【作者】 席子明；

【导师】 沈倍奋；

【作者基本信息】 河南大学 ， 高分子化学与物理， 2010， 博士

【摘要】 1背景肿瘤热疗(hyperthermia)是继手术、放疗、化疗之后一种全新的治疗肿瘤的“绿色疗法”,是肿瘤治疗的有效手段之一,但是其作用机制长期不甚明了,近年来,随着热疗设备的不断革新和技术的不断进步,热疗已成为研究的热点。热疗于1985年获美国食品及药物管理局认证,和手术、化疗、放疗、生物免疫治疗一起成为肿瘤综合治疗的有效手段。我国卫生部于2009年11月颁布了《肿瘤深部热疗和全身热疗技术管理规范(试行)》。为探讨DR5功能性抗体抗肿瘤作用以及TRAIL/DR5相互作用的分子机制,河南大学细胞与分子免疫学实验室利用DR5蛋白免疫BALB/c小鼠,制备出功能性抗DR5单克隆抗体,命名为mDRA-6。近年来,热作用于肿瘤细胞后凋亡基因表达的研究,多集中在P53、HSP-70、c-Myc等,热疗后肿瘤细胞凋亡与DR5的关系未见报道；基于热诱导细胞凋亡机制的联合治疗方法,主要集中在热疗与化疗药物、放疗等,热疗与mDRA-6、热疗与mDRA-6+化疗对肿瘤细胞凋亡的协同作用未见报道。本研究采用肝癌常见的SMMC-7721细胞系,探讨热疗对mDRA-6+热疗对肿瘤细胞的协同作用,mDRA-6+化疗+热疗对肿瘤细胞的影响,热疗导致肿瘤细胞凋亡的机制和热疗对细胞膜DR5表达、细胞内Ca2+的影响进行了初步观察。2目的探讨热疗对SMMC-7721肝癌细胞的影响,从而探讨热疗引起肿瘤细胞凋亡的可能机制；探讨热疗和人抗DR5单克隆抗体致肿瘤细胞凋亡的协同作用。3方法将SMMC-7721肝癌细胞系,置恒温循环水浴锅中42.5℃±0.5℃孵育1h,然后放入37℃,5%CO2的培养箱中继续培养,分别于热处理后0、6、12、24h,采用倒置显微镜LM×400观察细胞形态变化,采用MTT法检测细胞增殖,Annexin V/PI染色流式细胞仪检测细胞凋亡,抗DR5抗体366EC和FITC-羊抗鼠IgG处理细胞,流式细胞仪检测细胞DR5表达和Fluo-3/AM负载细胞检测胞内Ca2+浓度(平均荧光强度,MFI)的变化。MTT法检测抗人DR5单克隆抗体单独或联合应用顺铂或热疗对肝癌SMMC-7721细胞体外增殖的抑制作用；流式细胞仪检测不同药物对SMMC-7721细胞凋亡的影响。4结果热疗可导致肿瘤细胞形态改变,表现为细胞变小、变圆、脱落、出现空泡；热疗可抑制细胞增殖,细胞热疗1h后0h、6h、12h、24h细胞抑制率分别为：22.18%,26.76%,31.30%,36.62%。细胞凋亡率：细胞热疗1h后0h、6h、12h、24h Annexin V/PI双染,热疗后Oh细胞凋亡率为15%,其中早期凋亡率为2%,晚期凋亡率为13%;热疗后6h细胞凋亡率为65.45%,其中早期凋亡率为43.23%,晚期凋亡率为22.22%;热疗后12h,细胞凋亡率为12.32%,其中早期凋亡率为4.6%,晚期凋亡率为7.72%;热疗后24h细胞凋亡率为22.58%,其中早期凋亡率为7.15%,晚期凋亡率为15.43%。细胞热疗1h后0h、4h、8h、12h、24h细胞内钙离子浓度(平均荧光强度,MFI)分别为：18.13±0.20、13.44±0.84、7.87±3.35、13.01±2.38、37.24±1.31,对照组为14.28±3.65。细胞热疗1h后0h、6h、12h、24h细胞膜DR5表达率分别为：79.74%、83.22%、91.28%、92.52%,对照组为83.02%。未经热处理细胞增殖抑制率分别为：顺铂组32%;m-DRA-6组22%；顺铂与m-DRA-6联合组37%。热处理细胞增殖抑制率分别为：顺铂组58.06%;m-DRA-6组14.52%;顺铂与m-DRA-6联合组91.13%。细胞凋亡：未经热处理：顺铂组13.53%;m-DRA-6组13.66%;顺铂与m-DRA-6联合组84.24%。热处理组：对照组9.41%,顺铂组30.27%%,m-DRA-6组57.52%,顺铂与m-DRA-6联合组91.33%。5结论疗可提高肝癌SMMC-7721细胞膜DR5的表达,可提高肝癌SMMC-7721细胞内Ca2+；肿瘤细胞凋亡与细胞膜DR5的高表达、细胞内Ca2+的增加有关；细胞内Ca2+的增加可能是热疗致肿瘤细胞晚期凋亡的主要原因。抗人DR5单克隆抗体可增加化疗药物抗肿瘤作用,热疗与抗人DR5单克隆抗体和化疗药物有协同作用；热疗不仅可显著增加肿瘤细胞凋亡,还可增加抗人DR5单克隆抗体和化疗药物的抗肿瘤细胞作用。更多还原

【Abstract】 1 BackgroundHyperthermia of tumor is an effective and brand-new therapy following the surgery, radiotherapy and chemotherapy. Nevertheless, the mechanism is still elusive. Recently, it has been a research hot point owing to the continuous renovation of equipments and the rapid progress of technology for hyperthermia. Hyperthermia is one of the powerful methods for combined therapy of tumor as well as surgery, chemotherapy and biological therapy, approved by U S Food and Drug Administration (FDA) in 1985. The Chinese Ministry of Public Health issued "Standard Regulations of Technology on the Management of Hyperthermia for Deeper and Whole Body Tumor" in Nov.2009. mDRA-6 is a functional monoclonal antibody against death receptor 5(DR5), developed by inoculating the DR5 protein into BALB/c mouse in the Laboratory of Cellular and Molecular Immunology, Henan University. Fewer reports were on the relationship of apoptosis with DR5 after tumor hyperthermia in recent years. Much attention was paid to the p53, HSP-70 and c-Myc, et al. The combined therapy of tumor based on the apoptosis induced by hyperthermia was primarily concentrated on hyperthermia along with chemotherapy and radiotherapy. There was no report about the synergistic effect of hyperthermia with mDRA-6, or hyperthermia with mDRA-6 and radiotherapy on apoptosis in tumor cells. The current study will investigate the synergistic effect of mDRA-6 and hyperthermia or mDRA-6, radiotherapy and hyperthermia on tumor cells. We also addressed the mechanism of apoptosis induced by hyperthermia, the expression of DR5 on tumor cell surface and the effect on Ca2+ level in tumor cells after hyperthermia.2 ObjectiveTo investigate the effect of hyperthermia on the heptocarcinoma cell line SMMC-7721, the possible mechanism of apoptosis induced by hyperthermia and the synergistic effect of mDRA-6 and hyperthermia on apoptosis.3 MethodsHeptocarcinoma cell line SMMC-7721 was first heated in water bath at 42.5±0.5℃for lh and then was grown in 5% CO2 incubator with at 37℃. The morphological change was observed under inverted microscope(X400) at 0,6,12 and 24h after hyperthermia. MTT method was used to study the cellular proliferation. Annexin V/PI staining was performed for the detection of apoptosis with flow cytometry. The DR5 expression was measured with flow cytometry after treatment of the cells with antibody against DR5(366EC), and goat anti-mouse IgG-FITC. The intracellular Ca2+ concentration was also analysed by flow cytometry after loaded with Fluo-3. The MTT method was employed to test the inhibition of hepatocarcinoma cell line SMMC-7721 caused by DR5 antibody alone or combined with cisplatin or hyperthermia. The effect of varied drugs on the apoptotic rate of SMMC-7721 was also checked with flow cytometry.4 ResultsHyperthermia could lead to morphological alteration of cells, such as smaller size, rounder shape, exfoliation and vacuole formation. It also inhibited the cellular proliferation with inhibition rates 22.18%,26.76%,31.30% and 36.62% at 0h,6h,12h and 24h respectively after one hour’s hyperthermia. The cellular rate of apoptosis was calculated by double staining of Annexin V/PI after one hour’s hyperthermia. It was 15% at 0h(including earlier rate 2% and later rate 13%),65.45% at 6h(earlier 42.23% and later 22.22%),12.32% at 12h(earlier 4.6% and later 7.72%), and 22.58% at 24h(earlier 7.15% and 15.43%) respectively. The intracellular Ca2+ concentration (MFI) was 18.13±0.20、13.44±0.84、7.87±3.35、13.01±2.38、37.24±1.31 at 0,4, 8,12 and 24h respectively after one hour’s hyperthermia with 14.28±3.65 as control. The expression rate of DR5 on cell surface was 79.74,83.22,91.28 and 92.52% at 0,6, 12 and 24h respectively after one hour’s hyperthermia with 83.02% as control.The inhibition rate of proliferation without hyperthermia is 32% for cisplatin,22% for m-DRA-6 and 37% for cisplatin and m-DRA-6 combined vs 58.06% for cisplatin, 14.52% for m-DRA-6 and 91.13% for cisplatin and m-DRA-6 combined respectively in group with hyperthermia. The apoptotic rate is 13.53% 32% for cisplatin,13.66% for m-DRA-6 and 84.24% for cisplatin and m-DRA-6 respectively in group without hyperthermia vs 9.41% for hyperthermia only group,30.27% for cisplatin,57.52% for m-DRA-6 and 91.33% for cisplatin and m-DRA-6 combined respectively with hyperthermia. 5. ConclusionHyperthermia could increase the expression of DR5 on hepatocarcinoma cell line SMMC-7721. The apoptosis of SMMC-7721 was related to the higher expression of DR5 and intracellular Ca2+. The increase of intracellular Ca2+ was probably the key factor for the later apoptosis of hepatocarcinoma cells. Monoclonal antibody against DR5, m-DRA-6, could strengthen the anti-tumor functions of drugs in chemotherapy. The antibody was synergistic with drugs of chemotherapy. Hyperthermia could not only raised the apoptosis of carcinoma cells, but also stratified the anti-tumor role of anti-DR5 antibody and chemotherapical drugs.更多还原