【作者】 王宏芳；

【导师】 李娟；

【作者基本信息】 吉林大学 ， 劳动卫生与环境卫生学， 2004， 硕士

【摘要】 恶性肿瘤是危害人类生活的主要疾病之一，传统的治疗手段和药物对某些恶性肿瘤的治疗效果并不理想，寻找新的高效抗肿瘤药物成为当务之急。多金属氧酸盐高效、低毒的抗肿瘤活性已有报道，硼钨酸嘧啶盐（PBT）系硼钨酸盐与嘧啶经一定的化学合成而成的一类新的化合物，淡黄色，易溶于水。本文从其体内、外实验检测其抗肿瘤活性，初步探讨其抗肿瘤作用机制。1.1 体外MTT法检测PBT对SMMC-7721、SGC-7901、HeLa三种人癌细胞的抗肿瘤活性。IC50分别为57μg/ml、209μg/ml、193μg/ml。显示出较好的抗肿瘤活性，对恶性度较高的人肝癌SMMC-7721细胞效果尤为显著。1.2 MTT法检测PBT对FL人羊膜细胞的毒性作用，PBT在800～1600μg/ml时细胞抑制率在20%以上，最高抑制率为32.29%。表明PBT对人FL细胞有毒性作用。1.3 3H-TdR掺入法检测PBT对人肝癌SMMC-7721细胞DNA 合成的抑制作用，结果表明，随着浓度升高对DNA合成的抑制作用越强。1.4.1 流式细胞术法检测PBT对人肝癌SMMC-7721细胞凋亡诱导作用，10μg/ml、50μg/ml PBT作用于SMMC-7721细胞12小时，测得凋亡率分别为12.39%、10.68%，明显高于对照组；作用24小时，测得凋亡率分别为12.93%、11.04%，明显高于对照组，100μg/ml PBT未表现出诱导细胞凋亡作用。表明一定浓度PBT有诱导肿瘤细胞凋亡作用。对细胞周期的分析表明，两个时间段细胞经 10μg/ml、50μg/ml浓度 PBT处理后G0/G1期细胞百分率明显高于对照组，S期细胞百分率明显低于对照组，100μg/ml浓度组未表现出细胞周期阻滞作用。表明一定浓度PBT可使肿瘤生长受阻于G0/G1期。 <WP=50>1.4.2 PI荧光染色观察细胞核形态变化，表现为细胞核呈现红色荧光，经PBT处理的细胞呈现典型的凋亡形态学改变，细胞核边缘不规则，可见核碎裂现象，核仁减少或消失，有凋亡小体。2.1 经口急性毒性实验，计算PBT的半数致死剂量（LD50），LD50为1117.38mg/kg，95%可信限为911.59-1369.6 mg/kg。属于低毒化合物。 2.2 建立小鼠H22实体瘤模型。PBT 25mg/kg、50mg/kg、100mg/kg三个剂量对小鼠H22荷瘤的平均抑制率分别为20.32%、33.15%、52.94%，高剂量组抑瘤率与5-Fu组比较无显著性差异（P>0.05），表明PBT有较好的抗肿瘤活性，且高剂量作用效果可与临床阳性药物5-Fu相媲美。2.3 荷瘤小鼠尾静脉采血，计数白细胞数。结果表明PBT低剂量组白细胞数升高，中、高剂量组白细胞数未见降低，而5-Fu组白细胞数降为对照组细胞数的一半（P<0.05）。表明治疗剂量PBT无降外周血白细胞数的毒性作用，且低剂量PBT有升白细胞作用。2.4 荷瘤小鼠称重处死，无菌取出脾脏与胸腺并称重，计算脏器指数，结果显示，5-Fu组脾指数及胸腺指数均低于对照组（P<0.05），PBT各剂量组脾指数均高于5-Fu组（P<0.05），低、中剂量组脾指数均高于对照组（P<0.05）；低剂量组胸腺指数高于对照组（P<0.05），低、中剂量组胸腺指数均高于5-Fu组（P<0.05）。表明治疗范围内PBT对荷瘤小鼠脾与胸腺无明显影响，一定剂量PBT能促进脾与胸腺重量的增加。2.5 用脾细胞对有丝分裂原ConA的反应性来检测小鼠脾脏T淋巴细胞增殖反应。结果显示，低、中剂量PBT具有提高淋巴细胞转化功能的作用（P<0.05）。2.6 由FCM流式细胞术检测PBT对荷瘤小鼠脾与胸腺细胞凋亡率的影响。结果显示，低剂量PBT能抑制小鼠脾细胞凋亡。三个剂量PBT均能抑制小鼠胸腺细胞凋亡，从而保护大量胸腺细胞免于死亡，调节其免<WP=51>疫防护功能。本实验结果表明，PBT在体内、外均有较好的抗肿瘤活性，且属于低毒化合物，作用机制可能为：通过影响核酸代谢来抑制肿瘤细胞DNA合成；使肿瘤细胞周期阻滞于G0/G1期；诱导肿瘤细胞凋亡等，来抑制肿瘤细胞生长，达到抗肿瘤效果；刺激免疫细胞的活性，保护免疫器官，增进机体免疫功能，从而间接起到辅助抗肿瘤作用。其深入的作用机制有待进一步研究，能否开发成为新的抗肿瘤药物尚需完成大量工作。更多还原

【Abstract】 Malignant tumor is one of the major diseases affecting persons’ health and the invasion and metastasis of malignancy will threaten patients’ lives at last. Traditional therapenutic tools and drugs are not efficient to certain malignancy. So we must develop and utilize effective drugs against tumors. Polyoxometalates have been reported that have distinctive biology activity and anti-tumor activity. Pyrimidine borotungstate (PBT) is a new type compound of polyoxometalates with keggin structure. We study the effect of PBT on anti-tumor in vivo and vitro to disclose the mechanism of anti-tumor.1.1 MTT method was used to examine the anti-tumor activity of PBT against several human cancer cells ( SMMC-7721 cells, SGC-7901 cells,Hela cells ).The result showed that PBT could inhibit the growth and proliferation of human carcinoma cells. The IC50 were 57μg/ml、209μg/ml、193μg/ml respectively. Furthermore, it was able to kill SMMC-7721 cell significantly.1.2 MTT method was used to examine the cytotoxicity of PBT in human normal FL cell. The result showed that no toxic activities in low concentration but cytotoxicity was displayed in the range of 800～1600μg /ml ,the highest inhibition rate was 32.29%.1.3 3H-TdR method was assayed the inhibition of PBT on DNA synthesis of SMMC-7721 cell. The result displayed that the inhibition rate would become higher with the increase of concentration.1.4.1 We detected the apoptosis of SMMC-7721 cell by FCM. The apoptosis rate in low 、middle concentration groups were significant greater than control group (p<0.05), but the high concentration group had no effect of <WP=53>inducing apoptosis. we also analyzed the cell cycle and the result displayed that G0/G1 stage cells were significantly greater in low、middle concentration groups than that in control group and S stage cells were significant lower than that in control group. 1.4.2 The cell morphology of SMMC-7721 cell was observed under fluorescence microscope. The picture displayed the features of apoptosis, such as fragmented nuclei and apoptotic bodies.2.1 we counted the median lethal dose (LD50) of PBT by acute toxicity test by mouth in mice. The value was 1117.38mg /kg and the range of 95% confidence limit was 911.59-1369.6mg/kg. It indicated that PBT was a low toxic material.2.2 The bearing tumor mice with hepatoma-22 was established for anti-tumor experiment. PBT was administered by p.o and divided into 3 groups, that were 25mg/kg, 50mg/kg, 100mg/kg. After 12 days treatment with PBT, Low、 middle and high dose groups inhibition rate (IR) in H22 were 20.32%、33.15%、52.94% respectively. The IR in high dose group was not lower than 5-Fu group.2.3 Gotten blood from caudal vein of tumor mice and counted WBC value. The result showed that WBC value was higher in low dose group than that in control group（P<0.05）and the WBC value of other dose groups of PBT had no significant variation. But the WBC value of 5-Fu group was much lower than any other groups（P<0.05）. It indicated that PBT has no influence on blood function, no inhibition on spinal.2.4 The spleen index of low、 middle dose groups were higher than that of control group（P<0.05）, but the high dose group had no significant <WP=54>variation. The thymus index of low dose group was higher than that of control group（P<0.05）, but the middle and high dose groups had no significant variation. The both index in 5-Fu group were lower than that in other groups（P<0.05）. 2.5 The incremental reaction of mouse lymphocyte was observed by the activity of spleen cell to mitosis ConA. Result showed that PBT of low and middle dose groups had the effect of improving the lymphocyte conversion.2.6 FCM method was used to detect the apoptosis rate of spleen cells and thymus cells of tumor mice. The result showed that low dose PBT could inhibit the apoptotic of spleen cells. Three doses PBT can also inhibit the apoptosis of thymus cells of tumor mice to protect mass thymus cells not die.The result showed the efficient anti-tumor of PBT in vitro and in更多还原