异鼠李素抗肿瘤作用及其机理的研究
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摘要
目的：
从诱导肿瘤细胞分化和凋亡，抑制肿瘤细胞的增殖等方面对异鼠李素的抗肿瘤的作用及机制进行研究；采用现代时间生物学理论及其研究方法探讨肿瘤细胞的基因表达和与增殖有关的酶活性的时间特征，找出异鼠李素药物毒性反应最低、疗效作用最高的时间规律，为异鼠李素在临床上择时治疗提供理论基础。
方法与结果：
1．利用MTT法测定异鼠李素对细胞生长的抑制、光镜下计数绘制细胞生长曲线、光镜和电子显微镜对细胞形态观察、克隆形成和~3H-TdR掺入分析，基因组DNA片段分析、流式细胞术等方法研究异鼠李素对HeLa、Tca-8113、YTLMC-90、Eca-109细胞增殖和凋亡的作用。结果显示：异鼠李素能强烈抑制肿瘤细胞的增殖，对细胞生长的抑制作用具有浓度-时间和时间-效应关系；光镜和电镜下观察到异鼠李素导致肿瘤细胞结构改变，核染色质凝集，核啐裂形成凋亡小体，细胞核DNA降解，呈现典型区带特征-Ladder DNA；异鼠李素抑制肿瘤细胞增殖，S和G_2+M期细胞数减少，细胞增殖指数降低，凋亡率增高。
2．为了进一步证实异鼠李素的抑癌抗癌作用，对其抑制肿瘤生长增殖和诱导肿瘤细胞凋亡的分子机制进行了实验研究。用TRAP-PCR法检测异鼠李素处理后对人癌细胞端粒酶活性的影响；检测过氧化氢酶活性；免疫组化方法测定PCNA表达的变化：流式细胞术方法研究异鼠李素处理肿瘤细胞后对肿瘤相关基因表达的影响；用流式细胞术和免疫荧光法检测异鼠李素处理前后抑制分化因子Id-1表达。结果显示：处理组Hela、Tca-8113、Eca-109细胞株端粒酶活性较对照组差异显著(P＜0.05)；YTLMC-90细胞株端粒酶活性与对照组无明显差异(P＞0.05)：阳性药顺铂对上述几种肿瘤细胞株端粒酶活性无明显降低(P＞0.05)；经异鼠李素和顺铂处理后HeLa、Tca-8113、YTLMC-90、Eca-109细胞PCNA阳性率均低于对照组；过氧化氢酶活性处理组低于对照组；异鼠李素作用后，细胞凋亡率升高，bcl-2、bax、c-myc、H-ras、c-fos、P~(53)基因蛋白的表达改变，bcl-2、c-myc、H-ras表达降低，bax、c-fos和P~(53)表达升高。
3．观察异鼠李素对鼠移植瘤的抑制作用。以鼠Lewis肺癌细胞复制C57BL／6移植瘤模型，鼠随机分组用药，对照组、异鼠李素组、异鼠李素提前灌胃组、顺铂组。用药7天后收集标本，采用光镜、电镜观察，免疫组化分析PCNA、bcl-2、bax的表达。结果：异鼠李素组、提前灌胃组、顺铂组瘤重明显低于对照组(P＜0.05)；免疫组化显示：异鼠李素组、提前灌胃组、顺铂组PCNA表达明显低于对照组，差异有显著性P＜0.01)；bcl-2
四川大学博士学位论文
表达低于对照组，bax表达高于对照组，差异有显著性(P<0.05).结论:
异鼠李素可抑制鼠Lewis肺癌移植瘤生长，其机制可能与抑制肿瘤PCNA、
bc卜2表达，上调bax表达有关。
4.为了探讨人食道癌Eca一1 09细胞DNA合成与c一myc基因表达的近
日节律以及异鼠李素对二者的影响作用，达到有效治疗肿瘤的目的，我们
用3H一TdR掺入实验和流式细胞仪检测在24小时内不同时间点异鼠李素
对Eca一109细胞DNA合成和c一my。基因表达的近日节律的影响，其结果
用方差分析和Cosinor法进行统计学分析。结果发现人食道癌Eca一1 09细
胞DNA合成和c一myc基因的表达随近日节律变化，且异鼠李素对DNA
合成和c一myc基因的表达具有抑制作用，并影响其时间生物学特征。人食
道癌Eca一109细胞的生长与c一myc基因表达在20:oo至0:00达到高峰，且
异鼠李素在此时间点作用较强，这为制定肿瘤时辰化疗方案提供了有价值
的参考。
结论:
研究发现，异鼠李素可能主要通过抑制肿瘤细胞DNA合成、端粒酶、
过氧化氢酶活性，上调或下调分化和凋亡相关基因表达而抑制肿瘤细胞增
殖、生长;诱导癌细胞分化和凋亡抑制肿瘤，为临床化疗提供新的途径。
关键词:异鼠李素;增殖;凋亡;肿瘤;癌基因;抑癌基因;时间生物学
Objective:To study the inhibiting effect of isorhmnetin on human cervical cancer, tongue squamous epithelium cancer, Lung cancer and esophageal tumor cells, and to elucidate the molecular mechanisms of this compound inhibiting cancer cells.Methods and Resultes:1. Inhibiting growth in cancer cells were measured by microculture tetrazolium assay, growth curve, clone formation test and 3H-thymidine uptake assay, observed and analyzed by light microscopy, electronic microscopy, agarose gel electrophoresis, cell cycle analyzed by flow cytometry. Results: The growth of cancer cells were inhibited evidently after treated by isorhmnetin, the growth inhibition of cancer cells versus the concentration of isorhmnetin exhibited a dose-response and time-response relationship. The cells showed characters of apoptsis and differentiation observed by light and electronic microscopy, agarose gel electrophoresis of DNA showed change of "Ladder" pattern. The rates of apoptosis increased after treated with isorhmnetin, and the cell was inhibited in G0/G1 phase, the cellular number in S phase decreased (P<0.05).2.The telomerase activities of HeLa,Tca-8113,YTLMC-90 and Eca-109 cells in vivro were investigated by TRAP-ELIAS technique.The catalase activities of cancer cells were analyzed with UV-visible Recording Spectrophotometer. The expressions of genes were analyzed by flow cytometry. The expression of PCNA was detected by immunohistochemical method. The expression of Id-1 were inspected by flow cytometry and immunofluorescence method. Results: The telomerase and catalase activities of cancer were inhibited when treated with isorhmnetin compared to the negative control. Isorhmnetin decreased the expression of bcl-2, c-myc, H-ras oncogene and increased the expression of bax, c-fos and p53. The expression of PCNA and Id-1 were lower than the negative control group after treated with isorhmnetin.3.To investigate the effect of isorhmnetin on tumor growth, cell proliferation and apoptosis in transplantation rumor of lung cancer of Lewis cell line in C57BL/6 mice. Lewis cells were inoculated into the the C57BL/6 mice to establish Lewis lung cancer model, then fifty six C57B1/6 mice with Lewis tumor were randomized four groups: control group, isorhmnetin group, treated in advanced with isorhmnetin group, cisplatin group. After 7 days treatment,
samples of tumor were collected. The sections of tumor were observed under light microscope and electron microscope. The expression of PCNA, bcl-2 and bax were detected by immunohistochemistry. We found that, the tumor weight of control group was significally higher than those treated with medicines; immunohistochemical stainng of PCNA showed that the PCNA lable index displayed significally defference between negative control group and medicine groups, the expression of PCNA of medicine groups lower than that of negative control, and so did the staining of bcl-2; but the expression of bax of medicine groups higher than that of control group (P<0.05). 4. The circadian rhythms of DNA synthesis and the expression of c-myc gene in untreated and treated Eca-109 cells in human esophageal cancer with isorhmnetin were studied. The circadian rhythms of 3H-TdR incorporation and expression of c-myc gene in untreated and treated Eca-109 cells was measured by 3H-thymidine uptake assay and flow cytometry. Dates were documented by ANOVA and Cosinor analysis. The results showed that DNA synthesis and expression of c-myc gene in untreated group varied according to circadian time with statistical significance, the distribution curves of both DNA synthesis and the expression level of c-myc were fit for cosinor changes. The circadian rhythms of DNA synthesis and circadian parameters of c-myc expression in treated Eca-109 cells changed. The circadian parameters of DNA synthesis and expression level of c-myc were varied after treating by isorhmnetin. Conclusion:The results once more confirmed that isorhmnetin can inhibit growth, proliferation and induce differentiation of Hela, Tca-8113, YTLMC-90 and Eca-109 cells. The mechani
从诱导肿瘤细胞分化和凋亡，抑制肿瘤细胞的增殖等方面对异鼠李素的抗肿瘤的作用及机制进行研究；采用现代时间生物学理论及其研究方法探讨肿瘤细胞的基因表达和与增殖有关的酶活性的时间特征，找出异鼠李素药物毒性反应最低、疗效作用最高的时间规律，为异鼠李素在临床上择时治疗提供理论基础。
方法与结果：
1．利用MTT法测定异鼠李素对细胞生长的抑制、光镜下计数绘制细胞生长曲线、光镜和电子显微镜对细胞形态观察、克隆形成和~3H-TdR掺入分析，基因组DNA片段分析、流式细胞术等方法研究异鼠李素对HeLa、Tca-8113、YTLMC-90、Eca-109细胞增殖和凋亡的作用。结果显示：异鼠李素能强烈抑制肿瘤细胞的增殖，对细胞生长的抑制作用具有浓度-时间和时间-效应关系；光镜和电镜下观察到异鼠李素导致肿瘤细胞结构改变，核染色质凝集，核啐裂形成凋亡小体，细胞核DNA降解，呈现典型区带特征-Ladder DNA；异鼠李素抑制肿瘤细胞增殖，S和G_2+M期细胞数减少，细胞增殖指数降低，凋亡率增高。
2．为了进一步证实异鼠李素的抑癌抗癌作用，对其抑制肿瘤生长增殖和诱导肿瘤细胞凋亡的分子机制进行了实验研究。用TRAP-PCR法检测异鼠李素处理后对人癌细胞端粒酶活性的影响；检测过氧化氢酶活性；免疫组化方法测定PCNA表达的变化：流式细胞术方法研究异鼠李素处理肿瘤细胞后对肿瘤相关基因表达的影响；用流式细胞术和免疫荧光法检测异鼠李素处理前后抑制分化因子Id-1表达。结果显示：处理组Hela、Tca-8113、Eca-109细胞株端粒酶活性较对照组差异显著(P＜0.05)；YTLMC-90细胞株端粒酶活性与对照组无明显差异(P＞0.05)：阳性药顺铂对上述几种肿瘤细胞株端粒酶活性无明显降低(P＞0.05)；经异鼠李素和顺铂处理后HeLa、Tca-8113、YTLMC-90、Eca-109细胞PCNA阳性率均低于对照组；过氧化氢酶活性处理组低于对照组；异鼠李素作用后，细胞凋亡率升高，bcl-2、bax、c-myc、H-ras、c-fos、P~(53)基因蛋白的表达改变，bcl-2、c-myc、H-ras表达降低，bax、c-fos和P~(53)表达升高。
3．观察异鼠李素对鼠移植瘤的抑制作用。以鼠Lewis肺癌细胞复制C57BL／6移植瘤模型，鼠随机分组用药，对照组、异鼠李素组、异鼠李素提前灌胃组、顺铂组。用药7天后收集标本，采用光镜、电镜观察，免疫组化分析PCNA、bcl-2、bax的表达。结果：异鼠李素组、提前灌胃组、顺铂组瘤重明显低于对照组(P＜0.05)；免疫组化显示：异鼠李素组、提前灌胃组、顺铂组PCNA表达明显低于对照组，差异有显著性P＜0.01)；bcl-2
四川大学博士学位论文
表达低于对照组，bax表达高于对照组，差异有显著性(P<0.05).结论:
异鼠李素可抑制鼠Lewis肺癌移植瘤生长，其机制可能与抑制肿瘤PCNA、
bc卜2表达，上调bax表达有关。
4.为了探讨人食道癌Eca一1 09细胞DNA合成与c一myc基因表达的近
日节律以及异鼠李素对二者的影响作用，达到有效治疗肿瘤的目的，我们
用3H一TdR掺入实验和流式细胞仪检测在24小时内不同时间点异鼠李素
对Eca一109细胞DNA合成和c一my。基因表达的近日节律的影响，其结果
用方差分析和Cosinor法进行统计学分析。结果发现人食道癌Eca一1 09细
胞DNA合成和c一myc基因的表达随近日节律变化，且异鼠李素对DNA
合成和c一myc基因的表达具有抑制作用，并影响其时间生物学特征。人食
道癌Eca一109细胞的生长与c一myc基因表达在20:oo至0:00达到高峰，且
异鼠李素在此时间点作用较强，这为制定肿瘤时辰化疗方案提供了有价值
的参考。
结论:
研究发现，异鼠李素可能主要通过抑制肿瘤细胞DNA合成、端粒酶、
过氧化氢酶活性，上调或下调分化和凋亡相关基因表达而抑制肿瘤细胞增
殖、生长;诱导癌细胞分化和凋亡抑制肿瘤，为临床化疗提供新的途径。
关键词:异鼠李素;增殖;凋亡;肿瘤;癌基因;抑癌基因;时间生物学
Objective:To study the inhibiting effect of isorhmnetin on human cervical cancer, tongue squamous epithelium cancer, Lung cancer and esophageal tumor cells, and to elucidate the molecular mechanisms of this compound inhibiting cancer cells.Methods and Resultes:1. Inhibiting growth in cancer cells were measured by microculture tetrazolium assay, growth curve, clone formation test and 3H-thymidine uptake assay, observed and analyzed by light microscopy, electronic microscopy, agarose gel electrophoresis, cell cycle analyzed by flow cytometry. Results: The growth of cancer cells were inhibited evidently after treated by isorhmnetin, the growth inhibition of cancer cells versus the concentration of isorhmnetin exhibited a dose-response and time-response relationship. The cells showed characters of apoptsis and differentiation observed by light and electronic microscopy, agarose gel electrophoresis of DNA showed change of "Ladder" pattern. The rates of apoptosis increased after treated with isorhmnetin, and the cell was inhibited in G0/G1 phase, the cellular number in S phase decreased (P<0.05).2.The telomerase activities of HeLa,Tca-8113,YTLMC-90 and Eca-109 cells in vivro were investigated by TRAP-ELIAS technique.The catalase activities of cancer cells were analyzed with UV-visible Recording Spectrophotometer. The expressions of genes were analyzed by flow cytometry. The expression of PCNA was detected by immunohistochemical method. The expression of Id-1 were inspected by flow cytometry and immunofluorescence method. Results: The telomerase and catalase activities of cancer were inhibited when treated with isorhmnetin compared to the negative control. Isorhmnetin decreased the expression of bcl-2, c-myc, H-ras oncogene and increased the expression of bax, c-fos and p53. The expression of PCNA and Id-1 were lower than the negative control group after treated with isorhmnetin.3.To investigate the effect of isorhmnetin on tumor growth, cell proliferation and apoptosis in transplantation rumor of lung cancer of Lewis cell line in C57BL/6 mice. Lewis cells were inoculated into the the C57BL/6 mice to establish Lewis lung cancer model, then fifty six C57B1/6 mice with Lewis tumor were randomized four groups: control group, isorhmnetin group, treated in advanced with isorhmnetin group, cisplatin group. After 7 days treatment,
samples of tumor were collected. The sections of tumor were observed under light microscope and electron microscope. The expression of PCNA, bcl-2 and bax were detected by immunohistochemistry. We found that, the tumor weight of control group was significally higher than those treated with medicines; immunohistochemical stainng of PCNA showed that the PCNA lable index displayed significally defference between negative control group and medicine groups, the expression of PCNA of medicine groups lower than that of negative control, and so did the staining of bcl-2; but the expression of bax of medicine groups higher than that of control group (P<0.05). 4. The circadian rhythms of DNA synthesis and the expression of c-myc gene in untreated and treated Eca-109 cells in human esophageal cancer with isorhmnetin were studied. The circadian rhythms of 3H-TdR incorporation and expression of c-myc gene in untreated and treated Eca-109 cells was measured by 3H-thymidine uptake assay and flow cytometry. Dates were documented by ANOVA and Cosinor analysis. The results showed that DNA synthesis and expression of c-myc gene in untreated group varied according to circadian time with statistical significance, the distribution curves of both DNA synthesis and the expression level of c-myc were fit for cosinor changes. The circadian rhythms of DNA synthesis and circadian parameters of c-myc expression in treated Eca-109 cells changed. The circadian parameters of DNA synthesis and expression level of c-myc were varied after treating by isorhmnetin. Conclusion:The results once more confirmed that isorhmnetin can inhibit growth, proliferation and induce differentiation of Hela, Tca-8113, YTLMC-90 and Eca-109 cells. The mechani
引文
1. Wang Zheng-rong, Wang Ling, Yin Hua-hu et al, Effect of Total Flavonoids of Hippophae Rhamnoides on Contractile Mechanics and Calcium Transfer in Stretched Myocyte (J). Space Medicine & Medical Engineering ,2000; 12(1):6-9
王正荣，王玲，尹华虎等，沙棘总黄酮对牵张所致心衰心肌细胞的收缩力学和钙离子转运的影响(J)，航天医学与医学工程，2000，13(1)：6-9
2. Zhong Xiaogang, Wu Kainan, He Sheng et al. Effect of Quercetin on the Proliferation and Apoptosis in Transplation Tumor of Breast Cancer in Nude Mice (J). Sichuan Univ (Med Sci Edi), 2003;34(3):439-442
钟晓刚，吴凯难，何生等，檞皮素对人乳腺癌裸鼠移植瘤细胞增殖和凋亡的作用，四川大学学报(医学版)，2003；34(3)：439-442
3. Alcocer F, Whitley D, Salazar-Gonzalez JF, Jordan WD, et al. Quercetin inhibits human vascular smooth muscle cell proliferation and migration. Surgery 2002; 131 (2): 198-204.
4. Fan PS, GU Zhen-Lun, Sheng R et al. Inhinitory effect of quercetin on proliferation of human microvascular endothelial cells in vitro. Acta pharmacol Sin, 2003 24(12): 1231-1234.
5．成军，程序性细胞死亡与疾病．北京：北京医科大学中国协和医科大学联合出版社，1997
6．章静波．医学分子细胞生物学．北京：中国协和医科大学出版社，2002
7. Gong J, Traganos F, Darzynkiewicz Z. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry [J]. Anal Biochem, 1994, 218(2):314-319.
8. Balabhadrapthruni S, Thomas TJ, Yurkow EJ, et al. Effect of genistein and structurally related phytoestrogens on cell cycle kinetics and apoptosis in MDA-MB-468 human breast cancer cells. Oncol Res, 2000; 7 (1): 3
9. Wolff AC, Davidson NE. Primary systemic theraoy in operable breast cancer [J]. J clin Oncol, 2000, 18(7): 1558-1569.
10. Kaiser HE, Bodey B. The role of apoptosis in normal ontogenesis and solid human neoplasma [J].In Vivo, 2000,14(6):789-803
11．左连富，流式细胞术于生物医学[M]。沈阳：辽宁科学技术出版社，1996，240-374
12. D' Aprpa P, beardmore G, Liu If. Involvement of nucleic acid synthrsis in cell killing mechanism of topoisomease poison. Cancer Res 1990;50:6919-6924
13．何承伟，梁念慈，莫丽儿等，半边旗提取物6F诱导HL-60细胞凋亡[J]，肿瘤防治杂志，2002，9(1)：11-14
王正荣，王玲，尹华虎等，沙棘总黄酮对牵张所致心衰心肌细胞的收缩力学和钙离子转运的影响(J)，航天医学与医学工程，2000，13(1)：6-9
2. Zhong Xiaogang, Wu Kainan, He Sheng et al. Effect of Quercetin on the Proliferation and Apoptosis in Transplation Tumor of Breast Cancer in Nude Mice (J). Sichuan Univ (Med Sci Edi), 2003;34(3):439-442
钟晓刚，吴凯难，何生等，檞皮素对人乳腺癌裸鼠移植瘤细胞增殖和凋亡的作用，四川大学学报(医学版)，2003；34(3)：439-442
3. Alcocer F, Whitley D, Salazar-Gonzalez JF, Jordan WD, et al. Quercetin inhibits human vascular smooth muscle cell proliferation and migration. Surgery 2002; 131 (2): 198-204.
4. Fan PS, GU Zhen-Lun, Sheng R et al. Inhinitory effect of quercetin on proliferation of human microvascular endothelial cells in vitro. Acta pharmacol Sin, 2003 24(12): 1231-1234.
5．成军，程序性细胞死亡与疾病．北京：北京医科大学中国协和医科大学联合出版社，1997
6．章静波．医学分子细胞生物学．北京：中国协和医科大学出版社，2002
7. Gong J, Traganos F, Darzynkiewicz Z. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry [J]. Anal Biochem, 1994, 218(2):314-319.
8. Balabhadrapthruni S, Thomas TJ, Yurkow EJ, et al. Effect of genistein and structurally related phytoestrogens on cell cycle kinetics and apoptosis in MDA-MB-468 human breast cancer cells. Oncol Res, 2000; 7 (1): 3
9. Wolff AC, Davidson NE. Primary systemic theraoy in operable breast cancer [J]. J clin Oncol, 2000, 18(7): 1558-1569.
10. Kaiser HE, Bodey B. The role of apoptosis in normal ontogenesis and solid human neoplasma [J].In Vivo, 2000,14(6):789-803
11．左连富，流式细胞术于生物医学[M]。沈阳：辽宁科学技术出版社，1996，240-374
12. D' Aprpa P, beardmore G, Liu If. Involvement of nucleic acid synthrsis in cell killing mechanism of topoisomease poison. Cancer Res 1990;50:6919-6924
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