抗DR5单克隆抗体-mDRA-6对白血病细胞的凋亡诱导作用

目的:观察抗死亡受体5(DR5)单克隆抗体(mAb)-mDRA-6对白血病细胞的凋亡作用。方法:DR5蛋白免疫BALB/c小鼠,融合筛选抗DR5杂交瘤细胞,制备抗人DR5mAb-mDRA-6;荧光显微镜下观察mDRA-6作用下白血病细胞Jurkat、HL-60、K562的形态变化;MTT法测定不同浓度的mDRA-6对Jurkat、HL-60、K562细胞存活率的影响;通过FITC-Annexin V及PI标记细胞,以流式细胞术检测mDRA-6对Jurkat、HL-60、K562细胞凋亡率的影响。结果:mDRA-6导致Jurkat、HL-60细胞染色质浓缩、断裂,细胞出芽,凋亡小体形成;mDRA-6对Jurkat、HL-60细胞具有明显的杀伤作用,但对K562的杀伤作用较弱,25mg/L的mDRA-6作用12h,Jurkat、HL-60、K562细胞死亡率分别为88.76%,59.76%,5.18%。Annexin V及PI双染显示1mg/L的mDRA-6作用10h,Jurkat细胞凋亡率为86.06%,10mg/L的mDRA-6作用10h,HL-60细胞凋亡率为48.11%,但10mg/L的mDRA-6作用K562细胞10h,细胞凋亡不明显。结论:抗DR5mAb mDRA-6能够诱导白血病细胞凋亡,不同的白血病细胞株对mDRA-6的敏感性不同。     

TRAIL对多种肿瘤细胞的杀伤作用

目的： 研究肿瘤坏死因子相关凋亡诱导配体（TRAIL）作用下多种肿瘤细胞的生长抑制效应及凋亡诱导情况。方法： 利用大肠杆菌基因工程菌表达非融合rhsTRAIL，进行目的蛋白的分离纯化，得到rhsTRAIL样品，纯度为97%。通过倒置显微镜下观察、MTT法、流式细胞仪法检测其对细胞的生长抑制和诱导凋亡情况。结果： 一定浓度的TRAIL 可有效抑制LS174-T细胞、MCF-7细胞、GLC细胞、7402细胞、Jurkut T细胞生长，其生长抑制率具剂量依赖性，且各细胞对TRAIL敏感性不同，其中Jurkat T细胞最为敏感。用2 mg/L TRAIL作用Jurkat T细胞0-72 h，6 h后细胞即发生明显凋亡，其细胞凋亡率具时间依赖性。结论： 所制备的TRAIL可抑制多种肿瘤细胞生长，并诱导Jurkat T细胞凋亡。     

DR5在TRAIL诱导Jurkat细胞凋亡中的作用

目的：研究DR5在介导TRAIL凋亡信号中的作用。方法：用含人DR5细胞外全长结构域重组DR5免疫BALB／C小鼠，制备抗DR5单克隆抗体；流式细胞仪检测Jurkat细胞表面DR5表达水平；采用TRAIL凋亡检测试剂盒，检测Jurkat细胞凋亡率及抗DR5单克隆抗体对TRAIL诱导细胞凋亡的阻断率。结果：DR5在Jurkat细胞表面的表达率为94．83％，TRAIL和抗TRAIL单克隆抗体能够诱导Jurkat细胞凋亡，呈现明显的剂量相关性，TRAIL浓度在50-100ng/ml时，杀伤率达90％以上。预先用抗DR5单克隆抗体与Jurkat细胞作用后，TRAIL对Jurkat细胞的杀伤功能几乎完全被mAb所阻断，其平均阻断率达90．49％。结论：DR5在TRAIL诱导Jurkat细胞凋亡中起着十分关键的作用。     

重组人可溶性TRAIL的制备及其联合硼替佐米诱导肿瘤细胞的凋亡作用

目的:构建重组人肿瘤坏死因子相关的凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)原核表达质粒p ET-28a(+)-TRAIL114-281,优化蛋白表达和纯化条件,制备重组人可溶性TRAIL并鉴定其活性。方法:使用CCK-8初步验证TRAIL是否具有抑制肿瘤细胞生长的生物活性;将制备的TRAIL单独或联合50 nmol/L硼替佐米应用于H460细胞(对TRAIL敏感)和K562细胞(对TRAIL抵抗)24 h,流式细胞术检测细胞凋亡率,比色法检测caspase-8、-9、-3的活化程度,Western blot分析细胞中Bax、Bcl-2和c FLIP蛋白的表达。流式细胞术检测硼替佐米处理H460细胞和K562细胞24 h后DR4和DR5的表达量变化。结果:制备了具有生物学活性且性质稳定的重组人可溶性TRAIL,且成功诱导H460和K562细胞凋亡。不同浓度TRAIL处理H460细胞后其凋亡率随着TRAIL浓度升高而显著升高(P0.05),但K562细胞凋亡率并未随着TRAIL浓度明显升高。联合用药组的H460和K562细胞凋亡率均显著高于单独用药组(P0.05),凋亡过程中caspase-8、-9、-3均被活化,药物处理组的Bcl-2和c FLIP表达量均比对照组下降,尤其联合用药组表达量下降最为显著(P0.05),而Bax表达量无明显变化。硼替佐米处理H460和K562细胞后DR4和DR5表达量均上调(P0.05)。结论:硼替佐米能协同TRAIL启动内源性凋亡途径诱导H460和K562细胞凋亡,其可能机制是通过上调死亡受体DR4和DR5的表达量、下调抗凋亡蛋白Bcl-2和c FLIP的表达量来实现的。     

独蒜兰乙酸乙酯萃取物通过内源性线粒体通路诱导白血病K562细胞和HL-60细胞凋亡

目的:探讨独蒜兰乙酸乙酯萃取物对人慢性粒细胞白血病K562细胞和急性髓性白血病HL-60细胞增殖及凋亡的影响以及诱导凋亡的途径。方法:采用XTT法、台盼蓝拒染法、Annexin V-FITC/PI双染法、PI染色法、4',6-二脒基-2-苯基吲哚(DAPI)染色和Western blot法研究不同浓度独蒜兰乙酸乙酯萃取物对上述2种白血病细胞增殖、凋亡、细胞周期和凋亡相关蛋白表达等方面的影响。结果:独蒜兰正丁醇萃取物对K562细胞活力几乎没有抑制作用,而乙酸乙酯萃取物能显著抑制K562和HL-60细胞的活力和增殖。乙酸乙酯萃取物对于HL-60细胞作用24 h的半数抑制浓度(IC_(50))为(42.14±2.54)mg/L,对于K562细胞的IC_(50)为(51.28±3.12)mg/L。Annexin V-FITC/PI和DAPI染色结果显示,乙酸乙酯萃取物呈剂量依赖性诱导2种细胞凋亡,且50 mg/L的乙酸乙酯萃取物作用后K562细胞的凋亡率为33.1%,而HL-60细胞的凋亡率为63.1%,说明HL-60细胞对乙酸乙酯萃取物更加敏感,伴有典型的细胞核凋亡形态学改变。PI染色结果显示HL-60细胞和K562细胞都被阻滞于G_2期。Western blot结果显示,随着药物浓度的升高,细胞凋亡抑制蛋白Bcl-2的表达降低,而促凋亡蛋白Bax、cleaved caspase-3和活化的多腺苷二磷酸核糖聚合酶的表达逐渐升高,内源性线粒体凋亡的特征胞浆中细胞色素C和凋亡诱导因子表达也逐渐升高。结论:独蒜兰乙酸乙酯萃取物能显著抑制K562和HL-60细胞增殖,并通过触发内源性线粒体凋亡通路诱导这2种细胞凋亡。     

SeO2诱导白血病细胞凋亡过程中对细胞内活性氧和Ca2+水平影响

目的：研究SeO2对急性早幼粒细胞白血病细胞株NB4、红白血病细胞K562、急性粒细胞白血病细胞株HL-60的增生、凋亡、活性氧(ROS)及Ca2+水平等的影响。 方法： 采用不同浓度SeO2（3-30 μmol/L）分别处理3种白血病细胞，用流式细胞术测定细胞凋亡率、细胞中ROS和Ca2+的水平。 结果： 10和30 μmol/L SeO2能抑制3种细胞增生，30 μmol/L SeO2作用48 h能使54.0%的NB4细胞、46.5%的K562细胞和49.6%的HL-60细胞发生凋亡。同时下调细胞内ROS和Ca2+水平。NB4和HL-60细胞中ROS阳性细胞比例随SeO2浓度增加而减少，K562中只有30 μmol/L SeO2才能使ROS出现明显下降。10、30 μmol/L SeO2能使NB4和HL-60细胞内Ca2+水平逐步下降。K562中只有30 μmol/L SeO2才能使细胞内Ca2+水平出现明显下降。 结论： SeO2对3种白血病细胞均有诱导凋亡作用，在凋亡过程中涉及细胞内ROS及Ca2+水平下降。     

交联抗人DR5单抗诱导的Jurkat细胞凋亡的信号转导

目的探讨抗肿瘤坏死因子相关凋亡诱导配体（TRAIL）的死亡受体5（DR5）单抗——YM366EC对Jurkat细胞增殖的影响，阐明抗DR5抗体的抗肿瘤效应机制。方法MTF方法检测抗体对Jurkat细胞存活率，FrlE—AnnexinV／PI标记流式细胞仪检测交联YM366EC对Jurkat细胞凋亡率影响。交联YM366EC作用于Jurkat细胞2、4、6、8、10、12h收集细胞，提取蛋白Westernblot检测Bcl-2、Cyt—C、Caspase-8、Caspase-3、Bax、Caspase-9的变化。结果抗DR5抗体单独应用不能抑制高表达DR5分子的Jurkat细胞增殖，但应用抗小鼠IgG交联DR5抗体后可直接诱导TRAIL敏感的Jurkat细胞凋亡改变。并且Westernblot检测到随着抗体诱导时间的延长Jurkat细胞Bel-2蛋白表达减少；Cyt—C、Bax，有活性的Caspase-8、Caspase-3和Caspase-9蛋白的表达增加。结论交联抗DR5抗体YM366EC对Jur—kat细胞增殖有明显的抑制作用，诱导的Jurkat细胞凋亡的分子机制涉及到Cyt-C、Bax、Caspase．8、Caspase-3、Caspase-9。     

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.     

TRAIL联合长春新碱对HL-60细胞生长及凋亡的影响

目的研究肿瘤坏死因子相关的凋亡诱导配体（TRAIL）联合长春新碱（VCR）诱导急性早幼粒白血病细胞株HL-60细胞凋亡,探讨其应用于临床的可行性。方法在对数生长期的HL-60细胞中分别加入不同浓度的TRAIL和0.1mg/L的VCR,采用MTT比色法测定TRAIL和VCR单独和联合应用时对细胞的生长抑制率,并用流式细胞术检测细胞凋亡。结果TRAIL联合VCR可协同降低HL-60细胞活力,抑制细胞增殖,诱导细胞凋亡。且24h内当TRAIL浓度〈100g/L时促调亡作用随TRAIL浓度增加及培养时间延长而增强（P〈0.05）,而24h内当TRAIL浓度≥100μg/L时以及24h后虽抑制率和调亡率较高,但随浓度增高作用无显著差别（P〉0.05）。结论TRAIL与VCR具有协同作用,能高效杀灭白血病细胞。TRAIL是一种有望应用于白血病临床治疗的新型生物制剂。     

交联抗DR5单抗YM366EC诱导的Jurkat细胞凋亡机制探讨

为探讨抗肿瘤坏死因子相关凋亡诱导配体(TRAIL)的死亡受体5(DR5)单抗-YM366EC引起Jurkat细胞凋亡信号转导通路,阐明抗DR5抗体的抗肿瘤效应机制。观察了交联366EC对Jurkat细胞的形态变化,细胞增殖和细胞凋亡率影响。进一步用Western blot法检测YM366EC作用于Jurkat细胞不同时间Bcl-2、Cyt-C、Bax、Caspase 3、Caspase 9的表达变化。结果发现DR5抗体YM366EC单独应用无凋亡作用,但抗小鼠IgG交联366EC后可致Jurkat细胞染色质边集、断裂,细胞出芽,形成凋亡小体,并可直接诱导TRAIL敏感的Jurkat细胞凋亡。Western blot检测到随着抗体诱导时间的延长Jurkat细胞Bel-2蛋白表达减少;Cyt-C、Bax、有活性的Caspase 3和Caspase 9蛋白的表达增加。结果表明交联抗DR5抗体YM366EC对Jurkat细胞增殖有明显的抑制作用,诱导的Jurkat细胞凋亡的分子机制涉及到Cyt-C、Bax、Caspase 3、Caspase 9。     

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.     

慢性乙肝患者外周血单个核细胞TRAIL受体表达及其临床意义

目的探索慢性乙肝患者外周血单个核细胞(PBMC)肿瘤细胞坏死因子相关的凋亡诱导配体(TRAIL)受体在PBMC凋亡中的作用,及其与机体肝脏损伤的相关性。方法用RT-PCR和流式细胞术检测55例慢性乙肝患者(包括轻度、中度、重度)外周血单个核细胞TRAIL各受体(DR4、DR5、DcR1和DcR2)表达水平,同时检测肝功能相关指标,并进行相关性分析。以30例正常人作为对照。结果诱骗受体DcR1在慢性乙肝患者中的表达显著低于对照组(P<0.05)。DcR1的表达随慢性乙肝病情的加重而逐渐降低。慢性乙肝患者的DcR1表达与转氨酶(ALT)呈显著负相关,与血清白蛋白成显著正相关。结论慢性乙肝患者外周血单个核细胞DcR1表达下调可能是其细胞凋亡增加的机制之一,且DcR1表达情况可从一定程度上反映肝脏的损伤程度。     

小干扰RNA下调白血病细胞X连锁凋亡抑制蛋白的表达及其对化疗药物敏感性的影响

目的 探讨小干扰RNA(siRNA)下调白血病MOLT-4、HL-60、K562细胞的白血病细胞X连锁凋亡抑制蛋白(XIAP)的表达对化疗药物敏感性的影响.方法 采用实时荧光定量PCR和Western免疫印迹检测MOLT-4、HL-60、K562细胞及健康人外周血单个核细胞(PBMC)XIAP mRNA和XIAP蛋白表达水平.用NucleofectorTM核酸转染仪对高表达XIAP的K562细胞转染XIAP siRNA(实验组),同时以转染无同源性siRNA作阴性对照组,未转染任何siRNA的K562细胞作空白对照组,并检测3组细胞XIAP mRNA和XIAP蛋白表达水平.同时将实验组、阴性及空白对照组细胞暴露于不同浓度的依托泊苷(vp-16,0.01、0.1、1、10、100mg/L)及阿糖胞苷(Ara-c,0.1、1、10、100、1000、10 000mg/L),用CCK-8法检测细胞抑制率变化.结果 与健康人PBMC比较,MOLT-4、HL-60、K562细胞XIAPmRNA及蛋白表达均增高(均P＜0.05),其中K562细胞XIAPmRNA及蛋白表达水平最高,与MOLT-4、HL-60细胞比较差异有统计学意义(均P＜0.05).K562细胞转染XIAP siRNA48 h后,与阴性对照组、空白对照组比较,实验组细胞XIAP mRNA及蛋白表达水平均明显下降(mRNA:0.37±0.10比1.41±0.13比1.00±0.12,蛋白:0.37±0.03比0.99±0.08比0.98±0.07,均P＜0.05).在给予1 mg/L vp-16、10及100 mg/L的Ara-c后,实验组细胞抑制率与阴性对照组、空白对照组比较,差异有统计学意义(均P＜0.05);在给予其他浓度vp-16和Ara-c后,3组细胞抑制率差异均无统计学意义(均P＞0.05).结论 XIAP siRNA能特异性下调K562细胞XIAPmRNA和蛋白质水平的表达,增强K562细胞对化疗药物依托泊苷、阿糖胞苷的敏感性.     

Application of flow cytometry to molecular medicine: detection of tumor necrosis factor-related apoptosis-inducing ligand receptors in acute myeloid leukaemia blasts

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), a cytokine belonging to the TNF (tumor necrosis factor) family, is currently regarded as a potential anti-cancer agent. Nevertheless, several types of cancer cells display a low sensitivity to TRAIL or are completely resistant to this pro-apoptotic cytokine. TRAIL signalling is dependent on four receptors. Two of them, death receptors 4 and 5 (DR4 and DR5), induce apoptosis, whereas decoy receptors 1 and 2 (DcR1 and DcR2) are unable to evoke cell death upon TRAIL binding. TRAIL resistance may be related to the expression of TRAIL decoy receptors. TRAIL has been proposed as a novel therapeutic agent for the treatment of haematological disorders, including acute myeloid leukaemia (AML). Surprisingly, however, very limited information is available concerning the expression of TRAIL receptors in AML blasts. Here, we have evaluated, using flow cytometry, TRAIL receptor surface expression and sensitivity to TRAIL-dependent apoptosis of AML blasts from 30 patients. We observed frequent expression of TRAIL DcR1 and DcR2, while expression of DR4 and DR5 was less frequent. Nevertheless, the expression of DR4 or DR5 in leukaemic cells was always matched by a similar expression of one of the decoy receptors. Leukaemic blasts were invariably resistant, even to a high concentration (1000 ng/ml) of TRAIL. We suggest that AML blasts are resistant to TRAIL apoptosis in vitro. Therefore, it is unlikely that TRAIL alone might be used in the future as an innovative pharmacological agent for the treatment of AML.     

Osteoprotegerin (OPG) acts as an endogenous decoy receptor in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis of fibroblast-like synovial cells

We examined the role of osteoprotegerin (OPG) on tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in rheumatoid fibroblast-like synovial cells (FLS). OPG protein concentrations in synovial fluid from patients with rheumatoid arthritis (RA) correlated with those of interleukin (IL)-1beta or IL-6. A similar correlation was present between IL-1beta and IL-6 concentrations. Rheumatoid FLS in vitro expressed both death domain-containing receptors [death receptor 4 (DR4) and DR5] and decoy receptors [decoy receptor 1 (DcR1) and DcR2]. DR4 expression on FLS was weak compared with the expression of DR5, DcR1 and DcR2. Recombinant TRAIL (rTRAIL) rapidly induced apoptosis of FLS. DR5 as well as DR4 were functional with regard to TRAIL-mediated apoptosis induction in FLS; however, DR5 appeared be more efficient than DR4. In addition to soluble DR5 (sDR5) and sDR4, OPG administration significantly inhibited TRAIL-induced apoptogenic activity. OPG was identified in the culture supernatants of FLS, and its concentration increased significantly by the addition of IL-1beta in a time-dependent manner. Neither IL-6 nor tumour necrosis factor (TNF)-alpha increased the production of OPG from FLS. TRAIL-induced apoptogenic activity towards FLS was reduced when rTRAIL was added without exchanging the culture media, and this was particularly noticeable in the IL-1beta-stimulated FLS culture; however, the sensitivity of FLS to TRAIL-induced apoptosis itself was not changed by IL-1beta. Interestingly, neutralization of endogenous OPG by adding anti-OPG monoclonal antibody (MoAb) to FLS culture restored TRAIL-mediated apoptosis. Our data demonstrate that OPG is an endogenous decoy receptor for TRAIL-induced apoptosis of FLS. In addition, IL-1beta seems to promote the growth of rheumatoid synovial tissues through stimulation of OPG production, which interferes with TRAIL death signals in a competitive manner.     

Characterization of tumour necrosis factor-alpha-related apoptosis-inducing ligand and its receptors in the adult human testis

Tumour necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a member of the tumour necrosis factor-alpha (TNF-alpha) family of cytokines which is known to induce apoptosis upon binding to its death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. Two additional TRAIL receptors, DcR1/TRAIL-R3 and DcR2/TRAIL-R4, lack functional death domains and act as decoy receptors for TRAIL. In this study, the presence of TRAIL and its receptors was investigated by immunohistochemistry in adult human testes. In addition, TRAIL and its receptors were studied in terms of protein and mRNA using western blot analysis and RT-PCR respectively. TRAIL and its receptors were immunodetected according to the different testicular cell types: TRAIL, DR5/TRAIL-R2 and DcR2/TRAIL-R4 were localized in Leydig cells, DR4/TRAIL-R1 was seen in peritubular and Sertoli cells whereas ligand and all receptors were detected in germ cells. Proteins and mRNA corresponding to TRAIL and its receptors were also identified in adult human testes. In conclusion, TRAIL and its receptors DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1/TRAIL-R3 and DcR2/TRAIL-R4 are expressed in the human testis, and are predominantly localized in different germ cell types.     

杭白菊增强TRAIL基因诱导大肠癌细胞凋亡及机制的实验研究

目的：探讨杭白菊提取液对肿瘤坏死因子相关凋亡诱导配体基因（TRAIL）抑制人大肠癌细胞株DLD-1作用的影响及其可能机制。 方法： 杭白菊提取液联合重组腺病毒载体（Ad）介导的TRAIL基因作用于人大肠癌细胞株DLD-1，通过倒置显微镜、MTT比色法和流式细胞仪，研究分析其对DLD-1细胞抑制作用的效果。采用逆转录聚合酶链反应（RT-PCR）和流式细胞术检测杭白菊提取液作用前后DLD-1细胞TRAIL、TRAIL受体（TRAIL-Rs）mRNA以及细胞表面TRAIL蛋白表达的变化。 结果： Ad/hTERT-gTRAIL对DLD-1细胞的生长抑制率和凋亡率分别为31.4%和13.5%；联合杭白菊提取液后，生长抑制率和凋亡率均显著提高，达93.1%和45.4%（P<0.05）。杭白菊提取液作用后DLD-1细胞TRAIL mRNA的表达量从作用前的0.46上调至1.01, 细胞表面TRAIL蛋白表达的百分率从作用前的2.2%升高到5.0%（P<0.05）。TRAIL死亡受体（DR4、DR5）mRNA的表达量分别从0.70和0.22上调至1.10和0.83（P<0.05），而TRAIL诱骗受体（DcR1、DcR2）mRNA的表达量从0.60和1.15下调至0.19和0.78（P<0.05）。 结论： 杭白菊提取液联合重组腺病毒载体（Ad）介导的TRAIL基因(Ad/hTERT-gTRAIL)能有效诱导DLD-1细胞的凋亡。杭白菊提取液上调TRAIL死亡受体表达以及下调TRAIL诱骗受体的表达可能在增强TRAIL诱导的凋亡作用中起着重要作用。     

Ectopic expression of Bcl-2 and Bcl-xL inhibits apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) through suppression of caspases-8, 7, and 3 and BID cleavage in human acute myelogenous leukemia cell line HL-60.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of the latest members of the TNF superfamily known to induce apoptosis in a wide variety of tumor cells. Some cell types, however, are quite resistant to TRAIL. We investigated the effect of ectopic expression of Bcl-2 and Bcl-xL on TRAIL-induced apoptosis in human acute myelogenous leukemia HL-60 cells. We found that HL-60 cells, which express TRAIL receptors (also called death receptor, DR) DR4, DR5, and Dc (decoy) R2, are highly sensitive to TRAIL-induced cytotoxicity. Greater than 90% killing occurred within 24 h of TRAIL treatment. The expression of Bcl-2 and Bcl-xL, however, completely abolished the TRAIL-induced cytotoxic effects. Treatment of HL-60 cells with TRAIL induced caspase-8 activation within 2-4 h, but no activation could be seen in Bcl-2-expressing or Bcl-xL-expressing cells. TRAIL also induced cleavage of BID, which was also abolished by Bcl-2 and Bcl-xL. Similarly, TRAIL activated caspase-3 and caspase-7 in control cells but not in cells expressing Bcl-2 or Bcl-xL. Cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP), was abrogated by ectopic expression of Bcl-2 and Bcl-xL. Inhibition of caspases by the pan-caspase inhibitor, benzyloxycarbonyl-valine-alanine-aspartate-fluoromethylketone (zVAD-fmk) abolished the TRAIL-induced apoptosis. Overall, these results indicate that TRAIL-induced apoptosis involves activation of caspase-8, caspase-7, caspase-3, and BID cleavage, and Bcl-2 and Bcl-xL prevents TRAIL-induced apoptosis by abrogating caspase activation and BID cleavage.