TRAIL受体在人骨肉瘤组织中的表达

目的 研究肿瘤坏死因子相关凋亡诱导配体(TRAIL)受体在骨肉瘤细胞中的表达情况。方法 应用逆转录—聚合酶链反应(RT—PCR)对22例骨肉瘤组织标本、MG—63骨肉瘤细胞株、U251脑胶质瘤细胞株以及正常人外周血淋巴细胞TRAILR1—R4 mRNA表达进行检测。结果 22例骨肉瘤组织标本中15例同时表达TRAILR1、-R2和-R3，4例同时表达TRAIL-R1和-R2，只表达TRAIL-R1或-R2者3例，所有标本中均未检测到TRAIL，-R4表达；MG-63骨肉瘤细胞株、U251脑胶质瘤细胞株以及正常人外周血淋巴细胞则均检测到TRAILR1、-R2和-R3的联合表达。结论 死亡受体TRAIL-R1，R2在骨肉瘤中的普遍表达，是TRAIL诱导骨肉瘤细胞凋亡的分子基础；死亡受体和诱骗受体的差异性分布，并非TRAIL对选择性杀伤肿瘤细胞的关键性因素，可能还受其它因子调控。     

人可溶性TRAIL蛋白诱导骨肉瘤细胞凋亡的实验研究

目的 初步观察并探讨人重组可溶性TRAIL蛋白诱导MG-63骨肉瘤细胞凋亡的作用，并通过实验初步探讨TRAIL对肿瘤细胞的选择性杀伤作用。方法 MTT法测定TRAIL对MG-63细胞、MRC-5人肺二倍体细胞及L-02人肝细胞的抑制率；电镜观察与FACS分析TRAIL蛋白诱导MG-63骨肉瘤细胞凋亡的作用；用RT-PCR检测TRAIL受体在MG-63骨肉瘤细胞、MRC-5人肺二倍体细胞及L-02人肝细胞上的表达，并初步分析TRAIL诱导MG-63骨肉瘤细胞凋亡的机制。结果 MTT还原试验表明：作用24h后500ng/ml TRAIL的抑制率为10.1％，1μg/ml TRAIL的抑制率为24.3％，2μg/ml TRAIL的抑制率为50.6％，5μg／ml TRAIL的抑制率为95％以上，其半数抑制浓度为2μg/ml。而TRAIL对MRC-5人肺二倍体细胞株及L-02人肝细胞株无明显作用。细胞形态学观察显示：MG-63骨肉瘤细胞经TRAIL作用3～8h后即有部分细胞开始变小、变圆，24h后大量细胞变圆、脱落，漂浮于培养液中。透射电镜可观察到核固缩，染色质浓聚于核膜形成新月状小体。流式细胞仪检测显示2μg／ml的TRAIL处理MG-63骨肉瘤细胞6h，即可在二倍体峰前出现较明显的凋亡峰。结论 人可溶性TRAIL蛋白可以迅速地选择性杀伤MG-63骨肉瘤细胞，具有潜在的临床应用价值。     

TRAIL联合IFN-γ诱导骨肉瘤细胞凋亡实验研究

目的 观察干扰素(IFN)-γ协同肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导骨肉瘤细胞(MG-63)凋亡的作用,初步探讨协同作用机制,并观察联合用药时对成纤维细胞的毒性作用.方法 应用相差显微镜和电镜观察MG-63细胞经TRAIL、IFN-γ及TRAIL联合IFN-γ作用后细胞形态学变化;进行四唑盐比色试验观察不同实验组MG-63细胞和成纤维细胞抑制率;流式细胞仪观察细胞凋亡情况;逆转录-聚合酶链反应法观察IFN-γ作用后TRAIL膜受体表达情况.结果 TRAIL联合IFN-γ应用时,随着IFN-γ作用时间的延长,细胞悬浮、空泡化等现象逐渐明显,且在作用24小时最为显著,电镜显示细胞凋亡的特征性变化;联合用药组细胞抑制率较单独TRAIL组增加,两者有显著性差异(P<0.01);联合用药组细胞凋亡率显著增加(P<0.01);应用IFN-γ前后,TRAIL死亡受体(DR)4的相对表达量增加,IFN-γ作用24小时表达量最大,两者有显著性差异(P<0.01);联合用药组对成纤维细胞的毒性,与单独TRAIL组相比没有显著增加(P>0.05).结论 IFN-γ可协同TRAIL诱导MG-63细胞凋亡,DR4表达量增加可能是协同作用的缘故,联合用药不会增加对成纤维细胞的毒副反应.     

TRALL联合IFN-γ诱导骨肉瘤细胞凋亡实验研究

目的观察干扰素(IFN)-γ协同肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导骨肉瘤细胞(MG-63)凋亡的作用,初步探讨协同作用机制,并观察联合用药时对成纤维细胞的毒性作用。方法应用相差显微镜和电镜观察MG-63细胞经TRAIL、IFN-γ及TRAIL联合IFN-γ作用后细胞形态学变化;进行四唑盐比色试验观察不同实验组MG-63细胞和成纤维细胞抑制率;流式细胞仪观察细胞凋亡情况;逆转录-聚合酶链反应法观察IFN-γ作用后TRAIL膜受体表达情况。结果TRAIL联合IFN-γ应用时,随着IFN-γ作用时间的延长,细胞悬浮、空泡化等现象逐渐明显,且在作用24小时最为显著,电镜显示细胞凋亡的特征性变化;联合用药组细胞抑制率较单独TRAIL组增加,两者有显著性差异(P<0.01);联合用药组细胞凋亡率显著增加(P<0.01);应用IFN-γ前后,TRAIL死亡受体(DR)4的相对表达量增加,IFN-γ作用24小时表达量最大,两者有显著性差异(P<0.01);联合用药组对成纤维细胞的毒性,与单独TRAIL组相比没有显著增加(P>0.05)。结论IFN-γ可协同TRAIL诱导MG-63细胞凋亡,DR4表达量增加可能是协同作用的缘故。联合用药不会增加对成纤维细胞的毒副反应。     

塞来昔布协同顺铂P13K/Akt途径诱导骨肉瘤MG-63细胞凋亡的实验研究

目的 探讨环氧化酶-2(COX-2)选择性抑制剂塞来昔布对人骨肉瘤MG-63细胞增殖和凋亡的影响及作用机制.方法 塞来昔布(50 μmol/L或100 μmol/L)和(或)顺铂(10 μg/ml)作用骨肉瘤MG-63细胞48 h后,MTT法测定细胞增殖的抑制率,电子显微镜和流式细胞仪检测细胞凋亡,RT-PCR法检测基因水平COX-2表达变化,Western blot检测COX-2及凋亡相关蛋白表达变化.P13K抑制剂Wortmannin作用MG-63细胞48 h,检测蛋白表达变化.结果 塞来昔布导致MG-63细胞阻滞在G1期,通过激活半胱氨酸天冬氨酸蛋白酶(caspase)-9的内源性凋亡途径诱导细胞凋亡;顺铂单药作用后骨肉瘤MG-63细胞凋亡率为5.93%,而联合应用塞来昔布50μmol/L或100 μmol/L后,凋亡率分别为6.66%和37.15%,与顺铂联合具有明显的协同作用;COX-2蛋白表达未降低.塞来昔布联合顺铂明显降低P13K/Akt、survivin、Bcl-2的表达,检测到caspase-9、caspase-3的激活和PARP裂解片段.Wortmannin作用MG-63细胞48 h,检测到pAkt(Thr308)、Bcl-2、survivin表达下调.结论 塞来昔布可通过非COX-2途径诱导骨肉瘤MG-63细胞凋亡,与P13K/Akt、survivin、Bcl-2蛋白相关,并且PI3K/Akt途径在survivin、Bcl-2表达调控中发挥重要作用.这可能是塞来昔布药物干预的中心环节.     

藤黄酸联合顺铂对人骨肉瘤细胞作用的实验研究

[目的]本实验主要研究藤黄酸(gambogic acid,GA)在骨肉瘤中的抗肿瘤作用,检验藤黄酸与传统的骨肉瘤化疗药物顺铂(cisplatin,CDDP)联用是否具有协同作用,探讨其作用的机制。[方法]采用CCK-8法测定不同浓度藤黄酸、顺铂单用及联合应用对人骨肉瘤细胞MG-63的增殖抑制作用,PI染色流式细胞术检测细胞周期变化,Annexin V-FITC/PI双染色检测细胞凋亡变化。[结果]藤黄酸对MG-63具有明显生长抑制作用,且具有浓度依赖性,IC 25为0.52μg/ml,藤黄酸诱导MG-63细胞产生G2/M期细胞周期阻滞和凋亡,而顺铂则诱导产生G0/G1期细胞周期阻滞和凋亡。藤黄酸与顺铂联用时增殖抑制及诱导凋亡作用比单用时明显增强。[结论]藤黄酸通过诱导MG-63细胞周期阻滞和凋亡产生抗人骨肉瘤细胞增殖作用,藤黄酸与顺铂联用具有协同作用。     

TRAIL联合顺铂诱导肝癌细胞HepG2凋亡的研究

目的 探讨顺铂增加TRAIL诱导肝癌细胞HepG2凋亡的分子机制，提供TRAIL应用肝癌临床治疗的一种新的用药模式。方法 以肝癌细胞HepG2为研究对象，应用流式细胞仪分析顺铂联合应用TRAIL对细胞周期的影响，Western blot检测顺铂作用前后HepG2细胞在TRAIL诱导凋亡过程中Caspase-8、Caspase-3、DR4、DR、DcR，c—FLIP及RIP的表达变化。结果 顺铂能够增强TRAIL诱导HepG2细胞凋亡的能力，两者联合应用具有明显的协同作用，顺铂预先处理能够下调c-FLIP及RIP的表达及增加DR的表达。结论 TRAIL联合顺铂可以诱导肝癌细胞HepG2凋亡，其机制是通过下调c-FLIP及RIP的表达，解除Caspase-8受抑，恢复凋亡信号的传导来实现的。死亡受体D＆的表达上调也可能参与了这一变化过程。     

阿霉素协同TRAIL真核表达载体诱导前列腺癌细胞凋亡的研究

目的探讨阿霉素与肿瘤坏死因子相关凋亡诱导配体（TRAIL）真核表达载体诱导前列腺癌细胞凋亡的协同作用.方法 RT-PCR方法克隆人TRAIL全长基因,并插入真核表达载体pLXIN多克隆位点,经酶切和测序鉴定.将pLXIN-TRAIL质粒转染PC3-M细胞,RT-PCR方法检测外源性TRAIL表达率,TUNEL染色和流式细胞术比较单独表达TRAIL和联合应用阿霉素对PC3-M细胞凋亡率的影响,Western blot法检测经阿霉素诱导前后PC-3M细胞死亡受体-5（DR5）的表达变化.结果成功构建pLXIN-TRAIL真核表达载体,外源性TRAIL表达可以诱导PC-3M细胞凋亡,阿霉素能够增强TRAIL的凋亡诱导效应,6μg pLXIN-TRAIL质粒DNA组,加入1 mg/L阿霉素前后平均凋亡率分别为11.8%和20.7%,同时可提高PC-3M细胞DR5的表达. 结论 TRAIL真核表达载体介导的细胞凋亡是治疗前列腺癌的新途径,阿霉素可以通过提高DR5表达而增强PC-3M细胞对TRAIL诱导的凋亡敏感性.     

低剂量阿霉素联合TRAIL诱导人前列腺癌细胞LNCAP凋亡的实验研究

目的 研究低剂量阿霉素联合肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导人前列腺癌细胞LNCAP凋亡的作用和机制,为提高肿瘤治疗效果提供理论依据.方法 采用蛋白印迹、流式细胞术、MTT等实验技术,检测TRAIL和低剂量阿霉素分别或者联合处理LNCAP细胞后细胞的生存率变化、相关受体及其配体系统、凋亡相关蛋白(c-FLIP,BCL-2,XIAP)的变化.结果 低剂量阿霉素(0.86 μmol/L)可以增加细胞表面DR4和DR5的表达,同时减少凋亡抑制蛋白c-FLIP的表达,增强TRAIL诱导LNCAP细胞凋亡的敏感性.结论 低剂量阿霉素(0.86 μmol/L)可以增强TRAIL对LNCAP细胞的凋亡诱导效应.     

TRAIL和TRAILR在肝癌中表达及对肝癌的治疗作用

目的探讨肿瘤坏死因子相关凋亡诱导配体(tumornecrosisfactorrelatedapoptosis-inducingligand,TRAIL)与其受体(TRAILR)在肝细胞肝癌中的表达及TRAIL联合应用化疗药对肝癌细胞的杀伤作用。方法采用免疫组化和原位杂交检测100例肝癌及癌旁组织中TRAIL及TRAILR的表达。用MTT法检测TRAIL与化疗药联合应用对肝癌细胞的杀伤作用。结果TRAIL在正常肝组织中无表达,在癌旁组织中的表达明显高于癌组织;肝癌组织中死亡受体(DR5、DR4)的表达量显著强于正常肝组织,诱捕受体为低表达,与正常肝组织相比,两者差异显著(χ2=4·68,P<0·05)。Ⅲ/Ⅳ期肿瘤死亡受体的表达显著低于Ⅰ/Ⅱ期(χ2=6·17,P<0·05)。联合使用亚细胞毒性剂量的化疗药大幅度提高了TRAIL的细胞毒活性。结论TRAIL与化疗药物联合运用具有协同杀伤肝癌细胞的作用。     

Expression and function of TNF-family proteins and receptors in human osteoblasts

We studied how tumor necrosis-factor (TNF)-family proteins interact with osteoblasts to resolve several controversial points. We measured expression of TNFs, TNF-receptors, and nonsignaling (decoy) TNF receptors in human osteoblasts derived from mesenchymal stem cells and in MG63 human osteosarcoma cells using unamplified mRNA screening, with secondary Western or PCR analysis where indicated, and studied the effects of TNFs on osteoblasts in cell culture. Expression of TNFs and receptors was similar in MG63 cells and osteoblasts. TNF-R1 (p55), TRAIL receptor 1 and 2 (DR4 and 5), and Fas were expressed; RANK was undetectable. TNF-family ligands RANKL, TRAIL, and TNFalpha were expressed, but mRNAs were typically at low levels relative to receptors, suggesting that osteoblastic TNF signals, including RANKL, require specific stimuli. Flow cytometry of MG63 cells confirmed TNFalpha receptors and identified subpopulations with high surface-bound TNFalpha. Decoy receptors expressed included a novel soluble form of TNFRSF25 (formerly DR3 or Apo3), implicated in rheumatoid-arthritis linkage studies, as well as osteoprotegerin, a well-characterized osteoblast protein that binds TRAIL and RANKL, and DcR2, which binds TRAIL. Osteoblast apoptosis was studied using terminal deoxynucleotidyl transferase labeling and annexin V binding. MG63 cells were resistant to apoptosis by exogenous TNFalpha except when grown in media promoting osteoblast-like growth or matrix nodules. However, in media supporting osteoblast-like phenotype, apoptosis was induced by anti-Fas or TNF, in contrast to other studies with human osteoblasts. TRAIL caused cell retraction, supporting functional TRAIL response in cell differentiation, but did not cause apoptosis. We conclude that human osteoblasts have functional receptors for FasL, TNFalpha, TRAIL, but not RANKL, and that osteoblasts are protected by multiple nonsignaling TNF receptors against destruction by TNF-family proteins under conditions favoring cell growth.     

肿瘤坏死因子相关凋亡诱导配体联合阿霉素治疗骨肉瘤

目的 探讨肿瘤坏死因子相关凋亡诱导配体（TRAIL）与阿霉素（ADM）联用对骨肉瘤细胞株HOS是否具有协同杀伤作用，并对其机制进行初步研究。方法 MTT法检测细胞的抑制率和存活率，流式细胞术（FCM）检测细胞凋亡率，逆转录-聚合酶链反应（RT—PCR）检测TRAIL受体（receptor，R）的表达水平。结果 单用0．5mg／LTRAIL及1．0、10．0、100．0mg／LADM对HOS8603的抑制率分别为13．18％、5．56％、23．02％和76．72％，联合用药的抑制率分别为42．98％、53．12％、81．91％。0．5mg／LTRAIL和1．0mg／LADM有协同作用．TRAIL和ADM联用时TRAIL R2 mRNA表达较TRAIL单用时明显增强。结论 TRAIL与ADM联用对骨肉瘤细胞有明显的协同作用，其机制可能与ADM上调TRAILR2的表达有关。     

The death ligand TRAIL in diabetic nephropathy

Apoptotic cell death contributes to diabetic nephropathy (DN), but its role is not well understood. The tubulointerstitium from DN biopsy specimens was microdissected, and expression profiles of genes related to apoptosis were analyzed. A total of 112 (25%) of 455 cell death-related genes were found to be significantly differentially regulated. Among those that showed the greatest changes in regulation were two death receptors, OPG (the gene encoding osteoprotegerin) and Fas, and the death ligand TRAIL. Glomerular and proximal tubular TRAIL expression, assessed by immunohistochemistry, was higher in DN kidneys than controls and was associated with clinical and histologic severity of disease. In vitro, proinflammatory cytokines but not glucose alone regulated TRAIL expression in the human proximal tubular cell line HK-2. TRAIL induced tubular cell apoptosis in a dosage-dependant manner, an effect that was more marked in the presence of high levels of glucose and proinflammatory cytokines. TRAIL also activated NF-kappaB, and inhibition of NF-kappaB sensitized cells to TRAIL-induced apoptosis. It is proposed that TRAIL-induced cell death could play an important role in the progression of human DN.     

肿瘤坏死因子相关凋亡诱导配体治疗肝细胞癌的研究

目的探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)与其受体在肝细胞肝癌(hepatocellularcarcinoma,HCC)中的表达及意义,及利用TRAIL对HCC的治疗作用。方法采用免疫组化技术及原位杂交方法分别检测了100例肝癌组织,100例癌旁组织,40例正常肝组织中TRAIL及TRAILR的表达,并结合临床资料进行分析;采用不同浓度TRAIL处理肝癌细胞株HepG2、SMMC7721,观察经药物处理前后肿瘤细胞的凋亡发生率。结果TRAIL在正常肝组织中无表达,癌旁组织中的表达明显高于癌组织。86例肝癌组织不表达诱捕受体DcR1(86%),55例肝癌组织不表达诱捕受体DcR2(55%),40例正常肝组织两种诱捕受体均有表达。肝癌组织中死亡受体为高表达,诱捕受体为低表达,正常肝组织则相反,两者间有显著差异性(P<0.05)。肝癌组织中死亡受体的表达与肿瘤的分化呈正相关(P<0.01),与肿瘤分级呈负相关(P<0.05),与病人的性别、年龄、AFP水平、肿瘤的大小以及是否转移无关。经TRAIL(100ng/ml)处理24h,肝癌细胞凋亡发生率约10%,而Jurkat细胞凋亡率达70%以上,胆管癌细胞QBC939凋亡发生率约50%。结论HCC时,TRAILR普遍表达,但存在受体类型的表达差异,其中DcR1大多缺失,这为利用TRAIL治疗HCC提供了理论依据,然而,单一的TRAIL治疗只能有限的诱导肝癌细胞HepG2、SMMC7721发生凋亡,HCC对TRAIL诱导的凋亡存在耐药现象。     

转染肿瘤坏死因子相关凋亡诱导配体对膀胱癌细胞的抑制作用

目的探讨肿瘤坏死因子相关凋亡诱导配体（TRAIL）对膀胱癌细胞的抑制作用。方法构建TRAIL融合增强绿色荧光蛋白（EGFP）真核表达载体，脂质体法转染膀胱癌细胞株玎细胞。荧光显微镜下计算转染率。分别采用逆转录-聚合酶链反应（RT-PCR）、Western blot检测TRAIL的表达。采用流式细胞仪检测细胞凋亡，噻唑蓝（MTT）法、细胞倍增时间和集落形成率观察TRAIL的抑制作用。结果48h观察重组载体转染率为38．4％，空载体转染率为35．3％（P〉0．05）．RT-PCR和Western blot证实转染后EJ细胞中TRAIL的表达明显增加；凋亡率明显高于转染空白载体和未转染组（P〈0．01）；EJ细胞增殖受到明显抑制（P〈0．01）。结论TRAIL具有诱导膀胱癌细胞凋亡、抑制增殖的作用，有望成为治疗膀胱癌的新方法。     

Role of TNF-related apoptosis-inducing ligand (TRAIL) in the pathogenesis of varicocele-induced testicular dysfunction

The higher frequency of varicocele in men with infertility has drawn attention and resulted in increased research at the molecular level towards treatments. The aim of this study was to investigate the role of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptors in varicocele-induced testicular dysfunction in an experimental rat model. The rats were divided into three groups: control, sham and varicocele. Varicoceles in rats were induced by partial ligation of the left renal vein and left testes. The rats were analyzed 13 weeks after surgery. The degree of DNA fragmentation within cells in the testis was determined using terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay. Tubule degeneration was evaluated using the Johnsen score. The expression of TRAIL and its receptors was detected by immunohistochemical and Western blotting techniques. The apoptotic index, Johnsen score and the expression of TRAIL and TRAIL receptors were examined. The data are presented as the mean±s.d. and were analyzed using computer software. The Kruskal–Wallis and Dunn''s multiple comparison tests were used in the statistical analyses. The germ cell apoptotic index was increased in rats with varicoceles when compared with the sham and control groups (P=0.0031). The Johnsen score was significantly decreased in the varicocele group when compared with the sham and control groups (P<0.0001). Immunohistochemical and Western blotting analyses showed that after varicocele induction, the expression of TRAIL-R1 and TRAIL-R4 in germ cells was increased and the expression of TRAIL-R2 was decreased. There are no significant differences among the groups in terms of TRAIL and TRAIL-R3 receptor expression. The results of this study indicate that TRAIL and its receptors may have a potential role in the pathogenesis of varicocele-induced testicular dysfunction.     

TRAIL受体在前列腺癌中的表达

目的　探讨肿瘤坏死因子相关诱导凋亡配体 (TRAIL)受体在前列腺癌组织中的表达及其与前列腺癌恶性度的关系。方法　采用免疫组织化学方法检测前列腺癌组织及良性前列腺增生组织中DR4、DR5及DcR1的表达。结果　前列腺癌组织DcR1表达水平显著低于良性前列腺增生组织 (P <0 .0 1) ,DR4、DR5的表达水平两者无显著性差异。前列腺癌组织中DR4、DR5及DcR1的表达程度与前列腺癌组织的病理分级和临床分期无关 (P >0 .0 5 )。结论　TRAIL基因受体DcR1在前列腺癌凋亡机制中可能发挥重要作用