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DR5抗体诱导白血病细胞系凋亡与DR5表达
Leukemia Cell Apoptosis Induced by Anti-hdr5 Monoclonal Antibody and DR5 Expression on Leukaemia Cell
【作者】 王赟；
【作者基本信息】 河南大学 ， 生理学， 2010， 硕士
【摘要】 背景肿瘤坏死因子相关的凋亡诱导配体(TNF-related apoptosis inducing ligand, TRAIL)是1995年Wiley等发现的TNF超家族成员,能够诱导多种肿瘤细胞凋亡而对大多数正常细胞无影响。TRAIL通过与肿瘤细胞膜上的相应受体结合诱导细胞凋亡,目前发现TRAIL有5种类型的受体,其中TRAIL受体1(死亡受体4,death receptor 4, DR4)和RAIL受体2(死亡受体5,death receptor 5, DR5)胞内段含有死亡结构域,能够传递凋亡信号,其他三种受体:TRAIL受体3(DcR1)、TRAIL受体4(DcR2)和OPG(Osteoprotegerin)可阻断TRAIL诱导的凋亡,称为“诱骗”受体。虽然天然TRAIL有良好的抗肿瘤作用,但实验发现某些rhTRAIL对正常肝细胞等有细胞毒性,这使人们对其生物安全性产生了怀疑。研究发现一些抗DR4或DR5的功能性单克隆抗体(mAb)能够模拟TRAIL的作用,诱导肿瘤细胞凋亡,且无肝细胞毒性,因此抗人DR4和DR5的功能性单克隆抗体(mAb)成为目前TRAIL肿瘤生物治疗的新靶点。我们利用重组人DR5蛋白免疫BALB/c小鼠,制备了功能性抗DR5单克隆抗体-mDRA-6,前期研究表明mDRA-6可有效诱导多种肿瘤细胞凋亡,同时发现不同肿瘤细胞对其敏感性存有较大差异。虽然肿瘤细胞对mDRA-6敏感性的高低可能与细胞DR5表达、细胞内凋亡信号分子数量及激活状态、细胞内抗凋亡分子变化等诸因素有关,但mDRA-6与细胞DR5结合是启动细胞凋亡的前提,因此推测细胞DR5表达水平是影响mDRA-6诱导细胞凋亡的重要因素。本研究通过分析白血病细胞DR5表达与mDRA-6诱导细胞凋亡的关系,探讨提高细胞DR5表达是增强mDRA-6凋亡诱导作用的有效途径。目的检测不同白血病细胞系细胞表面DR5表达、细胞DR5蛋白表达、细胞DR5mRNA表达水平,分析抗人DR5单克隆抗体mDRA-6诱导白血病细胞凋亡与细胞DR5表达的关系。观察维生素E琥珀酸酯(Vitamin E Succinate; VES)对白血病细胞DR5表达的影响,探讨VES增加mDRA-6诱导白血病细胞凋亡作用及可能机制。方法MTT法检测mDRA-6对白血病细胞Jurkat、U937、HL-60、Raji和K562细胞的生长抑制作用;Annexin V–FITC/PI双染定量分析白血病细胞凋亡;流式细胞术检测细胞表面DR5表达;免疫印迹技术(Western blotting)检测细胞DR5蛋白表达情况;RT-PCR法检测细胞DR5mRNA表达。结果1. MTT检测表明,浓度为10μg /ml的mDRA-6作用12h,白血病Jurkat、U937、HL-60、Raji细胞死亡率分别为88.25%, 61.43%,59.76%,21.98%,K562的细胞死亡率仅为5.23%。5μmol/L、10μmol/L、20μmol/L的VES与mDRA-6分别共同作用于Raji和K562细胞,Raji细胞死亡率分别增加至24.67%(P﹥0.05)、35.65%(P<0.01)和40.22%(P<0.01);K562细胞死亡率增加至6.00%(P﹥0.05)、7.89%(P<0.01)、8.67%(P<0.01)。2. AnnexinⅤ/PI双染检测5μg/ml的mDRA-6作用白血病细胞12h,Jurkat、U937、HL-60、Raji和K562细胞凋亡率分别为85.96%、50.22%、48.11%、23.40%和7.20%。2μg/ml的mDRA-6单独作用Raji、K562细胞16h,细胞凋亡率分别为20.79%和7.74%,2μg/ml的mDRA-6与10μmol/L的VES共同作用Raji、K562细胞16h,细胞凋亡率分别增至43.18%和16.99%。3.白血病细胞Jurkat、U937、HL-60、Raji和K562细胞表面DR5表达分别为94.8%、74.71%、58.66%、42.35%和14.92%;Jurkat、U937、HL-60、Raji和K562细胞表面DR5表达与mDRA-6诱导的细胞凋亡呈明显正相关(r=0.9712,t=7.063,p<0.01)。10μmol/L的VES作用Raji、K562细胞12h后,细胞膜DR5表达率分别增加至70.08%和16.38%。4.免疫印迹技术检测显示,细胞DR5蛋白表达强弱依次为Jurkat、U937、HL-60、Raji和K562。Raji和K562细胞DR5蛋白基础表达量较少,5μmol/L、10μmol/L、20μmol/L的VES分别作用于Raji和K562细胞12h,Raji和K562细胞DR5蛋白表达量均增加,10μmol/L、20μmol/L的VES诱导DR5蛋白表达增加显著。5.白血病细胞系Jurkat、U937、HL-60、Raji和K562细胞DR5mRNA相对量分别为:306.82±106.36,214.48±93.56,198.47±78.45,123.68±67.23,45.29±26.39;Jurkat、U937、HL-60、Raji和K562细胞DR5mRNA表达与mDRA-6诱导的细胞凋亡呈明显正相关(r=0.9915,t=13.22,p<0.01)。20μmol/L的VES作用Raji细胞12h,细胞DR5mRNA表达显著增加(p<0.05),5μmol/L、10μmol/L的VES作用Raji细胞12h及5μmol/L、10μmol/L、20μmol/L的VES作用K562细胞12h,虽能使细胞DR5 mRNA表达增加,但无统计学意义。结论1. mDRA-6能够诱导人白血病Jurkat、U937、HL-60、Raji和K562细胞凋亡,但不同白血病细胞系细胞对mDRA-6的敏感性不同。Jurkat、U937、HL-60、Raji和K562细胞表面DR5表达及DR5mRNA表达与mDRA-6诱导的细胞凋亡呈明显正相关,白血病细胞系细胞对mDRA-6的敏感性不同与细胞DR5表达差异有关。2. VES增加Raji和K562细胞膜DR5表达、DR5蛋白表达及DR5mRNA表达,同时能够增强mDRA-6对白血病细胞系Raji和K562细胞的凋亡诱导作用。VES增加Raji和K562细胞DR5表达可能是其增强mDRA-6凋亡诱导作用的机制之一。
【Abstract】 BackgroundTumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily with the ability to induce apoptosis in a wide variety of transformed cell lines of diverse origin. At least five receptors for TRAIL have been identified so far. Two of them, TRAIL-R1 (DR4) and TRAIL-R2 (DR5), sharing a commom intracellular domain, called the death domain (DD), which is indispensable for initiation of the intracellular signaling cascade leading to cell death, are capable of transducing an apoptosis signal, whereas the other three, TRAIL-R3 (DcR1), TRAIL-R4 (DcR2) and osteoprotegerin (OPG), serve as decoy receptors to block TRAIL-mediated apoptosis. TRAIL has been known to induce apoptosis in a variety of tumor cells and some virally infected cells but not in most normal cells. However, increasing experimental evidences on hrTRAIL-inducing apoptosis of normal cells(especially hepatocytes) were reported in recent studies, arguing against the potential usefulness and safety of soluble TRAIL in cancer therapy. In addition to sTRAIL, monoclonal antibodies (mAbs) against TRAIL-R1 (DR4) or TRAIL-R2 (DR5) with tumoricidal activity are also potential candidates for cancer therapy. There are a number of agonistic mAbs against human DR4 or DR5 reported in previous studies, most of which need crosslinkers to ensure effective killing of tumor cells.We have cloned the human DR5 gene and prepared the soluble recombinant human DR5 protein (rhDR5), established hybridomas and produced an anti-hDR5 monoclonal antibody, mDRA-6. mDRA-6 showed strong tumoricidal activity in the absence of cross-linking, and had different sensitivity for tumor cells.ObjectiveThis study is to detect DR5 expression in leukaemia cells, and evaluate the correlation of DR5 expression with apoptosis induced by mDRA-6 in human leukemia cell. The synergistic effect of mDRA-6 and Vitamin E Succinate (VES) on Raji and K562 cells and its possible mechanism were studied.MethodsCytotoxic and apoptotic effects of mDRA-6 on Jurkat, U937, HL-60, Raji and K562 cells and the synergistic effect of mDRA-6 and vitamin E succinate (VES) on Raji and K562 cells were detected by MTT and flow cytometry with AnnexinV-FITC/PI staining. DR5 expression on leukaemic cell surface was determined by flow cytometric analysis of the binding of anti-DR5 mAb; DR5 total protein expression in leukaemic cells were detected by western blot. The RT-PCR(semi-quantitative reverse transcription polymerase chain reaction)method was used to detect the DR5 mRNA expression in leukaemic cells.Results①The specific cytotoxicity of the mDRA-6 against Jurkat cells was 88.25%, U937 cells was 61.43%,HL-60 cells was 59.76%, Raji cells was 21.98%, and K562 cells was 5.23% respectively at the concentration of 10mg/L for 12 hours by MTT analysis. The combination of mDRA-6 and VES exhibited synergistic effect on Raji cells and K562 cells. 10μg/mL of mDRA-6 and 20μmol/L of VES killed 40.22% Raji cells and 8.67% K562 cells.②The apoptotic rate of the Jurkat, U937, HL-60, Raji and K562 cells was 50.22%, 48.11%, 23.40% and 7.20%, respectively ,in the presence 5mg/L mDRA-6 for 12 hours determined by flow cytometry with Annexin V-FITC/PI staining. The apoptotic rate of Raji and K562 celsl was increased to 43.18% and 16.99% respectively, after treated with 2μg/mL of mDRA-6 and 10μmol/L of VES for 16 hours.③The DR5 expression level on leukaemia cell surface were 94.8% for Jurkat cells, 74.71% for U937 cells, 58.66% for HL-60 cells ,42.35%for Raji cells and 14.92% for K562 cells respectively. The DR5 expression level of Raji cells and K562 cells treated with 10μmol/L of VES for 12 hours increased to 70.08% and 16.38% respectively.④Western blotting revealed that expression levels of the DR5 protein in Jurkat, U937, HL-60, Raji and K562 cells were unequal.The DR5 protein expression levels in Raji cells and K562 cells were lower. However, the DR5 protein levels of Raji cells and K562 cells treated with VES increased .⑤The mRNA expression of DR5 in Jurkat,U937, HL-60, Raji and K562 cells were analyzed by RT-PCR. The mRNA expression of DR5 in Raji cells treated with 20μmol/L of VES for 12 hours increased (P<0.05) obviously.Conclusion①The expression level of DR5 in Jurkat, U937, HL-60, Raji and K562 cells were different. There is the correlation between DR5 expression and human leukemia apoptotic rate induced by mDRA-6 in human leukaemia cell.②Combination of mDRA-6 and vitamin E succinate has synergistic effect of apoptosis on Raji and K562 cells by increasing DR5 expression on Raji, K562 cells treated with vitamin E succinate.