海带硫酸多糖对人大肠癌Lovo细胞株增殖和凋亡的影响
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摘要
目的:大肠癌是常见的消化道恶性肿瘤之一,其发病率呈逐年上升趋势。近年来,尽管手术、放疗、化疗等综合治疗使大肠癌患者的术后生存得到了很大的改善,但疗效并不乐观。大肠癌患者早期常无明显症状,多数患者就诊时即已为中、晚期,还有15%-35%就诊时就已发生转移,而肿瘤转移是导致肿瘤患者死亡的重要原因之一。
早在70年代末,人们就发现肿瘤转移可能与一类肝素样物质作用有关,并对肝素在肿瘤转移中的作用进行了深入研究。80年代初期,乙酰肝素和乙酰肝素酶的发现为肿瘤转移研究开辟了新的领域。肿瘤侵袭转移必须突破细胞外基质(ECM)和血管内皮下基膜(BM)屏障,硫酸乙酰肝素蛋白多糖(HSPG)是一种蛋白聚糖类碳水化合物,为广泛存在于有脊椎动物和无脊椎动物组织细胞外基质(ECM)和细胞表面的生物大分子,是基底膜的主要构成成分。乙酰肝素酶(Heparanase, HPA)是一种葡萄糖醛酸内切酶,也是目前发现的唯一可以降解HSPG的酶,它通过切断硫酸乙酰肝素蛋白多糖侧链(HS)以降解细胞外基质和血管基膜,并通过释放和活化HSPG结合型的生长因子(如bFGF,VEGF等)诱导血管生成,从而促进肿瘤细胞的侵袭和转移。因此,对HPA抑制剂的研究将成为抗肿瘤药物治疗的新靶点。乙酰肝素酶抑制剂的设计主要根据乙酰肝素或其它类似物,Parish[1]等选择乙酰硫酸肝素盐的结构模拟物硫酸低聚糖作为侯选药物研究,因为这些成分可以识别乙酰硫酸肝素盐和抑制乙酰肝素酶裂解乙酰硫酸肝素盐、干扰促血管生成生长因子。在动物实验中已证实肝素可以抑制肿瘤的生长或转移、抑制血管生成或损害。虽然它们是抗凝剂,但研究发现消耗抗凝活性后它们仍然保留抗转移作用和抑制乙酰肝素酶活性的特性。Miao[2]等证实了肝素和某些硫酸多糖除抗凝作用以外的抗转移作用归功于它们的抑制乙酰肝素酶的作用,他们观察海带硫酸多糖对乙酰肝素酶活性和肿瘤转移的作用,发现表达乙酰肝素酶的B-16、BL-6鼠黑素瘤细胞和13762MAT鼠乳腺癌细胞乙酰肝素酶降解完整细胞外基质可能已被海带硫酸多糖有效抑制,而且在有效浓度时,海带硫酸多糖对肿瘤的增殖和原位生长有抑制作用。应用乙酰肝素酶抑制剂治疗的实验动物黑色素瘤、lewis肺癌和哺乳动物腺癌细胞肺转移率较对照组低,有显著差异。且发现海带硫酸多糖也抑制T细胞介导的迟发性过敏反应和实验性自身免疫反应性脑脊髓炎,因此对自身免疫性疾病也可能有一定作用。
海带硫酸多糖(Laminaria polysaccharidesulfate, LPS )是一种含有多种单糖和硫酸基的水溶性杂多糖,其生物学功能主要集中在抗肿瘤、免疫调节、血脂及血糖调节等方面。海带硫酸多糖从海带中提取,我国是海带(Laminaria japonica)生产大国,海带产量占世界产量一半,具有良好的开发应用前景。而海带是食药两用的褐藻类植物,其药用价值在本草纲目和嘉佑本草等古医书中已有记载。原材料易得,提取方法多种。Vlodavsky[3]等报道海带硫酸多糖作为乙酰肝素酶抑制剂在动物实验中能够有效的抑制肿瘤细胞的转移。有文献报道,体内、外实验证实LPS具有抑制肿瘤细胞增殖的作用,但其分子机制未见报道,本项目从LPS促进细胞凋亡,进而抑制细胞增殖及提高化疗药物的敏感性着手研究LPS的药物作用机理。bcl-2是抗凋亡基因,且与肿瘤的发展密切相关。它通过其转录产物bcl-2基因蛋白,参与细胞内抗氧化作用和抑制钙离子跨膜运动而调控细胞凋亡。NF-κBp65是一种重要的可诱导的基因转录调节因子,参与炎症、免疫、细胞的增殖和细胞凋亡等多种生理、病理过程的基因调控。近年来发现NF-κBp65调节许多黏附、侵袭、迁移相关因子的转录而参与肿瘤的侵袭转移,故降低NF-κBp65的表达将成为治疗癌症的新靶点。通过分析细胞周期变化及检测bcl-2基因和NF-κBp65基因表达的变化,观察海带硫酸多糖抗肿瘤的机制,即海带硫酸多糖通过抑制乙酰肝素酶的活性,从而抑制肿瘤细胞的生长和转移的机制。它与化疗药物联合应用,增强了肿瘤细胞对化疗药物的敏感性,可减少药物的用量,提高肿瘤患者的治愈率和生存质量。
方法:本研究以不同浓度的LPS处理大肠癌Lovo细胞,用四甲基偶氮蓝比色法(MTT)测定细胞增殖情况,细胞染色法观察细胞凋亡的形态,用流式细胞仪记录细胞周期改变、bcl-2及NF-κBp65基因表达情况,从而探讨海带硫酸多糖对人大肠癌Lovo细胞株的体外生长、凋亡及细胞周期的影响,并进行统计学分析。
结果:1.形态学观察:扫描镜下观察实验组及对照组:对照组细胞生长活跃,大小不等,细胞间有很多微丝样的结构互相连接。实验组的细胞形态变化较大,较多细胞变性及坏死,细胞由梭形变为圆形,表面形成许多小泡形的凋亡小体。
2. LPS对Lovo细胞的抑制率:MTT比色法检测LPS对Lovo细胞的增殖的抑制作用,从结果可见LPS对Lovo细胞具有较强的抑制作用,随着浓度增加与时间延长该抑制作用渐增强,呈剂量时效依赖关系,其间差异具统计学意义(P<0.05)。
3.细胞周期变化:实验组的细胞用碘化丙锭染色后,用流式细胞仪分析细胞周期,发现LPS可改变Lovo细胞的细胞周期,三种浓度S期细胞百分率增加,G0/G1期细胞百分率降低,细胞被阻滞于S期,有统计学意义(P<0.05)。
4. bcl-2与NF-κBp65基因表达的改变:Lovo细胞有bcl-2高表达,LPS可降低细胞中的bcl-2基因蛋白的表达,且随着LPS浓度的增加,改变愈加明显。LPS作用后,Lovo细胞内NF-κBp65的表达降低,随着LPS浓度的增加,降低愈明显,实验组与对照组比较及实验组间比较差异有统计学意义(P<0.05)。
结论
1硫酸海带多糖能有效抑制人大肠癌Lovo细胞的生长对大肠癌细胞有化疗增敏的作用。
2硫酸海带多糖能够下调NF-κBp65和bcl-2基因的表达,从而降低对大肠癌Lovo细胞凋亡的抑制。
3硫酸海带多糖有明显抑制乙酰肝素酶的作用,从而间接抑制肿瘤转移
Objective : Colorectal cancer is one of the common gastrointestinal neoplasms, and its incidence rate is rising year by year. Although the different available therapies, which including surgical operation ,radical therapy, and chemical therapy , have currently been used in the treatment of colorectal cancer, and all of these have markedly improved the prognosis of colorectal cancer. But results are not satisfied. As for the patients visiting, about 15%～35% already have tumor metastasis because there are not overt symptoms in the early period of colorectal cancer, and tumor metastasis is one of the majority reasons to result in death.
At the end of 70s, it is found that the metastasis of tumor was relativity with a sore of substance like heparin. Although these phenomena well-documented, it has takes 20years to isolate a heparanase gene, mainly because of instability in designing specific, quantitative assays. Cloning and functional characterization of the long sought-after heparanase opens a new chapter in the understanding and potential manipulation of metastasis processes.
Laminaria polysaccharidesulfate(LPS) is a watersolibility polysaccharide which include many types of monosaccharide and sulphuricacid. It’s biological function mainly concentrate on antitumor ,adjusting immunity, regularing blood fat and blood sugar and so on. Our country produced most laminaria japonica of the world. Laminaria japonica can eat and also can be used as medicine. It is reported that LPS can inhibit the transfusion of tumor cells in animal exprements. There is also some studying reported that LPS can inhibit the generation of tumor cells in vivo or in vitro, but it’s molecular organism biomechanism have not reported. Our item study that :LPS enhance the apoptosis of tumor cells ,inhibit the generation of the cells and enhance the sensitivity of chemical medicine. Bcl-2 which has an intimate relation with the progressing of tumor is an anti- apoptosis gene . Bcl-2 gene play it’s function through it’s transcript which called bcl-2 gene protein. Bcl-2 gene protein participate endo-cell anti-oxygen and inhibit the progress of Ca across memberance movement to adjust the apoptosis of tumor cells. We analysis cell cycle’s change and detect bcl-2 gene and NF-κBp65 gene in order to observe the antitumor mechanism of LPS.
Methods: 1 We use different density of LPS to deal with lovo cells of carcinoma of human large intestine. We use MTT method to detect cells’generation and use cell staining method to detect the apoptosis shape of cells.
2 We use flow cytometry method (FCM) to detect the change of cell cycles and the expression of bcl-2 gene and NF-κBp65 gene.
3 We use RT-PCR to detect the expression of heparanase. The purpose of all study methods are to study the influence on cell’s generation, apoptosis and cell cycles of lovo cells which belong to a type of carcinoma of human large intestine. At last use statistic method to analyze all data.
Results: 1 Morphology observation: we observe the cells of comparison groups and study groups through Scanning electron microscopy(SEM): the generation of cells in comparison groups is active,cells are in different shapes, cells have little cytoplasm ,cell’s membrane is ambiguity and nuclear membrane is integrated. Cell’s chromatin is thick and we also can observe caryocinesia phase.The study groups: it is great different in cells’shapes. Degeneration and necrosis have take placed in many cells. Decomposition have take placed in cytolymph. Cells’nuclear have broken up ,deliquesced or even disappeared.
2 We use MTT method to detect the inhibit condition of LPS on lovo cells. With the time extending and the increasing of the LPS’density , the inhibit function become stronger (P<0.05).
3 Cell cycle’s change: We use PI to deal with the study groups’cells and we find that LPS can change lovo cell’cell cycle in large extent.
4 The expression of bcl-2 gene and NF-κBp65 gene: LPS can lower the expression of bcl-2 gene and NF-κBp65 gene. The more increasing of LPS’density the lower the expression of those two genes.
Conclusions: 1 LPS can inhibit the generation of lovo cells and can enhance the sensitivity of chemical medicine.
2 LPS can down regular the expression of bcl-2 gene and NF-κBp65 gene. Through this method, LPS also can lower the inhibition to lovo cells.
3 LPS can obviously inhibit the function of heparanase and tumor transfusion indirectly.
早在70年代末,人们就发现肿瘤转移可能与一类肝素样物质作用有关,并对肝素在肿瘤转移中的作用进行了深入研究。80年代初期,乙酰肝素和乙酰肝素酶的发现为肿瘤转移研究开辟了新的领域。肿瘤侵袭转移必须突破细胞外基质(ECM)和血管内皮下基膜(BM)屏障,硫酸乙酰肝素蛋白多糖(HSPG)是一种蛋白聚糖类碳水化合物,为广泛存在于有脊椎动物和无脊椎动物组织细胞外基质(ECM)和细胞表面的生物大分子,是基底膜的主要构成成分。乙酰肝素酶(Heparanase, HPA)是一种葡萄糖醛酸内切酶,也是目前发现的唯一可以降解HSPG的酶,它通过切断硫酸乙酰肝素蛋白多糖侧链(HS)以降解细胞外基质和血管基膜,并通过释放和活化HSPG结合型的生长因子(如bFGF,VEGF等)诱导血管生成,从而促进肿瘤细胞的侵袭和转移。因此,对HPA抑制剂的研究将成为抗肿瘤药物治疗的新靶点。乙酰肝素酶抑制剂的设计主要根据乙酰肝素或其它类似物,Parish[1]等选择乙酰硫酸肝素盐的结构模拟物硫酸低聚糖作为侯选药物研究,因为这些成分可以识别乙酰硫酸肝素盐和抑制乙酰肝素酶裂解乙酰硫酸肝素盐、干扰促血管生成生长因子。在动物实验中已证实肝素可以抑制肿瘤的生长或转移、抑制血管生成或损害。虽然它们是抗凝剂,但研究发现消耗抗凝活性后它们仍然保留抗转移作用和抑制乙酰肝素酶活性的特性。Miao[2]等证实了肝素和某些硫酸多糖除抗凝作用以外的抗转移作用归功于它们的抑制乙酰肝素酶的作用,他们观察海带硫酸多糖对乙酰肝素酶活性和肿瘤转移的作用,发现表达乙酰肝素酶的B-16、BL-6鼠黑素瘤细胞和13762MAT鼠乳腺癌细胞乙酰肝素酶降解完整细胞外基质可能已被海带硫酸多糖有效抑制,而且在有效浓度时,海带硫酸多糖对肿瘤的增殖和原位生长有抑制作用。应用乙酰肝素酶抑制剂治疗的实验动物黑色素瘤、lewis肺癌和哺乳动物腺癌细胞肺转移率较对照组低,有显著差异。且发现海带硫酸多糖也抑制T细胞介导的迟发性过敏反应和实验性自身免疫反应性脑脊髓炎,因此对自身免疫性疾病也可能有一定作用。
海带硫酸多糖(Laminaria polysaccharidesulfate, LPS )是一种含有多种单糖和硫酸基的水溶性杂多糖,其生物学功能主要集中在抗肿瘤、免疫调节、血脂及血糖调节等方面。海带硫酸多糖从海带中提取,我国是海带(Laminaria japonica)生产大国,海带产量占世界产量一半,具有良好的开发应用前景。而海带是食药两用的褐藻类植物,其药用价值在本草纲目和嘉佑本草等古医书中已有记载。原材料易得,提取方法多种。Vlodavsky[3]等报道海带硫酸多糖作为乙酰肝素酶抑制剂在动物实验中能够有效的抑制肿瘤细胞的转移。有文献报道,体内、外实验证实LPS具有抑制肿瘤细胞增殖的作用,但其分子机制未见报道,本项目从LPS促进细胞凋亡,进而抑制细胞增殖及提高化疗药物的敏感性着手研究LPS的药物作用机理。bcl-2是抗凋亡基因,且与肿瘤的发展密切相关。它通过其转录产物bcl-2基因蛋白,参与细胞内抗氧化作用和抑制钙离子跨膜运动而调控细胞凋亡。NF-κBp65是一种重要的可诱导的基因转录调节因子,参与炎症、免疫、细胞的增殖和细胞凋亡等多种生理、病理过程的基因调控。近年来发现NF-κBp65调节许多黏附、侵袭、迁移相关因子的转录而参与肿瘤的侵袭转移,故降低NF-κBp65的表达将成为治疗癌症的新靶点。通过分析细胞周期变化及检测bcl-2基因和NF-κBp65基因表达的变化,观察海带硫酸多糖抗肿瘤的机制,即海带硫酸多糖通过抑制乙酰肝素酶的活性,从而抑制肿瘤细胞的生长和转移的机制。它与化疗药物联合应用,增强了肿瘤细胞对化疗药物的敏感性,可减少药物的用量,提高肿瘤患者的治愈率和生存质量。
方法:本研究以不同浓度的LPS处理大肠癌Lovo细胞,用四甲基偶氮蓝比色法(MTT)测定细胞增殖情况,细胞染色法观察细胞凋亡的形态,用流式细胞仪记录细胞周期改变、bcl-2及NF-κBp65基因表达情况,从而探讨海带硫酸多糖对人大肠癌Lovo细胞株的体外生长、凋亡及细胞周期的影响,并进行统计学分析。
结果:1.形态学观察:扫描镜下观察实验组及对照组:对照组细胞生长活跃,大小不等,细胞间有很多微丝样的结构互相连接。实验组的细胞形态变化较大,较多细胞变性及坏死,细胞由梭形变为圆形,表面形成许多小泡形的凋亡小体。
2. LPS对Lovo细胞的抑制率:MTT比色法检测LPS对Lovo细胞的增殖的抑制作用,从结果可见LPS对Lovo细胞具有较强的抑制作用,随着浓度增加与时间延长该抑制作用渐增强,呈剂量时效依赖关系,其间差异具统计学意义(P<0.05)。
3.细胞周期变化:实验组的细胞用碘化丙锭染色后,用流式细胞仪分析细胞周期,发现LPS可改变Lovo细胞的细胞周期,三种浓度S期细胞百分率增加,G0/G1期细胞百分率降低,细胞被阻滞于S期,有统计学意义(P<0.05)。
4. bcl-2与NF-κBp65基因表达的改变:Lovo细胞有bcl-2高表达,LPS可降低细胞中的bcl-2基因蛋白的表达,且随着LPS浓度的增加,改变愈加明显。LPS作用后,Lovo细胞内NF-κBp65的表达降低,随着LPS浓度的增加,降低愈明显,实验组与对照组比较及实验组间比较差异有统计学意义(P<0.05)。
结论
1硫酸海带多糖能有效抑制人大肠癌Lovo细胞的生长对大肠癌细胞有化疗增敏的作用。
2硫酸海带多糖能够下调NF-κBp65和bcl-2基因的表达,从而降低对大肠癌Lovo细胞凋亡的抑制。
3硫酸海带多糖有明显抑制乙酰肝素酶的作用,从而间接抑制肿瘤转移
Objective : Colorectal cancer is one of the common gastrointestinal neoplasms, and its incidence rate is rising year by year. Although the different available therapies, which including surgical operation ,radical therapy, and chemical therapy , have currently been used in the treatment of colorectal cancer, and all of these have markedly improved the prognosis of colorectal cancer. But results are not satisfied. As for the patients visiting, about 15%～35% already have tumor metastasis because there are not overt symptoms in the early period of colorectal cancer, and tumor metastasis is one of the majority reasons to result in death.
At the end of 70s, it is found that the metastasis of tumor was relativity with a sore of substance like heparin. Although these phenomena well-documented, it has takes 20years to isolate a heparanase gene, mainly because of instability in designing specific, quantitative assays. Cloning and functional characterization of the long sought-after heparanase opens a new chapter in the understanding and potential manipulation of metastasis processes.
Laminaria polysaccharidesulfate(LPS) is a watersolibility polysaccharide which include many types of monosaccharide and sulphuricacid. It’s biological function mainly concentrate on antitumor ,adjusting immunity, regularing blood fat and blood sugar and so on. Our country produced most laminaria japonica of the world. Laminaria japonica can eat and also can be used as medicine. It is reported that LPS can inhibit the transfusion of tumor cells in animal exprements. There is also some studying reported that LPS can inhibit the generation of tumor cells in vivo or in vitro, but it’s molecular organism biomechanism have not reported. Our item study that :LPS enhance the apoptosis of tumor cells ,inhibit the generation of the cells and enhance the sensitivity of chemical medicine. Bcl-2 which has an intimate relation with the progressing of tumor is an anti- apoptosis gene . Bcl-2 gene play it’s function through it’s transcript which called bcl-2 gene protein. Bcl-2 gene protein participate endo-cell anti-oxygen and inhibit the progress of Ca across memberance movement to adjust the apoptosis of tumor cells. We analysis cell cycle’s change and detect bcl-2 gene and NF-κBp65 gene in order to observe the antitumor mechanism of LPS.
Methods: 1 We use different density of LPS to deal with lovo cells of carcinoma of human large intestine. We use MTT method to detect cells’generation and use cell staining method to detect the apoptosis shape of cells.
2 We use flow cytometry method (FCM) to detect the change of cell cycles and the expression of bcl-2 gene and NF-κBp65 gene.
3 We use RT-PCR to detect the expression of heparanase. The purpose of all study methods are to study the influence on cell’s generation, apoptosis and cell cycles of lovo cells which belong to a type of carcinoma of human large intestine. At last use statistic method to analyze all data.
Results: 1 Morphology observation: we observe the cells of comparison groups and study groups through Scanning electron microscopy(SEM): the generation of cells in comparison groups is active,cells are in different shapes, cells have little cytoplasm ,cell’s membrane is ambiguity and nuclear membrane is integrated. Cell’s chromatin is thick and we also can observe caryocinesia phase.The study groups: it is great different in cells’shapes. Degeneration and necrosis have take placed in many cells. Decomposition have take placed in cytolymph. Cells’nuclear have broken up ,deliquesced or even disappeared.
2 We use MTT method to detect the inhibit condition of LPS on lovo cells. With the time extending and the increasing of the LPS’density , the inhibit function become stronger (P<0.05).
3 Cell cycle’s change: We use PI to deal with the study groups’cells and we find that LPS can change lovo cell’cell cycle in large extent.
4 The expression of bcl-2 gene and NF-κBp65 gene: LPS can lower the expression of bcl-2 gene and NF-κBp65 gene. The more increasing of LPS’density the lower the expression of those two genes.
Conclusions: 1 LPS can inhibit the generation of lovo cells and can enhance the sensitivity of chemical medicine.
2 LPS can down regular the expression of bcl-2 gene and NF-κBp65 gene. Through this method, LPS also can lower the inhibition to lovo cells.
3 LPS can obviously inhibit the function of heparanase and tumor transfusion indirectly.
引文
1. Parish CR,Freeman C,Brown KJ,et a1.Identification of sulfated oligosacchsride-based inhibitors of tumor growth and metastasis using novel vitro for angiogensis and heparanase activity[J].Cancer Res,1999,59(14): 3433~344
2. Miao HQ,Elkin M,Aingorn E,et al.Inhibition of heparanase activity and tumor meyastasis by laminarin sulfate and synthetic phosphorothioate [J].Int Cancer,1999,83(3):424~431
3. Vlodavsky I, Elkin M, Pappo O, et al. Mammalian heparanase as mediator of tumor metastasis and angiogenesis[J].Isr Med Assoc J,2000(Suppl):37~45
4. VlodavskyI,FriedmannY,ElinM.Mammalian.heparnanse:involvement in cancer metastasis ,angiogenesis and normal development. Swmin Cancer Biol. 2002,12(2):121~129
5. Vlodavsky I,Goleshmidt O,Zcharia E.Molicular properties and involvement of heparanase in cancer progression and normal development.Biochimie.2001, (8):831~839
6. Zcharia E,Metzger S, Chajek-Shaul T, Friedmann Y. Molicular properties and involvement of heparanase in cancer progression and mammary gland morphogenesis.J Mammary Gland Biol Neoplasia.2001,6(3):311~322
7. Vlodavsky I,Elink M,Friedmann Y.Mammalian as mediat-or of tumor metastasis and angiogenesis. Isr Med Assoc J.2000,2:37~45
8. El-Asssal ON,Yamanoi A,Ono T.The clinicopathological significance of heparanase and basic fibroblast growth factor expressions in hepatocellular carcinoma.Clin Canc-er Res.2001,7(5):1299~1305
9. Bame KJ, Venkatesan I, Dehdeshti J.Characterization of a novel intracellular heparanase that has Vlodavsky I, Elink M ,Pappo O, et al. Mammalian heparanase as mediator of tumor metastasis and angiogenesis .Isr Med Assoc J,200
10. Pillarisetti S,Paka L,Sasaki A,et al.Endothelial cell heparanase modulation of lipoprotein lipase activity. Evidence that heparin sulfate oligosaccharide and extracellular chaperone. J BiolChem,1997,272(25):15753~ 157590
11. Marchetti D, Reiland J, Erwin B. Ihibition of heparanase activity and heparanase-induced angiogenesis by suramin analogues. Iht J Cancer.2003 ,104(2):167~174
12. Lever R, Page CP.Novel drug development opportunities for heparin .Nat Rev Drug Didcov.2002,1(2):140~148
13. Irony-Tur-Sinai M,Vlodavsky I,Ben-Sasson SA.A synthetic heparin-mimicking polyanionic compound inhibits central nercous system inflammation. J Neurol Sci.2003,15,206(1):49~57
14. Harley R,Gruffydd-Jones TJ,Day MJ.Non-specific labeli ng of mast cells in feline oral muucosa-a potential problem in immunohistocchemical studies.J Comp PATHOL,2002, 127(2-3):228~231
15. Vlodavsky I,Friedmann Y,Elkin M.Mammalian heparri-nase: gene cloning, expression and function in tumor progression and metastasis. Nat Med. 1999,5(7): 793~802
16. Hernadez F,Perez M,Lucsa JJ, et al .Sulfo-glycosami- noglycan content affects PHF-tau solubility and alloes the identification of different types of PHFd.BrainRes, 2002, 935(1-2):65~72
17. Miao HQ,Elkin M,Aingorn E,et al. Inhibition of heparanse activity and tumor metastasis by laminarin sulfate and synthetic phosphorothioate. Int J Cancer,1999,83(3):424~ 431
18. D'Acquisto F, May MJ, Ghosh S. Inhibition of Nuclear Factor Kappa B (NF-B): An Emerging Theme in Anti-Inflammatory Therapies. Mol Interv. 2002 Feb;2 (1):22~35
1 徐宁,丁彦青,徐莉等.Familiar adenomatosis polyposis. Chinese Journal of Cancer Reseach, 1999,11(1):55
2 李学农,丁彦青,周军.3, 4' , 5-三羟基芪-3-β-单-D-葡萄糖苷对大肠癌细胞系蛋白激酶 C 的抑制效应.世界华人消化杂志,2001,9(2):198~201
3 孙青,丁彦青.肿瘤微转移检测方法研究进展,国外医学肿瘤学分册,1998,25(3):143
4 Vlodavsky I,Friedmann Y.Molecular properties and invol- vement of heparanase in cancer metastasis and angiogenes- is.J Clin Invest,2001 ,108(3):341~347
5 Vlodavsky I,Friedmann Y,Elkin M.Mammalian heparanase :gene cloning,expression and function in tumor progression and metastasis.Nat Med, 1999 ,5(7):793~802
6 Vlodavsky I, Elkin M, Peretz T, Friedmann Y. Mammalian heparanase: involvement in cancer metastasis, angiogenesis and normal development.Semin Cancer Biol, 2002 , 12 (2):121~129
7 Eccles SA.Heparanase: breaking down barriers in tumors.Nat Med,1999,5(7):735~736
8 Assal N,Yamanoi A,Ono T ,et al.The clinicopathological significance of heparanase and basic fibroblast growth factor expressions in hepatocellular carcinotma.Clin Cancer Res,2001,7(5):1299~1305
9 Fairbanks M,ildner AM,Leone JW.et al Processing hepara- nase precursor and evidence that the active. Enzyme is a hererodimer.J Biol Chem.1999,274(42):29587~27590
10 Iozzo RV Matrix proteoglycans: From molecular design to cellular function . Annu Rev Biochem, 1998, 67: 609~ 652
11 Piccolo P,Iqbal O,Demir M.et al.Global anticoagulant effects of a novel sulfated pentomanan ologosaccharide mixture.Clin Appl Thromb Hemost,2001,7(2):149~152
12 Montalto P. Vlachogiannakos J. Cox DJ. et al. Bacterial infection in cirrhosis impairs coagulation by a heparin effect:a prospective study.J Hepatol,2002,37(4):463~470
13 Eccles SA. Nat Med,1999,5(7):735~736
14 Kim HH,Lee WS,Yang JM, et al.Basic peptide system forefficient delivery of foreign genes.Biochim Biophys Acta,2003,1640(2-3):129~136
15 Chu JJH,Ng ML.Characterization of a 105-kDa plasma membrane associated glycoprotein,that is involved in West Nile virus bindin gandinfection .Virology,2003,312(2): 458~469
16 Toyoshima M,Nakajima M,Human heparanase Prificati- on,characterization,cloning,and expression.J Biol Ch- em,1999,274(34):241~253
17 Ihrcke NS ,Parker W,Reissner KJ,et al .Regulation of platelet hepatanase during inflammation:role of pH and proteinases. J Cell Physiol,1998,175(3):25
18 Gliat D,Hershkoviz R,Goldkorn I,et al.Molecular behawior adapts to context:heparanase functions as an extracellrlar matrixdegrading enzue or as a T cell adhesion molecule, depending on the local pH.J Exp Med,1995,181(5):19~29
19 Hulett MD,Freeman C,Hamdorf BJ,et al.Cloning of mama- alian heparanase an important enzyme in tumor invasion and metastasis Med,1999,5(7):803~809
20 Hulett MD,Freeman C,Hamdorf BJ,et al.Cloning of mammalian heparanase an important enzyme in tumor invasion and metastasis Med,1999,5(7):803~809
21 Winson WT.Glomerular matrix accumulation in experim- ental anti-GBMA bglomerulonephritis.Nephron,2002,84 (1):40~50
22 Bentolila A,Vlodavsky I. Ishai MR,Kovalchuk O,Haloun C, Domb AJ.Poly (N-acryl amino acids ): a new class of biologically active polyanions.J Med Chem, 2000,43(13): 2591~2600
23 Harley R,Gruffydd-Jones TJ,Day MJ.Non-specific labeling of mast cells in feline oral muucosa –a potential problem in immunohistochemical studies. J Comp PATHOL,2002, 127(2-3):228~231
24 Vlodavsky I,Elkin M,Pappo O,et al.Mammalian hepara- nase as mediator of tumor metastasis and angiogenesis.Isr Med Assoc J, 2002,2(Suppl):37~45
25 Hernadez F, Perez M, Lucsa JJ, et al. Sulfo-glycosam- inoglycan content affects PHF solubility and alloes the identification of different types of PHF.Brain Res,2002, 935(1-2):65~72
26 Miao HQ,Elkin M,Aingorn E,et al.Inhibition of heparanase activity and tumor metastasis by laminarin sulfate and synthetic phosphorothioate. Int J Cancer ,1999,83(3): 424~431
27 Ikuta M, Podyma KA, Maruyama K. Expression of hepar- anase in oral cancer cell lines and oral cancer tissues.Oral Oncol,2003,37(2):177~184
28 Walch ET,Marchetti D.Role of neurotrophins and neurotro- phins receptors in the in vitro invasion and heparanase prosuction of human prostate cancer cells.Clin Exp Metastais,1999,17(4):307~314
29 Demir ,M,Iqbal O,Hoppensteadt DA,et al.Anticoagualant andantiprotease profiles of a novel natural heparinomimetic mannopentaose phosphate sulfate(PI88) ClinAppl Thro- mb Hemost,2001,79(20);131~140
30 EI-Assal ON,Yamanoi A,Ono T,et al.The clinicopathologi- cal significance of heparanse and basic fibroblast growgh factor expression in hepatocellular carcinoma.Clin Canc- er Res,2001,7(5):1299~1305
31 Parish CR, Freeman C,Brown KJ,et al.Identification of sulfated oligosaccharidel-based inhibitors of tumor growth and metastasis using novel in vitor assays for angiogenesis and heparanase activity.Cancer Res,1999,59(14): 3433~3441
32 Mckenzie E,Tyson K,Stamps A,et al.Cloning and expres- sion profiling if hpa 2,a novel mammalian heparanase family member. Biochem Biophys Res Commun, 2000,27(6):110~117
33 Kuniyasu H,Chihara Y, Kubozoe T,et al. Co-expression of CD44v3 and heparanase is coorelated with mstastasis of human colon cancer.Int J Mol Med, 2002, 10 (3);333
34 Nobuhisa T,Naomoto Y, Ohkawa T, et al. Heparanase expression correlates with malignant potential in human coloncancer J Cancer Res Clin Oncol,2005,131(4): 229~237
35 Yael,Friedmann,Vlodavsky.et al. Expression of heparanase in normal,dysplastic and neoplastic human colonic mucosaand stroma.Evidence for its role in colonictumorigebesis American Jouranl of pathology,2000,157(4):116~117
36 Friedmann Y,Vlodavsky I,Aingorn H.et al.Expression of heparanase in normal. Dysplastic and neoplastic human colon mucosa and atroma.Am J Pathol ,2000,15(7): 1167~1175.
37 Bentolila A,Vlodavsky I,Ishiai MR,et al.Ploy (N-acryiami- on acids):A new class of biologically active polyanions J Med Chem,2000,43:2591~2600
38 Miao HQ,Elink M,Aingorn E,et al .Inhibition of hepara- anase activity and tumor metastasis by laminarin sulfate and synthetic phosphorothioate.In J Cancer,1999,83(3): 424~431
39 Bentolila A,Vlodavsky I. Ishai MR,Kovalchuk O,Haloun C, Domb AJ.Poly (N-acryl amino acids ): a new class of biologically active polyanions.J Med Chem ,2000,43(13): 2591~2600
2. Miao HQ,Elkin M,Aingorn E,et al.Inhibition of heparanase activity and tumor meyastasis by laminarin sulfate and synthetic phosphorothioate [J].Int Cancer,1999,83(3):424~431
3. Vlodavsky I, Elkin M, Pappo O, et al. Mammalian heparanase as mediator of tumor metastasis and angiogenesis[J].Isr Med Assoc J,2000(Suppl):37~45
4. VlodavskyI,FriedmannY,ElinM.Mammalian.heparnanse:involvement in cancer metastasis ,angiogenesis and normal development. Swmin Cancer Biol. 2002,12(2):121~129
5. Vlodavsky I,Goleshmidt O,Zcharia E.Molicular properties and involvement of heparanase in cancer progression and normal development.Biochimie.2001, (8):831~839
6. Zcharia E,Metzger S, Chajek-Shaul T, Friedmann Y. Molicular properties and involvement of heparanase in cancer progression and mammary gland morphogenesis.J Mammary Gland Biol Neoplasia.2001,6(3):311~322
7. Vlodavsky I,Elink M,Friedmann Y.Mammalian as mediat-or of tumor metastasis and angiogenesis. Isr Med Assoc J.2000,2:37~45
8. El-Asssal ON,Yamanoi A,Ono T.The clinicopathological significance of heparanase and basic fibroblast growth factor expressions in hepatocellular carcinoma.Clin Canc-er Res.2001,7(5):1299~1305
9. Bame KJ, Venkatesan I, Dehdeshti J.Characterization of a novel intracellular heparanase that has Vlodavsky I, Elink M ,Pappo O, et al. Mammalian heparanase as mediator of tumor metastasis and angiogenesis .Isr Med Assoc J,200
10. Pillarisetti S,Paka L,Sasaki A,et al.Endothelial cell heparanase modulation of lipoprotein lipase activity. Evidence that heparin sulfate oligosaccharide and extracellular chaperone. J BiolChem,1997,272(25):15753~ 157590
11. Marchetti D, Reiland J, Erwin B. Ihibition of heparanase activity and heparanase-induced angiogenesis by suramin analogues. Iht J Cancer.2003 ,104(2):167~174
12. Lever R, Page CP.Novel drug development opportunities for heparin .Nat Rev Drug Didcov.2002,1(2):140~148
13. Irony-Tur-Sinai M,Vlodavsky I,Ben-Sasson SA.A synthetic heparin-mimicking polyanionic compound inhibits central nercous system inflammation. J Neurol Sci.2003,15,206(1):49~57
14. Harley R,Gruffydd-Jones TJ,Day MJ.Non-specific labeli ng of mast cells in feline oral muucosa-a potential problem in immunohistocchemical studies.J Comp PATHOL,2002, 127(2-3):228~231
15. Vlodavsky I,Friedmann Y,Elkin M.Mammalian heparri-nase: gene cloning, expression and function in tumor progression and metastasis. Nat Med. 1999,5(7): 793~802
16. Hernadez F,Perez M,Lucsa JJ, et al .Sulfo-glycosami- noglycan content affects PHF-tau solubility and alloes the identification of different types of PHFd.BrainRes, 2002, 935(1-2):65~72
17. Miao HQ,Elkin M,Aingorn E,et al. Inhibition of heparanse activity and tumor metastasis by laminarin sulfate and synthetic phosphorothioate. Int J Cancer,1999,83(3):424~ 431
18. D'Acquisto F, May MJ, Ghosh S. Inhibition of Nuclear Factor Kappa B (NF-B): An Emerging Theme in Anti-Inflammatory Therapies. Mol Interv. 2002 Feb;2 (1):22~35
1 徐宁,丁彦青,徐莉等.Familiar adenomatosis polyposis. Chinese Journal of Cancer Reseach, 1999,11(1):55
2 李学农,丁彦青,周军.3, 4' , 5-三羟基芪-3-β-单-D-葡萄糖苷对大肠癌细胞系蛋白激酶 C 的抑制效应.世界华人消化杂志,2001,9(2):198~201
3 孙青,丁彦青.肿瘤微转移检测方法研究进展,国外医学肿瘤学分册,1998,25(3):143
4 Vlodavsky I,Friedmann Y.Molecular properties and invol- vement of heparanase in cancer metastasis and angiogenes- is.J Clin Invest,2001 ,108(3):341~347
5 Vlodavsky I,Friedmann Y,Elkin M.Mammalian heparanase :gene cloning,expression and function in tumor progression and metastasis.Nat Med, 1999 ,5(7):793~802
6 Vlodavsky I, Elkin M, Peretz T, Friedmann Y. Mammalian heparanase: involvement in cancer metastasis, angiogenesis and normal development.Semin Cancer Biol, 2002 , 12 (2):121~129
7 Eccles SA.Heparanase: breaking down barriers in tumors.Nat Med,1999,5(7):735~736
8 Assal N,Yamanoi A,Ono T ,et al.The clinicopathological significance of heparanase and basic fibroblast growth factor expressions in hepatocellular carcinotma.Clin Cancer Res,2001,7(5):1299~1305
9 Fairbanks M,ildner AM,Leone JW.et al Processing hepara- nase precursor and evidence that the active. Enzyme is a hererodimer.J Biol Chem.1999,274(42):29587~27590
10 Iozzo RV Matrix proteoglycans: From molecular design to cellular function . Annu Rev Biochem, 1998, 67: 609~ 652
11 Piccolo P,Iqbal O,Demir M.et al.Global anticoagulant effects of a novel sulfated pentomanan ologosaccharide mixture.Clin Appl Thromb Hemost,2001,7(2):149~152
12 Montalto P. Vlachogiannakos J. Cox DJ. et al. Bacterial infection in cirrhosis impairs coagulation by a heparin effect:a prospective study.J Hepatol,2002,37(4):463~470
13 Eccles SA. Nat Med,1999,5(7):735~736
14 Kim HH,Lee WS,Yang JM, et al.Basic peptide system forefficient delivery of foreign genes.Biochim Biophys Acta,2003,1640(2-3):129~136
15 Chu JJH,Ng ML.Characterization of a 105-kDa plasma membrane associated glycoprotein,that is involved in West Nile virus bindin gandinfection .Virology,2003,312(2): 458~469
16 Toyoshima M,Nakajima M,Human heparanase Prificati- on,characterization,cloning,and expression.J Biol Ch- em,1999,274(34):241~253
17 Ihrcke NS ,Parker W,Reissner KJ,et al .Regulation of platelet hepatanase during inflammation:role of pH and proteinases. J Cell Physiol,1998,175(3):25
18 Gliat D,Hershkoviz R,Goldkorn I,et al.Molecular behawior adapts to context:heparanase functions as an extracellrlar matrixdegrading enzue or as a T cell adhesion molecule, depending on the local pH.J Exp Med,1995,181(5):19~29
19 Hulett MD,Freeman C,Hamdorf BJ,et al.Cloning of mama- alian heparanase an important enzyme in tumor invasion and metastasis Med,1999,5(7):803~809
20 Hulett MD,Freeman C,Hamdorf BJ,et al.Cloning of mammalian heparanase an important enzyme in tumor invasion and metastasis Med,1999,5(7):803~809
21 Winson WT.Glomerular matrix accumulation in experim- ental anti-GBMA bglomerulonephritis.Nephron,2002,84 (1):40~50
22 Bentolila A,Vlodavsky I. Ishai MR,Kovalchuk O,Haloun C, Domb AJ.Poly (N-acryl amino acids ): a new class of biologically active polyanions.J Med Chem, 2000,43(13): 2591~2600
23 Harley R,Gruffydd-Jones TJ,Day MJ.Non-specific labeling of mast cells in feline oral muucosa –a potential problem in immunohistochemical studies. J Comp PATHOL,2002, 127(2-3):228~231
24 Vlodavsky I,Elkin M,Pappo O,et al.Mammalian hepara- nase as mediator of tumor metastasis and angiogenesis.Isr Med Assoc J, 2002,2(Suppl):37~45
25 Hernadez F, Perez M, Lucsa JJ, et al. Sulfo-glycosam- inoglycan content affects PHF solubility and alloes the identification of different types of PHF.Brain Res,2002, 935(1-2):65~72
26 Miao HQ,Elkin M,Aingorn E,et al.Inhibition of heparanase activity and tumor metastasis by laminarin sulfate and synthetic phosphorothioate. Int J Cancer ,1999,83(3): 424~431
27 Ikuta M, Podyma KA, Maruyama K. Expression of hepar- anase in oral cancer cell lines and oral cancer tissues.Oral Oncol,2003,37(2):177~184
28 Walch ET,Marchetti D.Role of neurotrophins and neurotro- phins receptors in the in vitro invasion and heparanase prosuction of human prostate cancer cells.Clin Exp Metastais,1999,17(4):307~314
29 Demir ,M,Iqbal O,Hoppensteadt DA,et al.Anticoagualant andantiprotease profiles of a novel natural heparinomimetic mannopentaose phosphate sulfate(PI88) ClinAppl Thro- mb Hemost,2001,79(20);131~140
30 EI-Assal ON,Yamanoi A,Ono T,et al.The clinicopathologi- cal significance of heparanse and basic fibroblast growgh factor expression in hepatocellular carcinoma.Clin Canc- er Res,2001,7(5):1299~1305
31 Parish CR, Freeman C,Brown KJ,et al.Identification of sulfated oligosaccharidel-based inhibitors of tumor growth and metastasis using novel in vitor assays for angiogenesis and heparanase activity.Cancer Res,1999,59(14): 3433~3441
32 Mckenzie E,Tyson K,Stamps A,et al.Cloning and expres- sion profiling if hpa 2,a novel mammalian heparanase family member. Biochem Biophys Res Commun, 2000,27(6):110~117
33 Kuniyasu H,Chihara Y, Kubozoe T,et al. Co-expression of CD44v3 and heparanase is coorelated with mstastasis of human colon cancer.Int J Mol Med, 2002, 10 (3);333
34 Nobuhisa T,Naomoto Y, Ohkawa T, et al. Heparanase expression correlates with malignant potential in human coloncancer J Cancer Res Clin Oncol,2005,131(4): 229~237
35 Yael,Friedmann,Vlodavsky.et al. Expression of heparanase in normal,dysplastic and neoplastic human colonic mucosaand stroma.Evidence for its role in colonictumorigebesis American Jouranl of pathology,2000,157(4):116~117
36 Friedmann Y,Vlodavsky I,Aingorn H.et al.Expression of heparanase in normal. Dysplastic and neoplastic human colon mucosa and atroma.Am J Pathol ,2000,15(7): 1167~1175.
37 Bentolila A,Vlodavsky I,Ishiai MR,et al.Ploy (N-acryiami- on acids):A new class of biologically active polyanions J Med Chem,2000,43:2591~2600
38 Miao HQ,Elink M,Aingorn E,et al .Inhibition of hepara- anase activity and tumor metastasis by laminarin sulfate and synthetic phosphorothioate.In J Cancer,1999,83(3): 424~431
39 Bentolila A,Vlodavsky I. Ishai MR,Kovalchuk O,Haloun C, Domb AJ.Poly (N-acryl amino acids ): a new class of biologically active polyanions.J Med Chem ,2000,43(13): 2591~2600