借鉴。     

死亡受体及线粒体途径与激活诱导T细胞凋亡

免疫系统形成了一系列确保激活的淋巴细胞能被有效清除的分子及信号途径，称之为激活诱导的细胞死亡。激活诱导的细胞死亡主要负责调节免疫细胞稳态及清除自身反应性淋巴细胞。死亡受体途径及线粒体死亡途径分别是细胞凋亡的外在途径和内在途径也参与了激活诱导的T细胞凋亡，本文就外周成熟T细胞凋亡相关的死亡受体途径，线粒体死亡途径及其相关调控机制研究进展作一综述。     

诱导细胞凋亡的抗hDR5单抗的研究

目的 研制能诱导肿瘤细胞凋亡的抗人DR5单克隆抗体（McAb）。方法 以可溶性人死亡受体（death receptor，DR）5胞外段免疫小鼠，采用杂交瘤技术制备抗人DR5的McAb；MTT方法筛选分泌有细胞毒活性McAb的杂交瘤细胞；亲和层析方法纯化McAb；夹心ELISA法测定McAb亚型；Western blot和斑点ELISA法检测McAb的抗原表位类型；间接ELISA法检测McAb的特异性；流式细胞仪检测FITC-annexinⅤ／PI双色标记的Jurkat细胞的凋亡率；DNA琼脂糖凝胶电泳测定凋亡细胞的DNA片段化。结果 获得1株杂交瘤细胞，其分泌的McAb命名为mDRA-6，为IgG1；其抗原表位类型为构象表位；其特异性识别hDR5，与hFas、hDR4等无交叉反应。mDRA-6对Jurkat细胞具有细胞毒作用；经McAb mDRA-6处理后，Jurkat细胞膜表面高表达丝氨酸磷脂，并导致Jurkat细胞中的DNA片段化。结论 mDRA-6是一个具有诱导细胞凋亡活性的新的抗人DR5功能性抗体，在以TRAIL／DR5系统进行肿瘤治疗和探讨DR5的功能结构域研究方面具有广泛应用前景。     

死亡受体及线粒体途径与激活诱导T细胞凋亡

免疫系统形成了一系列确保激活的淋巴细胞能被有效清除的分子及信号途径 ,称之为激活诱导的细胞死亡。激活诱导的细胞死亡主要负责调节免疫细胞稳态及清除自身反应性淋巴细胞。死亡受体途径及线粒体死亡途径分别是细胞凋亡的外在途径和内在途径 ,也参与了激活诱导的T细胞凋亡 ,本文就外周成熟T细胞凋亡相关的死亡受体途径、线粒体死亡途径及其相关调控机制研究进展作一综述。     

009 死亡受体及线粒体途径与激活诱导T细胞凋亡

免疫系统形成了一系列确保激活的淋巴细胞能被有效清除的分子及信号途径,称之为激活诱导的细胞死亡.激活诱导的细胞死亡主要负责调节免疫细胞稳态及清除自身反应性淋巴细胞.死亡受体途径及线粒体死亡途径分别是细胞凋亡的外在途径和内在途径,也参与了激活诱导的T细胞凋亡,本文就外周成熟T细胞凋亡相关的死亡受体途径、线粒体死亡途径及其相关调控机制研究进展作一综述.     

阿司匹林促进宫颈癌HeLa细胞凋亡

阿司匹林(acetysalicylicacid,ASA)是非类固醇抗炎药(nonsteroidal antiinflammatory drugs,NSAIDs)的代表药物.最近研究证实,阿司匹林具有高效降低直肠结肠肿瘤发病率的作用~([1]);文献报道普通阿司匹林应用在半年以上,可有效降低宫颈癌的发病率~([2]).本研究对阿司匹林抑制宫颈癌HeLa细胞增殖作用及相关机制进行研究,为宫颈癌的预防与治疗提供有益的探索.     

赖氨大黄酸通过激活JNK诱导宫颈癌HeLa细胞凋亡

目的研究赖氨大黄酸(RHL)对人宫颈癌HeLa细胞增殖、凋亡的影响,并探讨其作用机制。方法用MTT法检测细胞增殖;用流式细胞仪检测细胞凋亡,用Western blot检测凋亡相关蛋白及JNK蛋白与蛋白磷酸化水平。结果 RHL能有效抑制宫颈癌HeLa细胞增殖,并能诱导其凋亡,随药物浓度的增加,细胞凋亡率也逐渐升高;RHL能够激活caspase-3、caspase-7和PARP,并激活磷酸化的JNK表达,磷酸化的JNK表达增加在RHL的诱导凋亡中起主要作用。结论 RHL通过激活JNK-caspase-PARP信号通路抑制HeLa细胞增殖,并诱导其凋亡,赖氨大黄酸解决了大黄酸不溶于水的问题,有望成为临床肿瘤辅助化疗药物。     

人截短型凋亡诱导因子的表达对HeLa细胞的促凋亡作用

目的：观察人截短型AIF基因的表达对HeLa细胞的促凋亡作用。方法：用RT-PCR法分段克隆全长人AIF基因，经改造截去其N-端线粒体定位信号及部分Spacer区域（1-120位氨基酸）的编码序列，从而获得人截短型AIF（AIF△1-120）基因。将其克隆入pIRES2-EGFP绿色荧光蛋白（EGFP）共表达载体，用脂质体法转染HeLa细胞，通过荧光显微镜观察。免疫组织化学检测，间接免疫荧光检测，电镜观察等方法。检测目的基因在转染细胞中的表达，以及对转染细胞的形态及生长状况的影响。结果：成功地构建了人截短型AIF（AIF△1-120)基因的真核表达载体。转染HeLa细胞后，可检测到人截短型AIF分子的表达，随着转染后时间的延长，可观察到表达人截短型AIF分子的HeLa细胞呈现典型的凋亡特征。结论：人截短型AIF基因的表达可诱导HeLa细胞凋亡。     

银杏叶提取物对LPS诱导HeLa细胞凋亡的影响

目的研究银杏叶提取物（Ginkgo biloba leaf extract,GbE）对细菌脂多糖（LPS）诱导的人宫颈癌细胞HeLa细胞凋亡的影响。结论将HeLa细胞分成LPS组、GbE＋LPS干预组和对照组,各组细胞加入相应药物干预8～72h。采用MTT法检测GbE对细胞的毒性作用;计算各组细胞不同时间段细胞数,测定各组细胞的增殖情况;通过细胞形态学检测（AO/EB染色）测定细胞凋亡发生情况。结果在72h内不同浓度GbE对HeLa细胞无明显细胞毒性作用,细胞存活率在90%～100%之间,差异无统计学意义（P〉0.05）;LPS组经LPS诱导8h后细胞在各时间段增殖均较其他两组活跃,细胞数明显高于其他两组（tGbE＋LPS=6.423,P〈0.001;t对照组=3.976,P〈0.005）;GbE＋LPS组培养48h后细胞凋亡数明显高于LPS组和对照组。结论 GbE具有抑制LPS诱导的HeLa细胞增殖及促进细胞凋亡的作用。     

白藜芦醇诱导人宫颈癌细胞凋亡的作用

目的观察白藜芦醇对人宫颈癌Hela细胞凋亡的影响。方法采用TUNEL法检测不同浓度的白藜芦醇诱导体外培养的Hela细胞凋亡情况。结果浓度大于50μmol／L的白藜芦醇处理Hela细胞后,细胞凋亡特征明显可见,并呈时间和剂量依赖关系。结论白藜芦醇对人宫颈癌Hela细胞有明显的促凋亡作用。     

Complement Activation by Apoptotic Cells Occurs Predominantly via IgM and is Limited to Late Apoptotic (Secondary Necrotic) Cells

Apoptotic cells activate complement via various molecular mechanisms. It is not known which of these mechanisms predominate in a physiological environment. Using Jurkat cells as a model, we investigated complement deposition on vital, early and late apoptotic (secondary necrotic) cells in a physiological medium, human plasma, and established the main molecular mechanism involved in this activation.

Upon incubation with recalcified plasma, binding of C3 and C4 to early apoptotic cells was similar to background binding on vital cells. In contrast, late apoptotic (secondary necrotic) cells consistently displayed substantial binding of C4 and C3 and low, but detectable, binding of C1q. Binding of C3 and C4 to the apoptotic cells was abolished by EDTA or Mg-EGTA, and also by C1-inhibitor or a monoclonal antibody that inhibits C1q binding, indicating that complement fixation by the apoptotic cells was mainly dependent on the classical pathway. Late apoptotic cells also consistently bound IgM, in which binding significantly correlated with that of C4 and C3. Depletion of plasma for IgM abolished most of the complement fixation by apoptotic cells, which was restored by supplementation with purified IgM.

We conclude that complement binding by apoptotic cells in normal human plasma occurs mainly to late apoptotic, secondary necrotic cells, and that the dominant mechanism involves classical pathway activation by IgM.     

Phagocytosis of Apoptotic Trophoblast Cells by Human Endometrial Endothelial Cells Induces Proinflammatory Cytokine Production

Citation Peng B, Koga K, Cardenas I, Aldo P, Mor G. Phagocytosis of apoptotic trophoblast cells by human endometrial endothelial cells induces proinflammatory cytokine production. Am J Reprod Immunol 2010; 64: 12–19 Problem Apoptosis is a normal constituent of trophoblast turnover in the placenta; however in some cases, this process is related to pregnancy complications such as preeclampsia. Recognition and engulfment of these apoptotic trophoblast cells is important for clearance of dying cells. The aim of this study was to show the cross talk between human endometrial endothelial cells (HEECs) and apoptotic trophoblast cells in an in vitro coculture model and its effect on cytokine production by HEECs. Method of study Fluorescent‐labeled HEECs were cocultured with fluorescent‐labeled apoptotic human trophoblast cells. Confocal microscopy and flowcytometry were used to show the interaction between these two types of cells. Cytokine profiles were determined using multiplex analysis. Results HEECs are capable to phagocytose apoptotic trophoblasts. This activity is inhibited by the phagocytosis inhibitor cytochalasin B. Phagocytosis of apoptotic trophoblast cells induced the secretion of the proinflammatory cytokines interleukin‐6 and monocyte chemoattractant protein‐1 by HEECs. Conclusion This study provides the first evidence that HEECs have an ability to phagocytose apoptotic trophoblasts. Furthermore, we demonstrated an inflammatory response of HEECs after phagocytosing the apoptotic trophoblast cells. This event may contribute to the inflammatory response in both normal pregnancy and pathologic pregnancy such as preeclampsia.     

Uptake of Apoptotic K562 Leukaemia Cells by Immature Dendritic Cells is Greatly Facilitated by Serum

Dendritic cells (DCs) are potent antigen-presenting cells and play an important role in T-cell-mediated immunity. DCs have been shown to induce strong antitumour immune responses both in vitro and in vivo. One way of providing the DCs with all relevant tumour antigens would be to incubate the DCs with material from dead tumour cells. We have examined the uptake of apoptotic and necrotic K562 leukaemia cells by DCs under different culture conditions. Results from coincubation experiments strongly suggested that uptake of apoptotic K562 cells was dependent upon the addition of autologous serum (AS). Under these conditions, 47-79% of all DCs were shown to ingest apoptotic material. AS also seemed to be important for the expression of functionally important markers, most notably HLA class I, CD86, CCR7 and CD83. The vast majority of DCs were shown to ingest necrotic material from K562 cells, with no additional effect of AS. The results suggest that incubation of DCs with apoptotic material for cell therapeutic purposes may best be performed in the presence of AS.     

凋亡肿瘤细胞负载的树突状细胞对抗原特异性CTL的激活

为探讨凋亡Daudi细胞负载的树突状细胞 (DC )激发诱导肿瘤抗原特异性CTL及其生物学特性 ,采用常规方法从人外周血单个核细胞 (PBMC )诱导DC ,激发型CD40mAb联合 50Gyγ射线辐照诱导Daudi凋亡后负载DC ,然后与自体T细胞共育 ,并联合IL 2激发诱导肿瘤特异性CTL ;采用免疫荧光标记和流式细胞仪测定分析膜分子的表达 ;ELISA检测细胞因子的产生 ;Dextran FITC内吞实验分析DC的抗原摄取能力 ;3H掺入试验和51 Cr释放试验分别测定CTL的增殖和细胞毒效应。结果 :(1 )细胞因子序贯体外诱导 7d的DC ,对Dextran吞噬能力最强 ;(2 )CD40mAb诱导联合γ射线辐照 ,能有效介导Daudi细胞的凋亡 ;(3 )抗原负载联合CD40mAb激发可使DC上调表达CD1a、CD80、CD86和HLA DR ,并能促进IL 1 2的分泌 ;(4)凋亡Daudi负载后的DC在激发型CD40mAb作用下 ,能激发和扩增对Daudi细胞具有高效和特异杀伤作用的细胞毒性T细胞。因而认为凋亡肿瘤细胞负载联合CD40mAb刺激的DC可有效激活和扩增肿瘤特异性CTL。     

In Vitro Cytotoxicity of Mokko Lactone in Human Leukemia HL-60 Cells: Induction of Apoptotic Cell Death by Mitochondrial Membrane Potential Collapse

We studied the effect of mokko lactone (ML) isolated from the roots of Saussurea lappa (Compositae), a plant that is used for medicinal purposes in Korea, on the induction of apoptosis in human leukemia HL-60 cells. ML was cytotoxic to HL-60 cells, and this cytotoxic effect of ML appears to be attributable to its induction of apoptotic cell death, as ML induced nuclear morphologic changes and internucleosomal DNA fragmentation and increased the proportion of Annexin V-positive cells and the activity of caspase-3. Further studies revealed that the induction of apoptosis by ML was associated with the loss of mitochondrial membrane potential. Collectively, our results suggest that apoptosis induced by ML in HL-60 cells was executed by a collapse of mitochondrial membrane potential followed by the activation of caspase-3. This is the first report on the mechanism of apoptosis-inducing effect of ML.     

Induction of L-Variants in Human Diploid Cells Infected by Group A Streptococci

Human diploid cells in culture, infected with a balanced amount of living group A streptococci, were able to survive the infection and could be divided and propagated normally thereafter. The streptococci were rapidly phagocytized by the tissue culture cells. At the beginning, they kept their typical appearance, as well as their ability to fix dyes and group-specific immunoglobulins. After 1 to 2 days, the number of detectable streptococci decreased and they underwent important morphological changes. After some subsequent divisions of the cell line, streptococci persisted in cells only as large, isolated, swollen cocci, and no longer grew on suitable media. After six to eight divisions, a noticeable percentage of the tissue culture cells were very similar in appearance to the same cell line experimentally infected with “stable” L-variants. Cultures on L-phase media of supernatant fraction and cells, made 24 to 48 hr after inoculation, showed typical L-colonies. These grew well on media without antibiotics, as well as on media containing penicillin or vancomycin. They could be propagated on media with penicillin for months and were able to revert to group A streptococci after several subcultures on antibiotic-free media. Controls of uninoculated tissue culture cells never showed the presence of any microorganism. Group A streptococci inoculated into Eagle's basal medium, which was used for the tissue cultures, did not grow and never gave rise to L-colonies, even though the medium contained penicillin. Previous data suggest a biochemical explanation for this conversion, which otherwise is an occasional phenomenon.     

Blood Group Antigens on HeLa Cells shown by Mixed Agglutination

The mixed agglutination reaction has been used for investigating the presence of blood group antigens on the surface of human cervical carcinoma cells (HeLa) cultured for eight years in vitro.

The H antigen was demonstrated in the absence of A and B. The MN-type antigen has been found as well as Tj^a.

Treatment of HeLa cells with ficin greatly enhanced the reaction of anti-H and anti-Tj^a with the corresponding antigens on HeLa cells. The authors failed to show the following antigens: Rh(D) and Rh(c), S, P, Le^a, Le^b, Lu^a and Lu^b.

     

Accelerated Cytopathology in HeLa Cells Induced by Reovirus and Cycloheximide

The addition of the protein inhibitor cycloheximide, at concentrations which suppress virus replication, to HeLa cell monolayers infected with reovirus type 2 results in the appearance of accelerated cytopathic effects (CPE). At high multiplicity of infection, CPE appeared after a lag period of 2 to 3 hr and increased progressively, until by 12 hr the entire monolayer had rounded and sloughed off. During this same period, both the uninfected cycloheximide-treated and untreated virus-infected controls exhibited no CPE. The phenomenon is affected by the kind of cell species employed and can be reversed if the antibiotic is removed within 1 hr after exposure. The evidence obtained through studies in which specific metabolic inhibitors and direct biochemical analyses were used suggests that the accelerated CPE observed in cycloheximide-treated reovirus-infected cells is the consequence of the combined inhibition of the synthesis of both cellular protein and ribonucleic acid. The accelerated CPE is also induced in the antibiotic-treated cells by reovirus serotypes 1 and 3.