【作者】 王德林；

【导师】 米粲；

【作者基本信息】 重庆医科大学 ， 外科学， 2004， 博士

【摘要】 目 的肾癌是泌尿系统常见的恶性肿瘤，手术仍是治疗原发性肾癌唯一有效的方法。近年来，某些实体肿瘤的化学药物治疗已经取得长足进步，但现有的化学药物和放射治疗对肾癌疗效不明显，大宗病例统计化学药物治疗肾癌的总反应率仅6%。故应进一步探索有效的肾癌治疗方案。20世纪90年代，我国学者成功应用三氧化二砷（arsenic trioxide，As2O3）治疗复发性急性早幼粒细胞白血病（acute promyelocytic leukemia, APL），引起了国内外医学界的极大兴趣。对于As2O3的抗癌作用的研究，已经从血液系统肿瘤进入实体肿瘤。但关于As2O3对肾细胞癌的药理作用和抗肿瘤机制的研究罕见报道。本课题选择人肾癌786-0细胞系为实验对象，研究As2O3对786-0细胞的抑制作用及其分子机制，为临床应用As2O3治疗肾癌提供理论依据。方 法 与 结 果第一部分 采用细胞培养、光学显微镜、电子显微镜、MTT和肿瘤细胞克隆形成等方法观察As2O3处理前后786-0细胞生长和细胞形态学的改变、用免疫细胞化学方法检测As2O3处理前后786-0细胞PCNA和ki67蛋白表达的变化。2.0μmol/L以上浓度As2O3可显著抑制786-0细胞的增殖、降低细胞核分裂指数(P <0.01)，使786-0细胞内PCNA和ki-67蛋白表达降低(P<0.05)，肿瘤细胞克隆形成明显减少，其作用随药物浓度升高和时间延长而增加。第二部分 采用细胞培养、FCM观察As2O3作用786-0细胞后细胞周期的改变，用免疫组化检测As2O3致细胞Cyclin蛋白和CDKI基因的改变，RT-PCR检测Egr-1的改变。以2.0μmol/L或5.0μmol/LAs2O3作用786-0细胞<WP=9>24h、48h、72h，使其细胞周期停滞于G2/M和G0/G1期，周期素蛋白CyclinA、B1、D1、E表达均不同程度降低(P<0.05)，而其CDKI基因p16、p21WAF!、p27CIPI表达升高(P<0.05)，Egr-1在2.0μmol/L As2O3处理786-0细胞0.5 h后，开始表达，并随时间延长而增加，4 h达高峰后下降，24 h后消失。第三部分 采用细胞培养、形态学观察、DNA琼脂糖凝胶电泳、流式细胞技术（FCM）和TUNEL技术等方法观察As2O3诱导786-0细胞凋亡，用免疫细胞化学和RT-PCR技术等检测凋亡相关基因p53、bcl-2、c-myc及c-fos的mRNA和蛋白表达的改变。2.0μmol/L以上浓度As2O3作用786-0细胞24h后开始发生细胞凋亡，出现凋亡的形态学改变、DNA片段化和特征性亚二倍体峰（凋亡峰），使786-0细胞内p53活性升高(P<0.05)，而bcl-2、c-myc和c-fos基因表达降低(P<0.05)。第四部分 采用免疫细胞化学方法检测As2O3处理前后786-0细胞VEGF、nm-23、MMP-9、TIMP-1蛋白表达的改变；RT-PCR分析TGF-β1、TβR-I和TβR-II mRNA表达的改变。2.0μmol/L As2O3处理肾癌786-0细胞系72h后可显著抑制786-0细胞中VEGF、MMP-9的蛋白表达(P <0.01)，而促进786-0细胞中TIMP-1和nm-23蛋白的表达(P<0.05)。786-0细胞中缺乏TβR-II mRNA，As2O3处理后，786-0细胞中TGF-β1和TβR-I mRNA表达无改变（P>0.05）。第五部分 采用免疫细胞化学、ISH、Western blot和EMSA等技术检测肾癌组织和As2O3作用786-0细胞前后的p65 mRNA含量、NF-κB DNA结合活性及p65、IKKα/β、IκBα和ICAM-1蛋白的表达。结果肾癌组织中NF-κB DNA结合活性及p65、IKKα/β和ICAM-1蛋白表达量升高(P <0.01)，但IκBα蛋白明显降低(P <0.01)；经2.0μmol/L浓度As2O3处理 786-0细胞72 h后，p65 mRNA含量、NF-κB DNA结合活性及p65、IKKα/β、IκBα和ICAM-1蛋白的表达量均降低(P <0.01)。结 论1． As2O3通过降低786-0细胞内PCNA和ki-67蛋白的表达，肿瘤细胞克隆形成明显减少，对786-0细胞具有显著的增殖抑制作用，并具有浓度和时间依赖<WP=10>性特点；2． As2O3通过降低CyclinA、B1、D1、E蛋白的表达和增强p16、P21WAF!、p27CIPI活性，将786-0细胞阻滞于G2/M和G0/G1期；3． As2O3可能通过增加p53基因的表达和下调其bcl-2、c-myc和c-fos基因表达诱导人肾癌786-0细胞凋亡；4． As2O3通过抑制VEGF和MMP-9的蛋白表达，增加TIMP-1和 nm-23蛋白的表达，抑制786-0细胞的血管生成和转移；5． NF-κB DNA结合活性及p65、IKKα/β和ICAM-1蛋白增加可能与肾癌的发生和进展有关，2.0μmol/L以上浓度As2O3可以全面抑制786-0细胞中p65、IKKα/β、IκBα和ICAM-1蛋白和p65 mRNA的表达，抑制NF-κB DNA结合活性，可能是As2O3抑制786-0细胞增殖并诱导细胞凋亡的另一种机制。总之，As2O3通过抑制786-0细胞增殖、阻滞细胞周期、诱导细胞凋亡、抑制血管生成和NF-κB信号传递通路的激活等多种途径，发挥对肾癌786-0细胞的抑制作用，NF-κB可能成为抗肾癌治疗的新靶点。更多还原

【Abstract】 1．Objective Renal cell carcinoma is common malignant tumor in urinary system. Up to date, surgery remains the only effective method of treatment of primary renal cancer. The traditional modern management of advanced solid tumors has been with cytotoxic agents, despite the remarkable advances realized with other tumors, renal cancer has remained refractory to these agents and radiotherapy, chemotherapy involving more than 4000 patients with cancer of kidney was found a total response rate of 6%. So, it is necessary for us to explore the more effective chemotherapy for patients with renal carcinoma. In nineties last century, As2O3 was successfully used to cure the acute promyelocytic leukemia (APL) in our country, and its anti-cancer effect immediately attracted the great interest of scientists in the world. Now, the studies on anti-tumor effect of As2O3 have been undertaken not only in hemological tumor, but also in solid tumor. However, there have been few reports about the effect of As2O3 in renal cancer. In orde to provide the theoretical basis for clinical application of As2O3 in therapy of the kidney cancer, the inhibiting effects of As2O3 on human renal cell carcinoma 786-0 cells and its mechanisms were studied in this paper. 2．Methods and ResultsPart One Effect of As2O3 on the proliferation inhibition of 786-0 cells were analysed by cell culture, light microscopy, electronic microscopy, MTT and tumor colony formation, and the changes of protein expression of PCNA and ki-67 were studied by immunocytochemistry. The result showed that ≥2.0μmol/L As2O3 could effectively inhibit the cell proliferation of 786-0(P <0.01) , decrease it’s mitosis index (P <0.01), down-regulate expression of PCNA and ki-67 protein in cells(P <0.05), which was the time and dose-dependent. Part Two In this study, The change of 786-0 cell cycle was measured by cell culture flow cytometric assay, immunocytochemistry and RT-PCR.We found that <WP=12>treatment with 2.0μmol/L or 5 μmol/L As2O3 for 24h, 48h and 72h, could obviously induce a G2/M and G0/G1 phase arrest in 786-0 cells, decreased protein expression of cyclinA, B1, D1, E (P <0.05) and increased activities of CDKI genes p16, p21WAF! and p27 CIPI (P <0.01), the expression of Egr-1 mRNA was observed 786-0 cells treated with As2O3 at half an hour, increased to a peak at 4 hours , and then decreased quickly.Part Three This study was to explored the onset of apoptosis in 786-0 cells treated with As2O3 by cell morphology, DNA gel electrophoresis, flow cytometric assay and TUNEL, immunocytochemistry and RT-PCR. The characteristic apoptotic morphological changes and DNA fragmentation were observed after treated with ≥2.0μmol/L As2O3 for 48h, and FCM revealed that 2.0μmol/L As2O3 can induce a sub-G1 peak in 786-0 cells after treated with As2O3 for 24h. The expression of p53 gene was up-regulatied (P <0.05), but bcl-2, c-myc and c-fos were down regulated (P <0.05).Part Four The changes of VEGF、nm-23、MMP-9、TIMP-2 protein expression were studied by immunocytochemistry , and TGF-β1, TβR-I and TβR-II mRNA were analysed by RT-PCR in 786-0 cells treated with As2O3. Results showed that 2.0μmol/L As2O3 could effectively inhibit the protein expression of VEGF and MMP-9 in 786-0 cells (P <0.05), but increase nm-23 and TIMP-1 protein expression (P <0.05). In this study, absence of expression of transforming growth factor-β type II receptor mRNA was found in 786-0 cells, but we could not detected the change of mRNA expression of transforming growth factor-β1 and transforming growth factor-β type II receptor in 786-0 cells treated with As2O3 for 72 h.Part Five In this study, we investigated NF-κB DNA binding activity, p65 mRNA, p65, IKKα/β,IκBαand ICAM-1 protein in reanl cell cancer tissues and human renal cell carcinoma 786-0 cells by immunocytochemistry, in situ hybridization, Western blot and EMSA. Results showed that in renal cell cancer tissues, NF-κB DNA binding activity, p65, IKKα/βand ICAM-1 protein were increased , compared to normal更多还原