蒺藜皂苷和沙棘籽渣黄酮对乳腺癌和肝癌细胞的抑制作用
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摘要
本文的一大板块研究了蒺藜皂苷对乳腺癌和肝癌细胞的抑制效应，选用蒺藜全草提取皂苷，在细胞水平和分子水平层面上着重研究了蒺藜皂苷对人乳腺癌细胞Bcap-37和肝癌细胞BEL-7402的体外抑制效应极其机理。
首先应用MTT法、考马斯亮蓝测蛋白法、SRB、生长曲线法观察了蒺藜皂苷(STT)体外对于Bcap-37和BEL-7402细胞生长的抑制作用，发现STT能够在体外显著抑制人乳腺癌细胞Bcap-37和肝癌细胞BEL-7402的增殖，且该作用具有显著的剂量依赖效应。
为了深入评价STT体外抗肿瘤作用的效果，采用MTT法进行了STT对正常肝细胞和肝癌细胞抑制作用的比较实验并观察了其与盐酸阿霉素对肿瘤细胞的协同抑制效应，结果显示：STY对于正常肝细胞LO2的抑制效应要小于肝癌细胞BEL-7402；高浓度的STT与盐酸阿霉素对于抑制肿瘤细胞具有协同效应，但在低浓度时表现为拮抗效应。
采用wright染色法、丫啶橙染色法和透射电子显微镜进行形态学观察发现：两种癌细胞经STT作用后，均可见核固缩、染色质边集以及出芽的凋亡细胞；碘化丙啶(PI)染色的流式细胞术法检测了STT对Bcap-37和BEL-7402细胞周期的影响，应用免疫荧光与流式细胞术结合法检测STT对BEL-7402细胞的抗细胞凋亡蛋白Bcl-2含量以Bcap-37细胞的细胞周期蛋白cyclin D1和cyclin E水平的影响，结果表明：STT能够促进大量BEL-7402细胞的凋亡，但未见细胞周期阻滞效应，STT诱导Bcap-37细胞的凋亡率较低，却可将Bcap-37细胞周期阻滞在G0／G1期。而且STT能够降低BEL-7402细胞的抗凋亡蛋白Bcl-2表达，并能够降低Bcap-37细胞的细胞周期蛋白cyclin D1和cyclin E水平。
采用活性氧(ROS)检测试剂盒观察发现STT可提高Bcap-37细胞内活性氧的含量，促发细胞的氧化损伤，但对BEL-7402细胞无显著影响，该项研究结果显示：促发活性氧所诱导的氧化损伤也是STT抑制Bcap-37细胞增殖效应的机制之一。
综上所述：STT可抑制人乳腺癌细胞Bcap-37和人肝癌细胞BEL-7402的增殖，对人正常肝细胞的具有较低的抑制效应，高浓度的STT与ADM对Bcap-37和BEL-7402细胞具有协同抑制效应，STT抑制Bcap-37细胞增殖的主要机理
摘要
有:降低cyclinE和cyclin Dl水平诱导细胞周期阻滞以及促进细胞凋亡和诱发
氧化损伤，而STT对肝癌细胞BEL一7402抑制效应的主要机理之一是通过下调
BCL一2蛋白表达诱导肝癌细胞凋亡。
本文的第二大板块是上海市西部开发科技合作项目(编号014358009)子
课题的一项内容，继前一板块完成羡黎皂普对肿瘤细胞作用影响的基础上，为了
解沙棘籽渣黄酮的生物学活性，研究从天然产物化学的提取，分离纯化的现代技
术切入，并对有效成分的生药学原理采用离体肿瘤细胞和基因芯片等技术进行了
后续研究。
研究首先采用0.8 mol几的盐酸80℃水解lh作为水解条件，酸水解法处理
沙棘籽渣中提取的黄酮昔。其后以甲醇一水(60:40)为流动相，紫外检测器的
检测波长为368nxn，流速为1 .0 ml/ min作为色谱条件，用HPLC测定黄酮昔元
含量。结果显示:每1009沙棘籽渣样品中棚皮素、山奈素、异鼠李素的含量分
别为18.58 mg，122.50 mg，27.64 mg;而回收率分别为99.47%，103.700，0，95.51%。
在此基础上，采用黄酮纯度和得率为指标比较了传统的化学纯化法与聚酞胺
柱层析法，进而对沙棘籽渣黄酮的提取和分离纯化工艺作了研究:结果发现两种
方法所得黄酮纯度相当(50一55%)，但聚酞胺柱层析后黄酮得率(24%)大大高
于化学纯化法，后者的工艺的优势明显优于前者。
分离纯化工艺研究结果不仅有助于中药的现代化，同时奠定了探索生物学活
性的后续试验基础。
离体细胞试验首先采用了MTT法测定FsH对乳腺癌细胞Bc即一37的生长抑
制作用，发现，200一1 000 09/ml的FSH能显著抑制BcaP一37的增殖生长，且随
浓度的升高，抑制作用增强，具有一定的剂量依赖关系。为了探讨FsH对BcaP一37
细胞的生长抑制作用的机理，采用Wright染色法和Tu呵EL法观察FSH对
Bcap一37细胞的致凋亡作用;采用PI染色的流式细胞术法检测凋亡率和细胞周期
变化，结果表明:FSH能够诱导Bcap一37细胞的凋亡，凋亡率最高达到2.63%，
并对Bcap一37细胞具有GZ舰期阻滞效应。
为了探讨FSH对Bcap一37细胞生长抑制作用的分子水平的机制，采用基因
芯片检测技术检测了细胞增殖、细胞周期和细胞凋亡相关基因的表达，发现FSH
能够下调Bcap一37细胞内细胞周期蛋白依赖激酶1(C DKI)、DNA复制相关基因
摘要
(DNA聚合酶Q、拓扑异构酶H、PCNA)以及c一myc、C一fos等癌基因的表达;
而能够上调caspaseg、BAKI，FADD以及MADD等促凋亡基因的表达:FsH
抑制Bcap一37细胞增殖分子机理主要是通过调控相关基因表达阻滞细胞周期，抑
制细胞DNA复制以及抑制癌基因所启动的DNA复制及转录，并能通过增强
Bc即一37细胞的凋亡敏感性间接促进细胞凋亡。
由于活性氧在细胞增殖、死亡中所起的重要作用，继而研究了FSH对BcaP一37
细胞内活性氧水平的影响，发现FSH能显著增加Bcap一37细胞内RoS水平，表
明FSH能通过促进细胞氧化损伤抑制Bcap一37癌细胞的增殖。
此外，研究还就FSH对人肝癌BEL一74
Firstly, MTT, SRB, cell growth curve assay, and protein content assay were used to determine the inhibitory effects of STT on the growth of Bcap-37 and BEL-7402 cell line, and then it is shown that STT has potent inhibitory effects on both Bcap-37 cell line and BEL-7402 cell line in a concentration-dependent manner.
In order to evaluate the in vitro antitumor effect of STT, we used MTT assay to compare the inhibitory effect of STT on human hepatocarcinoma cells line BEL-7402 with that on human normal liver cells line L02 through MTT assay, and used the MTT and Webb coefficient assay to judge the co-effect of STT and adriamycin(ADM) on Bcap-37 and BEL-7402 cells, the results show that compared with its inhibition on the growth of BEL-7402 cell line, STT has less inhibitory effect on the proliferation of the normal liver cell line L02, and moreover the synergetic effects of STT and ADM on the two cancer cell line can be achieved at a high concentration of STT, however, when STT is at a low concentration, the antagonism of the two drugs will be produced.
We applied several methods of morphological observation such as wright staining, acridine orange staining and electron microscope to observe the effect of STT on the morphological alterations of the two cancer cell line, to find that after treatment with STT, both of the two cancer cell lines will exhibit typical
morphological alterations of apoptosis, such as rounding of cells, nucleus pyknosis, apoptotic body and so on.
The flow cytometry detection with PI staining was used to detect cell cycle arrest and apoptotic peak of the two cell line after treatment with STT, and therefore it can be seen from the results that The apoptotic peaks can be induced by STT on both of the two cancer cell lines, however, STT can induce apoptosis of a large percent of BEL-7402 cell, while the apoptotic rate of Bcap-37 cell is much lower than that of BEL-7402 cell. In addition, STT has GO/G1 arrest effect on cell cycle of Bcap-37 cells.
In addition, the molecular mechanism of cell cycle arrest and apoptosis induction have been studied. The immunological fluorescent assay followed by flow cytometry detection was applied to determine BCL-2 protein expression of BEL-7402 cells and cyclin Dl, cyclin E protein expression of Bcap-37 cells. The results demonstrate that STT can reduce BCL-2 protein level of BEL-7402 cell line; in addition, the cyclin E and cyclin Dl expression of Bcap-37 cell line can be down-regulated by STT.
Moreover, we have quantified ROS in the two cancer cell lines after being treated by STT by using the reactive oxygen species (ROS) detection kit. The data shows that the quantity of ROS in Bcap-37 cell line, which can result in oxygenic damage, increases remarkably after treatment with STT, but there is no significant change of the ROS level in BEL-7402 cells.
In summary, it can be inferred that STT has potent inhibitory effects on both Bcap-37 cell line and BEL-7402 cell line in a concentration-dependent manner, but has less inhibitory effect on the proliferation of the normal liver cell line L02. The synergetic effect on the two cell lines between STT with ADM can be seen at a high concentration of STT. The mechanism of inhibitory effect of STT on Bcap-37 cells are not only to induce apoptosis, arrest cell cycle of Bcap-37 cells at GO/G1 by decreasing the level of cyclin Dl and cyclin E, but also to give rise to oxygenic damage in cells. One of the possible causes of the anti-proliferating effect of STT on BEL-7402 cells is induces apoptosis by decreasing the level of BCL-2.
After finishing those experiments mentioned above, another series of research were done to investigate the influence of flavonoids from the seed residues of Hippophae rhamnoides L. on the two cancer cell lines.
In order to determinate the content of total flavonoid aglycones in the seed residues of Hippophae rhamnoides L., the HPLC followed by acid hydration was applied. The mobile phase was Methanol-H2O (60:40), the wavelength for detection was 368 nm and the flow rate was 1.0 ml/m
首先应用MTT法、考马斯亮蓝测蛋白法、SRB、生长曲线法观察了蒺藜皂苷(STT)体外对于Bcap-37和BEL-7402细胞生长的抑制作用，发现STT能够在体外显著抑制人乳腺癌细胞Bcap-37和肝癌细胞BEL-7402的增殖，且该作用具有显著的剂量依赖效应。
为了深入评价STT体外抗肿瘤作用的效果，采用MTT法进行了STT对正常肝细胞和肝癌细胞抑制作用的比较实验并观察了其与盐酸阿霉素对肿瘤细胞的协同抑制效应，结果显示：STY对于正常肝细胞LO2的抑制效应要小于肝癌细胞BEL-7402；高浓度的STT与盐酸阿霉素对于抑制肿瘤细胞具有协同效应，但在低浓度时表现为拮抗效应。
采用wright染色法、丫啶橙染色法和透射电子显微镜进行形态学观察发现：两种癌细胞经STT作用后，均可见核固缩、染色质边集以及出芽的凋亡细胞；碘化丙啶(PI)染色的流式细胞术法检测了STT对Bcap-37和BEL-7402细胞周期的影响，应用免疫荧光与流式细胞术结合法检测STT对BEL-7402细胞的抗细胞凋亡蛋白Bcl-2含量以Bcap-37细胞的细胞周期蛋白cyclin D1和cyclin E水平的影响，结果表明：STT能够促进大量BEL-7402细胞的凋亡，但未见细胞周期阻滞效应，STT诱导Bcap-37细胞的凋亡率较低，却可将Bcap-37细胞周期阻滞在G0／G1期。而且STT能够降低BEL-7402细胞的抗凋亡蛋白Bcl-2表达，并能够降低Bcap-37细胞的细胞周期蛋白cyclin D1和cyclin E水平。
采用活性氧(ROS)检测试剂盒观察发现STT可提高Bcap-37细胞内活性氧的含量，促发细胞的氧化损伤，但对BEL-7402细胞无显著影响，该项研究结果显示：促发活性氧所诱导的氧化损伤也是STT抑制Bcap-37细胞增殖效应的机制之一。
综上所述：STT可抑制人乳腺癌细胞Bcap-37和人肝癌细胞BEL-7402的增殖，对人正常肝细胞的具有较低的抑制效应，高浓度的STT与ADM对Bcap-37和BEL-7402细胞具有协同抑制效应，STT抑制Bcap-37细胞增殖的主要机理
摘要
有:降低cyclinE和cyclin Dl水平诱导细胞周期阻滞以及促进细胞凋亡和诱发
氧化损伤，而STT对肝癌细胞BEL一7402抑制效应的主要机理之一是通过下调
BCL一2蛋白表达诱导肝癌细胞凋亡。
本文的第二大板块是上海市西部开发科技合作项目(编号014358009)子
课题的一项内容，继前一板块完成羡黎皂普对肿瘤细胞作用影响的基础上，为了
解沙棘籽渣黄酮的生物学活性，研究从天然产物化学的提取，分离纯化的现代技
术切入，并对有效成分的生药学原理采用离体肿瘤细胞和基因芯片等技术进行了
后续研究。
研究首先采用0.8 mol几的盐酸80℃水解lh作为水解条件，酸水解法处理
沙棘籽渣中提取的黄酮昔。其后以甲醇一水(60:40)为流动相，紫外检测器的
检测波长为368nxn，流速为1 .0 ml/ min作为色谱条件，用HPLC测定黄酮昔元
含量。结果显示:每1009沙棘籽渣样品中棚皮素、山奈素、异鼠李素的含量分
别为18.58 mg，122.50 mg，27.64 mg;而回收率分别为99.47%，103.700，0，95.51%。
在此基础上，采用黄酮纯度和得率为指标比较了传统的化学纯化法与聚酞胺
柱层析法，进而对沙棘籽渣黄酮的提取和分离纯化工艺作了研究:结果发现两种
方法所得黄酮纯度相当(50一55%)，但聚酞胺柱层析后黄酮得率(24%)大大高
于化学纯化法，后者的工艺的优势明显优于前者。
分离纯化工艺研究结果不仅有助于中药的现代化，同时奠定了探索生物学活
性的后续试验基础。
离体细胞试验首先采用了MTT法测定FsH对乳腺癌细胞Bc即一37的生长抑
制作用，发现，200一1 000 09/ml的FSH能显著抑制BcaP一37的增殖生长，且随
浓度的升高，抑制作用增强，具有一定的剂量依赖关系。为了探讨FsH对BcaP一37
细胞的生长抑制作用的机理，采用Wright染色法和Tu呵EL法观察FSH对
Bcap一37细胞的致凋亡作用;采用PI染色的流式细胞术法检测凋亡率和细胞周期
变化，结果表明:FSH能够诱导Bcap一37细胞的凋亡，凋亡率最高达到2.63%，
并对Bcap一37细胞具有GZ舰期阻滞效应。
为了探讨FSH对Bcap一37细胞生长抑制作用的分子水平的机制，采用基因
芯片检测技术检测了细胞增殖、细胞周期和细胞凋亡相关基因的表达，发现FSH
能够下调Bcap一37细胞内细胞周期蛋白依赖激酶1(C DKI)、DNA复制相关基因
摘要
(DNA聚合酶Q、拓扑异构酶H、PCNA)以及c一myc、C一fos等癌基因的表达;
而能够上调caspaseg、BAKI，FADD以及MADD等促凋亡基因的表达:FsH
抑制Bcap一37细胞增殖分子机理主要是通过调控相关基因表达阻滞细胞周期，抑
制细胞DNA复制以及抑制癌基因所启动的DNA复制及转录，并能通过增强
Bc即一37细胞的凋亡敏感性间接促进细胞凋亡。
由于活性氧在细胞增殖、死亡中所起的重要作用，继而研究了FSH对BcaP一37
细胞内活性氧水平的影响，发现FSH能显著增加Bcap一37细胞内RoS水平，表
明FSH能通过促进细胞氧化损伤抑制Bcap一37癌细胞的增殖。
此外，研究还就FSH对人肝癌BEL一74
Firstly, MTT, SRB, cell growth curve assay, and protein content assay were used to determine the inhibitory effects of STT on the growth of Bcap-37 and BEL-7402 cell line, and then it is shown that STT has potent inhibitory effects on both Bcap-37 cell line and BEL-7402 cell line in a concentration-dependent manner.
In order to evaluate the in vitro antitumor effect of STT, we used MTT assay to compare the inhibitory effect of STT on human hepatocarcinoma cells line BEL-7402 with that on human normal liver cells line L02 through MTT assay, and used the MTT and Webb coefficient assay to judge the co-effect of STT and adriamycin(ADM) on Bcap-37 and BEL-7402 cells, the results show that compared with its inhibition on the growth of BEL-7402 cell line, STT has less inhibitory effect on the proliferation of the normal liver cell line L02, and moreover the synergetic effects of STT and ADM on the two cancer cell line can be achieved at a high concentration of STT, however, when STT is at a low concentration, the antagonism of the two drugs will be produced.
We applied several methods of morphological observation such as wright staining, acridine orange staining and electron microscope to observe the effect of STT on the morphological alterations of the two cancer cell line, to find that after treatment with STT, both of the two cancer cell lines will exhibit typical
morphological alterations of apoptosis, such as rounding of cells, nucleus pyknosis, apoptotic body and so on.
The flow cytometry detection with PI staining was used to detect cell cycle arrest and apoptotic peak of the two cell line after treatment with STT, and therefore it can be seen from the results that The apoptotic peaks can be induced by STT on both of the two cancer cell lines, however, STT can induce apoptosis of a large percent of BEL-7402 cell, while the apoptotic rate of Bcap-37 cell is much lower than that of BEL-7402 cell. In addition, STT has GO/G1 arrest effect on cell cycle of Bcap-37 cells.
In addition, the molecular mechanism of cell cycle arrest and apoptosis induction have been studied. The immunological fluorescent assay followed by flow cytometry detection was applied to determine BCL-2 protein expression of BEL-7402 cells and cyclin Dl, cyclin E protein expression of Bcap-37 cells. The results demonstrate that STT can reduce BCL-2 protein level of BEL-7402 cell line; in addition, the cyclin E and cyclin Dl expression of Bcap-37 cell line can be down-regulated by STT.
Moreover, we have quantified ROS in the two cancer cell lines after being treated by STT by using the reactive oxygen species (ROS) detection kit. The data shows that the quantity of ROS in Bcap-37 cell line, which can result in oxygenic damage, increases remarkably after treatment with STT, but there is no significant change of the ROS level in BEL-7402 cells.
In summary, it can be inferred that STT has potent inhibitory effects on both Bcap-37 cell line and BEL-7402 cell line in a concentration-dependent manner, but has less inhibitory effect on the proliferation of the normal liver cell line L02. The synergetic effect on the two cell lines between STT with ADM can be seen at a high concentration of STT. The mechanism of inhibitory effect of STT on Bcap-37 cells are not only to induce apoptosis, arrest cell cycle of Bcap-37 cells at GO/G1 by decreasing the level of cyclin Dl and cyclin E, but also to give rise to oxygenic damage in cells. One of the possible causes of the anti-proliferating effect of STT on BEL-7402 cells is induces apoptosis by decreasing the level of BCL-2.
After finishing those experiments mentioned above, another series of research were done to investigate the influence of flavonoids from the seed residues of Hippophae rhamnoides L. on the two cancer cell lines.
In order to determinate the content of total flavonoid aglycones in the seed residues of Hippophae rhamnoides L., the HPLC followed by acid hydration was applied. The mobile phase was Methanol-H2O (60:40), the wavelength for detection was 368 nm and the flow rate was 1.0 ml/m
引文
[1] 江苏新药学院编，中药大词典[M]上海，上海科学技术出版社，1999；1274-1276；2452-2453
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[3] 曹惠玲，陈浩宏，许士凯．蒺藜及其有效成分的药理与临床研究进展，[J]中成药2001；23(8)：602-605
[4] 宿燕岗，杨学义，陈珠．心脑舒通的临床应用及研究状况，[J]深圳中西医结合杂志 1999；9(1)：39-40
[5] 李明娟，瞿伟菁．蒺藜皂苷的降血糖作用．[J]中药材，2002：25(6)：
[6] 褚书地，瞿伟菁，逄秀凤等．蒺藜皂苷的降血脂作用．[J]中药材 2003；26(5)：341-344
[7] N.A. Awadh Ali a, W.D. J(?) lich b, C. Kusnick c, et al. Screening of Yemeni medicinal plants for antibacterial and cytotoxic activities. [J] Journal of Ethnopharmacology 2001, 74:173-179
[8] Bedir E, Khan IA, Walker LA. Biologically active steroidal glycosides from Tribulus terrestris. [J] Pharmazie 2002; 57:491-3.
[9] 金京玲、任东鲜、金哲洙，等．大孔吸附树脂法提取蒺藜皂苷．[J]延边大学医学学报 1999，22(1)：29-30
[10] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. [J] J Immunol Methods 1983; 65: 55-63.
[11] 张均田等．现代药理学实验方法，[M]北京：北京医科大学和中国协和医科大学联合出版社，1999：820-821
[12] 张如松，叶益萍，刘雪莉．自首乌甾体总苷的体外抗肿瘤作用，[J]中草药 2000；31(8)：599-601
[13] 王青青．余海．SRB显色法用于抗癌药物敏感性试验的研究，[J]科技通报 2000；16(2)：104-107
[14] 薛妍，刘宝瑞，慕利梅．姜黄素和阿霉素联合应用对人肝癌细胞SMMC-7721抑制作用的研究，[J]肿瘤研究与临床 2000；12(6)：372-374
[15] Yeh YA, Herenyiova M, Weber G. Quercetin: synergistic action with carboxyam idotriaxolein human breast carcinoma cells, [J] Life Sci 1995; 57:1285.
[16] 钱曼，张秀华，梅兵等．棘阿米巴滋养体对HeLa细胞的细胞毒性作用，[J]中国寄生虫学与寄生虫病杂志 2001；19(1)：37-40
[17] 姜泊，张亚历，周殿元．分子生物学常用实验方法．[M]北京：人民军医出版社，1996：1746
[18] Nicoletti I, Migliorati G, Pagliacci MC, et al. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. [J] J. Immunol Methods 1991, 139:271-279
[19] Gong JP, Ardelt B, Traganos F, et al. Unscheduled expression of cyclin B1 and cyclin E in several leukemic and solid tumor cell line, [J]Cancer Res 1994; 54:4285-4288
[20] 温汉平，王小宁，孙华蕴等．肿瘤细胞体外药敏试验MTT分析法，[J]中华肿瘤杂志 1993；15：470-471
[21] 谭卫东．金红．罗弟祥．抗肿瘤药物筛选中MTT法和SRB法的比较，[J]天然产物研究与开发 1999；(11)3：17-22
[22] Ganey PE, Carter LS, Mueller RA, et al. Doxorubicin toxicity in perfused tat heart.Decereased cell death at low oxygen tension, [J]Circ Res, 1991; 68:1610-1613
[23] 魏一生，叶庆林，金静君等．雷藤甲素和亚硒酸钠对人白血病细胞系HL60生长抑制作用的协同效应，[J]福建医科大学学报 1999；33(2)：129-132
[24] Kerr JER, Wyllie AH, CurrieAH, et al. Apoptosis, a basic biological phenomenon with wider implications in tissue kinetics.[J] Br J Cancer 1972; 26: 239-.
[25] 欧阳高亮，李祺福、洪水根．细胞凋亡与肿瘤的发生发展和治疗．[J]国外医学肿瘤学分册．2000，27(5)：266-268
[26] Kerr JF, Winterord CM, Harmon BV. Apoptosis its significance in cancer and cancer therapy. [J] Cancer 1994,73: 2013-2026
[27] Sima SM, Schindler M. Cell biological mechanisms in multidrug resistance in tumors. [J] Proc Natl Acad Sci USA 1994; 91 : 3497-3504
[28] 周剑峰，陈燕，李崇渔等．阿糖胞苷诱导HL-60细胞凋亡的研究，[J]中华肿瘤杂志 1997；19(2)：107-110
[29] Tsujimoto Y, Cossman J, Jaffe E, et al. Invovlement of the Bcl-2 gene in human follicular lymphoma, [J] Science 1985, 228: 1440-1443.
[30] Rosse T, Oliver R, Monney L, et al. Bcl-2 prolongs cell survival after bax induced release of
cytochrome c, [J] Nature 1998; 391 : 496-499.
[31] Schwarze MM, Hawley RG. Prevention of myeloma cell apoptosis by ectopic bcl-2 expression or interleukin 6-mediated up-regulation of bcl-xL. [J]Cancer Res 1995; 55:2262-2265
[32] Thomas A, El Rouby S, Reed JC, et al. Drug-induced apoptosis in B-cell chronic lymphocytic leukemia: relationship between p53 gene mutation and bcl-2/bax proteins in drug resistance. [J] Oncogene 1996; 12: 1055-1062.
[33] Hartwell LH, Kastan MB. Cell cycle control and cancer. [J]Science. 1994; 266: 1821-1828.
[34] Molinari M. Cell cycle checkpoints and their inactivation in human cancer. [J]Cell Prolif. 2000; 33(5): 261-274.
[35] 刘芳．细胞周期与抗肿瘤治疗展望．[J] 国外医学临床生物化学与检验学分册 2002；23(04)：202-203
[36] Wedgwood S, Dettman RW, Black SH. ET-1 stimulate pul-monary arterial smooth muscle cell proliferation viain duction of reactive oxygen species. [J]Am J Physiol Lung Cell Mol Physiol, 2001; 281 : 1058-1067.
[37] 陈诗书，汤雪明主编．细胞生长与细胞分裂．医学细胞与分子生物学．上海：上海医科大学出版社，1995，3132
[38] Hunter T, Pines J. Cyclins and cancer: cyclin D and CDK inhibitors come of age. Cell, 1994,79:573-582
[39] Ohtsubo M, Theodoras AM, Schumacher J, et al. Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. [J]. Mol Cell Biol, 1995, 15:2612-2624.
[40] GengY, Eaton E N, Picon M,et al. Regulation of cyclin E transcription by E2F and retinoblastoma protein [J]. Oncogene, 1996, 12(6): 1173-1180.
[41] Klaunig JE, Xu Y, Isenberg JS, et al. The role of oxidative stress in chemical carcinogenesis. [J] Environ Health Perspect 1998; 106(suppl1): 289-295.
[41] Arnold RS, Shi J, Murad E, et al. Hydrogen peroxide mediates the cell growth and transformation caused by the mitogeni-coxidase NOX1, [J] Proc Natl Acad Sci USA 2001; 98: 5550-5555
[42] 秦达明．自由基的生物学作用和平衡，江西教育学院院报(自然科学版) 2000；21(3)：41-47
[43] Phillips DC, Allen K, Griffiths HR. Synthetic ceramids induce growth arrest or apoptosis by
altering cellular redoxsta-tus. [J]Arch Biochem Biophys 2002; 407:15-24.
[41] Chung YW, Jeong DW, Won JY, et al. H_2O_2-induced AP-1 activation and its effect on p21(WAF1/CIP1)-mediated G2/M arrest in a p53-deficient human lung cancer cell. [J] Biochem Biophys Res Commun 2002; 293: 1248-1253.
[2] 褚书地，瞿伟菁，李穆等．蒺藜化学成分及其药理作用研究进展，[J]中国野生植物资源 2003；22(4)：4-7
[3] 曹惠玲，陈浩宏，许士凯．蒺藜及其有效成分的药理与临床研究进展，[J]中成药2001；23(8)：602-605
[4] 宿燕岗，杨学义，陈珠．心脑舒通的临床应用及研究状况，[J]深圳中西医结合杂志 1999；9(1)：39-40
[5] 李明娟，瞿伟菁．蒺藜皂苷的降血糖作用．[J]中药材，2002：25(6)：
[6] 褚书地，瞿伟菁，逄秀凤等．蒺藜皂苷的降血脂作用．[J]中药材 2003；26(5)：341-344
[7] N.A. Awadh Ali a, W.D. J(?) lich b, C. Kusnick c, et al. Screening of Yemeni medicinal plants for antibacterial and cytotoxic activities. [J] Journal of Ethnopharmacology 2001, 74:173-179
[8] Bedir E, Khan IA, Walker LA. Biologically active steroidal glycosides from Tribulus terrestris. [J] Pharmazie 2002; 57:491-3.
[9] 金京玲、任东鲜、金哲洙，等．大孔吸附树脂法提取蒺藜皂苷．[J]延边大学医学学报 1999，22(1)：29-30
[10] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. [J] J Immunol Methods 1983; 65: 55-63.
[11] 张均田等．现代药理学实验方法，[M]北京：北京医科大学和中国协和医科大学联合出版社，1999：820-821
[12] 张如松，叶益萍，刘雪莉．自首乌甾体总苷的体外抗肿瘤作用，[J]中草药 2000；31(8)：599-601
[13] 王青青．余海．SRB显色法用于抗癌药物敏感性试验的研究，[J]科技通报 2000；16(2)：104-107
[14] 薛妍，刘宝瑞，慕利梅．姜黄素和阿霉素联合应用对人肝癌细胞SMMC-7721抑制作用的研究，[J]肿瘤研究与临床 2000；12(6)：372-374
[15] Yeh YA, Herenyiova M, Weber G. Quercetin: synergistic action with carboxyam idotriaxolein human breast carcinoma cells, [J] Life Sci 1995; 57:1285.
[16] 钱曼，张秀华，梅兵等．棘阿米巴滋养体对HeLa细胞的细胞毒性作用，[J]中国寄生虫学与寄生虫病杂志 2001；19(1)：37-40
[17] 姜泊，张亚历，周殿元．分子生物学常用实验方法．[M]北京：人民军医出版社，1996：1746
[18] Nicoletti I, Migliorati G, Pagliacci MC, et al. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. [J] J. Immunol Methods 1991, 139:271-279
[19] Gong JP, Ardelt B, Traganos F, et al. Unscheduled expression of cyclin B1 and cyclin E in several leukemic and solid tumor cell line, [J]Cancer Res 1994; 54:4285-4288
[20] 温汉平，王小宁，孙华蕴等．肿瘤细胞体外药敏试验MTT分析法，[J]中华肿瘤杂志 1993；15：470-471
[21] 谭卫东．金红．罗弟祥．抗肿瘤药物筛选中MTT法和SRB法的比较，[J]天然产物研究与开发 1999；(11)3：17-22
[22] Ganey PE, Carter LS, Mueller RA, et al. Doxorubicin toxicity in perfused tat heart.Decereased cell death at low oxygen tension, [J]Circ Res, 1991; 68:1610-1613
[23] 魏一生，叶庆林，金静君等．雷藤甲素和亚硒酸钠对人白血病细胞系HL60生长抑制作用的协同效应，[J]福建医科大学学报 1999；33(2)：129-132
[24] Kerr JER, Wyllie AH, CurrieAH, et al. Apoptosis, a basic biological phenomenon with wider implications in tissue kinetics.[J] Br J Cancer 1972; 26: 239-.
[25] 欧阳高亮，李祺福、洪水根．细胞凋亡与肿瘤的发生发展和治疗．[J]国外医学肿瘤学分册．2000，27(5)：266-268
[26] Kerr JF, Winterord CM, Harmon BV. Apoptosis its significance in cancer and cancer therapy. [J] Cancer 1994,73: 2013-2026
[27] Sima SM, Schindler M. Cell biological mechanisms in multidrug resistance in tumors. [J] Proc Natl Acad Sci USA 1994; 91 : 3497-3504
[28] 周剑峰，陈燕，李崇渔等．阿糖胞苷诱导HL-60细胞凋亡的研究，[J]中华肿瘤杂志 1997；19(2)：107-110
[29] Tsujimoto Y, Cossman J, Jaffe E, et al. Invovlement of the Bcl-2 gene in human follicular lymphoma, [J] Science 1985, 228: 1440-1443.
[30] Rosse T, Oliver R, Monney L, et al. Bcl-2 prolongs cell survival after bax induced release of
cytochrome c, [J] Nature 1998; 391 : 496-499.
[31] Schwarze MM, Hawley RG. Prevention of myeloma cell apoptosis by ectopic bcl-2 expression or interleukin 6-mediated up-regulation of bcl-xL. [J]Cancer Res 1995; 55:2262-2265
[32] Thomas A, El Rouby S, Reed JC, et al. Drug-induced apoptosis in B-cell chronic lymphocytic leukemia: relationship between p53 gene mutation and bcl-2/bax proteins in drug resistance. [J] Oncogene 1996; 12: 1055-1062.
[33] Hartwell LH, Kastan MB. Cell cycle control and cancer. [J]Science. 1994; 266: 1821-1828.
[34] Molinari M. Cell cycle checkpoints and their inactivation in human cancer. [J]Cell Prolif. 2000; 33(5): 261-274.
[35] 刘芳．细胞周期与抗肿瘤治疗展望．[J] 国外医学临床生物化学与检验学分册 2002；23(04)：202-203
[36] Wedgwood S, Dettman RW, Black SH. ET-1 stimulate pul-monary arterial smooth muscle cell proliferation viain duction of reactive oxygen species. [J]Am J Physiol Lung Cell Mol Physiol, 2001; 281 : 1058-1067.
[37] 陈诗书，汤雪明主编．细胞生长与细胞分裂．医学细胞与分子生物学．上海：上海医科大学出版社，1995，3132
[38] Hunter T, Pines J. Cyclins and cancer: cyclin D and CDK inhibitors come of age. Cell, 1994,79:573-582
[39] Ohtsubo M, Theodoras AM, Schumacher J, et al. Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. [J]. Mol Cell Biol, 1995, 15:2612-2624.
[40] GengY, Eaton E N, Picon M,et al. Regulation of cyclin E transcription by E2F and retinoblastoma protein [J]. Oncogene, 1996, 12(6): 1173-1180.
[41] Klaunig JE, Xu Y, Isenberg JS, et al. The role of oxidative stress in chemical carcinogenesis. [J] Environ Health Perspect 1998; 106(suppl1): 289-295.
[41] Arnold RS, Shi J, Murad E, et al. Hydrogen peroxide mediates the cell growth and transformation caused by the mitogeni-coxidase NOX1, [J] Proc Natl Acad Sci USA 2001; 98: 5550-5555
[42] 秦达明．自由基的生物学作用和平衡，江西教育学院院报(自然科学版) 2000；21(3)：41-47
[43] Phillips DC, Allen K, Griffiths HR. Synthetic ceramids induce growth arrest or apoptosis by
altering cellular redoxsta-tus. [J]Arch Biochem Biophys 2002; 407:15-24.
[41] Chung YW, Jeong DW, Won JY, et al. H_2O_2-induced AP-1 activation and its effect on p21(WAF1/CIP1)-mediated G2/M arrest in a p53-deficient human lung cancer cell. [J] Biochem Biophys Res Commun 2002; 293: 1248-1253.
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