重楼、白头翁等中药与汉防己甲素抗血管生成作用的筛选及机理研究
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摘要
第一部分具有抗血管生成作用的中药筛选
摘要：目的筛选重楼、白头翁等14种中药醇提物体外对人肝癌HepG2、SMC7721细胞株，人肠癌Lovo、SW-116细胞株，人胃癌AGS、BGC-823细胞株，人食管癌CaEs-17细胞株和人脐静脉内皮细胞有明显差异细胞毒性的中药。方法采用MTT法测定了14种中药醇提物对人肝癌、胃癌、大肠癌、食管癌细胞和人脐静脉内皮细胞增殖的抑制作用，采用生长曲线测定法检测14种中药醇提物体外培养肿瘤细胞和内皮细胞的差异性细胞毒作用。筛选出中药最敏感的肿瘤细胞株及对内皮细胞有差异细胞毒性的中药。结果人大肠癌Lovo细胞对于药物最为敏感。重楼、白头翁醇提物和汉防己甲素在对肿瘤细胞亚细胞毒浓度下作用于内皮细胞有明显的增殖抑制作用(P值分别为0．003、0．006、0．024)，且药物对内皮细胞增殖抑制48h时作用最明显，并有较好的剂量依赖关系。结论重楼醇提物在15-60μg/ml浓度、白头翁醇提物在2-8μg/ml浓度和汉防己甲素在2-8μg/ml浓度对肿瘤细胞和内皮细胞有明显的差异性细胞毒作用，药物对内皮细胞增殖作用的抑制48小时最明显，并有较好的剂量依赖关系。
第二部分重楼、白头翁醇提物和汉防己甲素体外对血管内皮细胞生物学行为的影响
目的研究重楼、白头翁醇提物和汉防己甲素对血管内皮细胞迁移、细胞周期、细胞凋亡等方面的作用。方法利用transwell小室趋化实验、体外小管形成实验观察药物对HUVEC迁移、成血管能力的影响。应用AnnexinV荧光染色和流式细胞仪(FCM)检测药物作用后内皮细胞凋亡的发生和细胞周期的变化。结果重楼醇提物(15-60μg／ml)、白头翁醇提物(2-8μg／ml)和汉防己甲素(2-8μg／ml)作用24h后内皮细胞小管数目减少，而且管腔不完整，与对照组相比有显著性差异，且对人脐静脉内皮细胞小管形成能力呈剂量依赖性抑制，相关系数分别为-0.934、-0.982、-0.931，P值分别为0.000、0.000、0.000。药物作用12h后细胞迁移数与对照组相比有显著性差异，且对人脐静脉内皮细胞的迁移能力呈剂量依赖性抑制，相关系数分别为-0.933、-0.929、-0.933，P值分别为0.000、0.000、0.000。重楼(60μg／ml)、白头翁醇提物(8μg／ml)和汉防己甲素(8μg／ml)在48h时可明显诱导人脐静脉内皮细胞凋亡。在药物作用24h时重楼醇提物、汉防己甲素能阻滞细胞周期停滞在G_0-G_1期，白头翁醇提物阻滞细胞周期停滞在G_2-M期。结论重楼、白头翁醇提物和汉防己甲素在体外能有效抑制血管生成，其机制可能与阻断内皮细胞迁移、小管形成和诱导内皮细胞凋亡、阻滞内皮细胞增殖周期有关。
第三部分重楼、白头翁醇提物和汉防己甲素对鸡胚体内血管生成的作用研究
目的进一步了解重楼醇、白头翁醇提物和汉防己甲素体内抗血管生成作用。方法引用鸡胚尿囊膜((chickchorio allantoic membrane，CAM)模型观察重楼醇、白头翁醇提物和汉防己甲素对血管生成的作用。结果CAM中加入药物后72h载体周围血管生长受抑制，出现大小不等的无血管区。重楼醇提物在15-60μg／ml浓度、汉防己甲素在2-8μg／ml浓度、白头翁醇提物在2-8μg／ml浓度时对鸡胚尿囊膜血管形成具有抑制作用。结论重楼醇、白头翁醇提物和汉防己甲素在体内有抑制鸡胚尿囊膜血管生成作用。
第四部分重楼、白头翁醇提物和汉防己甲素对KM小鼠H22细胞移植瘤的作用
目的研究重楼、白头翁醇提物和汉防己甲素体内对KM小鼠皮下移植瘤的作用。方法体外在鼠H22肝癌细胞株和S180肉瘤细胞株中筛选出对上述中药敏感的H22肝癌细胞，建立H22荷瘤KM小鼠模型，分别给予重楼、白头翁醇提物和汉防己甲素灌胃治疗10天，观察用药后肿瘤的重量及肿瘤微血管密度(MVD)的变化。结果2g／kg、4g／kg重楼醇提物治疗组H22移植瘤重与生理盐水(NS)对照组相比明显减轻(P值分别为0.012、0.001)。2g／kg、4g／kg重楼醇提物治疗组H22移植瘤微血管计数与NS对照组相比明显减少(P值分别为0.015、0.001)。80mg／kg汉防己甲素治疗组H22移植瘤重与NS对照组相比明显减轻(P值为0.017)。40mg／kg、80mg／kg汉防己甲素治疗组H22移植瘤微血管计数与NS对照组相比明显减少(P值分别为0.037、0.003)。20g／kg白头翁醇提物治疗组H22移植瘤重与生理盐水对照组相比明显减轻(P值为0.010)。10／kg、20g／kg白头翁醇提物治疗组H22移植瘤微血管计数与NS对照组相比明显减少(P值分别为0.037、0.010)。结论重楼醇提物、白头翁醇提物及汉防己甲素体内对KM小鼠H22移植瘤具有明显抗肿瘤作用；同时可明显抑制肿瘤组织微血管密度，推测其抗肿瘤作用可能与血管生成抑制有关。
第五部分重楼、白头翁醇提物和汉防己甲素对裸鼠Lovo细胞移植瘤的作用
目的研究重楼、白头翁醇提物和汉防己甲素体内对裸鼠Lovo细胞皮下移植瘤的作用。方法建立人大肠癌细胞Lovo移植瘤裸鼠模型，分别给予重楼、白头翁醇提物和汉防己甲素灌胃治疗14天，观察用药后肿瘤体积、肿瘤重量、肿瘤微血管密度(MVD)和裸鼠体重变化。结果4g／kg重楼醇提物治疗组Lovo移植瘤重与NS对照组相比明显减轻(P值为0.017)。2g／kg、4g／kg重楼醇提物治疗组Lovo移植瘤微血管计数与NS对照组相比明显减少(P值分别为0.035、0.003)。20g／kg白头翁醇提物治疗组Lovo移植瘤重与NS对照组相比明显减轻(P值为0.036)。10g／kg、20g／kg白头翁醇提物治疗组Lovo移植瘤的微血管计数与NS对照组相比明显减少(P值分别为0.043、0.033)。80mg／kg汉防己甲素治疗组Lovo移植瘤重与NS对照组相比明显减轻(P值为0.040)。80mg／kg汉防己甲素治疗组Lovo移植瘤的微血管计数与NS对照组相比明显减少(P值为0.035)。10g／kg、20g／kg白头翁醇提物与2g／kg、4g／kg重楼醇提物和80mg／kg汉防己甲素用药后Lovo荷瘤裸鼠体重无明显改变。结论重楼醇提物、白头翁醇提物及汉防己甲素体内对裸鼠Lovo移植瘤具有明显抗肿瘤作用；且可明显抑制肿瘤组织微血管密度，推测其抗肿瘤作用可能与血管生成抑制有关。
第六部分重楼、白头翁醇提物和汉防己甲素对裸鼠Lovo移植瘤血管生成基因表达的芯片分析
目的了解重楼、白头翁醇提物和汉防己甲素对裸鼠Lovo细胞移植瘤肿瘤组织血管生成相关的基因表达的变化。方法采用基因芯片技术，检测重楼、白头翁醇提物和汉防己甲素对裸鼠Lovo细胞移植瘤组织血管生成相关基因的影响，从基因水平进一步探讨其作用的靶点。结果在裸鼠Lovo细胞移植瘤肿瘤组织中，13种血管生成相关基因受到了调节，其中表达下调的基因有10种，表达上调的基因有3种。4g／kg的重楼醇提物作用后的裸鼠Lovo细胞移植瘤肿瘤组织上调的抑血管基因有3种：Ⅳ型胶原α3(COL4A3)上调3倍，趋化因子配体9(CXCL9)上调4倍，血小板反应蛋白1(THBS1)上调10倍；下调的促血管基因有3种：趋化因子配体3(CXCL3)下调2.7倍，肝素酶(HPSE)下调113倍，分化抑制因子1(ID1)下调12倍。80mg／kg的汉防己甲素作用后的裸鼠Lovo细胞移植瘤肿瘤组织下调的促血管基因有7种：血管生成素样蛋白4(ANGPTL4)下调3.8倍。碱性成纤维细胞生长因子2(FGF2)下调5.5倍，成纤维细胞生长因子6(FGF6)下调2.4倍，c-fos引导生长因子(FIGF)下调3.8倍，肝素酶(HPSE)下调4.8倍，层粘连蛋白α5(LAMA5)下调2.9倍，血管内皮生长因子B(VEGFB)下调7.9倍。20g／kg的白头翁醇提物作用后的裸鼠Lovo细胞移植瘤肿瘤组织下调的促血管基因有3种：血管生成素样蛋白4(ANGPTL4)下调3.6倍，趋化因子配体2(CCL2)下调4.5倍，血管肉皮生长因子B(VEGFB)下调7.7倍。结论重楼、白头翁醇提物和汉防己甲素可通过不同靶点调控裸鼠Lovo细胞移植瘤肿瘤组织血管生成相关基因的表达。
Part One Screen Traditional Chinese Medicine Which Having Anti-angiogenesis Effect
Objectives To screen Traditional Chinese Medicine composition of14 alcohol extracts of Traditional Chinese Medicine, paridis and pulsatillae, obviouslydifference cyotoxic on human hepatoma HepG2, SMC7721 cell strain; human colon carcinomaLovo, SW-116 cell strain; human gastric carcinoma AGS, BGC-823 cell strain; humanesophageal carcinoma CaEs-17 cell strain and endothelial cell (EC) of human umbilical veinin vitro. Methods The MTT assay had been developed for quantitative evaluation of theinhibited effect of 14 alcohol extracts of Traditional Chinese Medicine on proliferation ofhuman cell strains of hepatoma、colon carcinoma、gastric carcinoma、esophageal carcinomaand EC of human umbilical vein. Used growth curve assay to detect the variability cell toxicaction to tumour cell and EC which was cultured in vitro by 14 alcohol extracts of TraditionalChinese Medicine. Screened for the most sensitive cell strain to the Traditional ChineseMedicine and for the Traditional Chinese Medicine which had different cytotoxic to EC.Results The most sensitive cell to the medicine was human colon carcinoma Lovo cell.Alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae could obviously inhibitthe proliferation of EC in deuto-cytotoxic concentration (P=0.003、0.006、0.024). Effect wasmost obviously at 48h, and had fine dose-dependent relationship. Conclusion Alcoholextract of paridis in 15-60μg/ml, tetrandrine in 2-8μg/ml and alcohol extract of pulsatillae in2-8μg/ml had obviously variability cell toxic action to tumour cell and EC, the inhibitory effectto EC proliferation was most obviously at 48h, and had fine dose-dependent relationship.
Part Two Alcohol Extract of Paridis, Puisatillae and Tetrandrine Effect to The BiologicalBehaviour of Vascular Endothelial Cell in Vitro
Objectives To investigate the effect of migration, cell cycle andapoptosis mechanism of alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillaeto vascular endothelial cell. Methods Used transwell cabin test and out-body canaliculizationtest to observe the alcohol extract of paridis impact the migrational and vaso-formed ability ofHUVEC. And used Annexin V fluorescein stain and flow cytometry to detect the developmentof apoptosis and the variation of the cell cycle after drugs used to the EC. Results While thealcohol extract of paridis in 15-60μg/ml, tetrandrine in 2-8μg/ml and alcohol extract ofpulsatillae in 2-8μg/ml effected at 24 hours to the EC, the number of the tubule was reducedand lumina became unintegrated. There was significant difference compared with the controlgroup, and the inhibition was dose-dependent to canaliculization of human umbilical veinendothelial cell, R=-0.934,-0.982,-0.931; P=0.000,0.000,0.000. The number of cell migrationhad obviously different compared with the control group after medicine effected for 12 hours,and the capability of HUVEC migration was dose-dependent, R=-0.933,-0.929,-0.933;P=0.000,0.000,0.000. And when alcohol extract of paridis in 60μg/ml, tetrandrine in 8μg/mland alcohol extract of pulsatillae in 8μg/ml at 48h, it could obviously induce human umbilicalvein EC apoptosis. The alcohol extract of paridis and tetrandrine could block the cell cycle andarrest at G0-G1 after 24 hours, and the alcohol extract of pulsatillae arrest at G2-M after 24hours. Conclusion The alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillaecould actively suppress angiogenesis in vitro and the mechanism may related to block themigration, canaliculization of the EC and induce the EC apoptosis, suppress the endothelial cellcycle.
Part Three Research The Mechanism of Alcohol Extract of Paridis, Pulsatillae and Tetrandrine toAnti-angiogenesis of The Chick Embryo in Vivo
Objectives To further understand the effect of anti-angiogenesis of thealcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae in vivo. MethodsUsed chickchorio allantoic membrane (CAM) model to observe the effect of anti-angiogenesisof the alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae. ResultsSurrounding the carrying agent of CAM the blood vessel was suppressed after added medicinefor 72h, then there appeares avascular area that inequality of size. There was inhibitory actionto the CAM angiogenesis when the density of the alcohol extract of paridis is between15-60ng/ml, tetrandrine between 2-8ng/ml and alcohol extract of pulsatillae between2-8ng/ml.Conclusion The alcohol extract of paridis, tetrandrine and alcohol extract ofpulsatillae could inhibit CAM angiogenesis in vivo.
Part Four The Effect of Alcohol Extract of Paridis, Pulsatillae and Tetrandrine on Bearing H22Tumor Mice
Objectives To study the antiangiogenic effect of alcohol extract ofparidis, pulsatillae and tetrandrine in vivo on bearing tumor Mice. Methods We discoveredthat the drugs were more sensitive to H22 cell line than S180 cell line in vitro and selected H22cell line to establish H22 transplantation tumor mice models, then intragastric administrationthe alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae. To observe weightmagnitude and microvessel density of the tumor after management. Results The tumorweight of the alcohol extract of paridis in 2g/kg and 4g/kg were lighter than NS control group(P=0.012, 0.001). The microvessel density of the alcohol extract of paridis in 2g/kg and4g/kg were fewer than NS control group (P=0.015, 0.001). The tumor weight of tetrandrinein 80mg/kg were lighter than NS control group (P=0.017). The microvessel density oftetrandrine in 40mg/kg and 80mg/kg were fewer than NS control group(P=0.015,0.001). Thetumor weight of alcohol extract of pulsatillae in 20g/kg wre lighter than NS control group (P=0.01). The microvessel density of alcohol extract of pulsatillae in 10g/kg and 20g/kg werefewer than NS control group(P=0.037,0.010). Conclusion This experiment showed that thealcohol extract of paridis, alcohol extract of pulsatillae and tetrandrine had antitumor ability invivo on H22 transplantation tumor. They also inhibited tumor MVD. So we presume that theantitumor ability was correlate with the antiangiogenic effect.
Part Five The Effect of Alcohol Extract of Paridis, Pulsatillae and Tetrandrine on Nude MiceBearing Human Colon Carcinoma (Lovo)
Objectives To study the effect of alcohol extract of paridis,pulsatillae and tetrandrine in vivo on nude mice bearing Lovo transplantation tumor. MethodsEstablished Lovo transplantation tumor nude mouse models, then intragastric administrationthe alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae. To observe grosstumor volume, weight magnitude, microvessel density of the tumor and body weight aftermanagement. Results The tumor weight of the alcohol extract of paridis in 4g/kg was lighterthan NS control group (P=0.017). The microvessel density of the alcohol extract of paridis in2g/kg and 4g/kg were fewer than NS control group (P=0.035, 0.003). The tumor weight ofalcohol extract of pulsatillae in 20g/kg was lighter than NS control group (P=0.036). Themicrovessel density of alcohol extract of pulsatillae in 10g/kg and 20g/kg were fewer than NScontrol group (P=0.043,0.033). The tumor weight of tetrandrine in 80mg/kg was lighter thanNS control group (P=0.040). The microvessel density oftetrandrine in 80mg/kg was fewerthan NS control group (P=0.035).The body weight of bearing Lovo transplantation tumornude mouse post-treatment with alcohol extract of pulsatillae (10g/kg,20g/kg), alcohol extractof paridis (2g/kg,4g/kg) and tetrandrine (80mg/kg) had no significant change. ConclusionThis experiment showed that the alcohol extract of paridis, alcohol extract of pulsatillae andtetrandrine had antitumor ability in vivo on Lovo transplantation tumor. They also inhibitedtumor MVD. So we presume that the antitumor ability was correlate with the antiangiogenic effect.
Part Six Differential Angiogenesis Related Genes Expression of Alcohol Extract of Paridis,pulsatillae and Tetrandrine on nude mice bearing Lovo transplantation tumor
Objectives To study the differential angiogenesis related genesexpression of alcohol extract of paridis, pulsatillae and tetrandrine in vivo on nude micebearing Lovo transplantation tumor. Methods We used microarray analysis to detect thedifferential genes expression of alcohol extract of paridis, pulsatillae and tetrandrine in vivo onnude mice bearing Lovo transplantation tumor.Then we approached the effective target on thelevel of gene deeply. Results 13 angiogenesis related genes were found regulated above 2folds or more by the arrays on the tissue of nude mice bearing Lovo transplantation tumor, andamong them 3 up and 10 down. 3 genes were found up regulated effected by alcohol extract ofparidis (4g/kg): COL4A3 up regulated above 3 folds, CXCL9 up regulated above 4 folds,THBS1 up regulated above 10 folds. 3 genes were found down regulated effected by alcoholextract of pulsatillae (4g/kg): CXCL3 down regulated above 2.7 folds, HPSE down regulatedabove 11.3 folds, ID1 down regulated above 12 folds. 7 genes were found down regulatedeffected by tetrandrine (80mg/kg): ANGPTL4 down regulated above 3.8 folds, FGF2 downregulated above 5.5 folds, FGF6 down regulated above 2.4 folds, FIGF down regulated above3.8 folds, HPSE down regulated above 4.8 folds, LAMA5 down regulated above 2.9 folds,VEGFB down regulated above 7.9 folds. 3 genes were found down regulated effected byalcohol extract of pulsatillae (20g/kg): ANGPTL4 down regulated above 3.6 folds, CCL2down regulated above 4.5 folds, VEGFB down regulated above 7.7 folds. Conclusion Basedon our nude mice bearing Lovo transplantation tumor model, and by oligonucleotide arrays, wefound paridis, pulsatillae and tetrandrine can ragulate the expression of angiogenesis relatedgenes through different target in vivo on nude mice bearing Lovo transplantation tumor.
摘要：目的筛选重楼、白头翁等14种中药醇提物体外对人肝癌HepG2、SMC7721细胞株，人肠癌Lovo、SW-116细胞株，人胃癌AGS、BGC-823细胞株，人食管癌CaEs-17细胞株和人脐静脉内皮细胞有明显差异细胞毒性的中药。方法采用MTT法测定了14种中药醇提物对人肝癌、胃癌、大肠癌、食管癌细胞和人脐静脉内皮细胞增殖的抑制作用，采用生长曲线测定法检测14种中药醇提物体外培养肿瘤细胞和内皮细胞的差异性细胞毒作用。筛选出中药最敏感的肿瘤细胞株及对内皮细胞有差异细胞毒性的中药。结果人大肠癌Lovo细胞对于药物最为敏感。重楼、白头翁醇提物和汉防己甲素在对肿瘤细胞亚细胞毒浓度下作用于内皮细胞有明显的增殖抑制作用(P值分别为0．003、0．006、0．024)，且药物对内皮细胞增殖抑制48h时作用最明显，并有较好的剂量依赖关系。结论重楼醇提物在15-60μg/ml浓度、白头翁醇提物在2-8μg/ml浓度和汉防己甲素在2-8μg/ml浓度对肿瘤细胞和内皮细胞有明显的差异性细胞毒作用，药物对内皮细胞增殖作用的抑制48小时最明显，并有较好的剂量依赖关系。
第二部分重楼、白头翁醇提物和汉防己甲素体外对血管内皮细胞生物学行为的影响
目的研究重楼、白头翁醇提物和汉防己甲素对血管内皮细胞迁移、细胞周期、细胞凋亡等方面的作用。方法利用transwell小室趋化实验、体外小管形成实验观察药物对HUVEC迁移、成血管能力的影响。应用AnnexinV荧光染色和流式细胞仪(FCM)检测药物作用后内皮细胞凋亡的发生和细胞周期的变化。结果重楼醇提物(15-60μg／ml)、白头翁醇提物(2-8μg／ml)和汉防己甲素(2-8μg／ml)作用24h后内皮细胞小管数目减少，而且管腔不完整，与对照组相比有显著性差异，且对人脐静脉内皮细胞小管形成能力呈剂量依赖性抑制，相关系数分别为-0.934、-0.982、-0.931，P值分别为0.000、0.000、0.000。药物作用12h后细胞迁移数与对照组相比有显著性差异，且对人脐静脉内皮细胞的迁移能力呈剂量依赖性抑制，相关系数分别为-0.933、-0.929、-0.933，P值分别为0.000、0.000、0.000。重楼(60μg／ml)、白头翁醇提物(8μg／ml)和汉防己甲素(8μg／ml)在48h时可明显诱导人脐静脉内皮细胞凋亡。在药物作用24h时重楼醇提物、汉防己甲素能阻滞细胞周期停滞在G_0-G_1期，白头翁醇提物阻滞细胞周期停滞在G_2-M期。结论重楼、白头翁醇提物和汉防己甲素在体外能有效抑制血管生成，其机制可能与阻断内皮细胞迁移、小管形成和诱导内皮细胞凋亡、阻滞内皮细胞增殖周期有关。
第三部分重楼、白头翁醇提物和汉防己甲素对鸡胚体内血管生成的作用研究
目的进一步了解重楼醇、白头翁醇提物和汉防己甲素体内抗血管生成作用。方法引用鸡胚尿囊膜((chickchorio allantoic membrane，CAM)模型观察重楼醇、白头翁醇提物和汉防己甲素对血管生成的作用。结果CAM中加入药物后72h载体周围血管生长受抑制，出现大小不等的无血管区。重楼醇提物在15-60μg／ml浓度、汉防己甲素在2-8μg／ml浓度、白头翁醇提物在2-8μg／ml浓度时对鸡胚尿囊膜血管形成具有抑制作用。结论重楼醇、白头翁醇提物和汉防己甲素在体内有抑制鸡胚尿囊膜血管生成作用。
第四部分重楼、白头翁醇提物和汉防己甲素对KM小鼠H22细胞移植瘤的作用
目的研究重楼、白头翁醇提物和汉防己甲素体内对KM小鼠皮下移植瘤的作用。方法体外在鼠H22肝癌细胞株和S180肉瘤细胞株中筛选出对上述中药敏感的H22肝癌细胞，建立H22荷瘤KM小鼠模型，分别给予重楼、白头翁醇提物和汉防己甲素灌胃治疗10天，观察用药后肿瘤的重量及肿瘤微血管密度(MVD)的变化。结果2g／kg、4g／kg重楼醇提物治疗组H22移植瘤重与生理盐水(NS)对照组相比明显减轻(P值分别为0.012、0.001)。2g／kg、4g／kg重楼醇提物治疗组H22移植瘤微血管计数与NS对照组相比明显减少(P值分别为0.015、0.001)。80mg／kg汉防己甲素治疗组H22移植瘤重与NS对照组相比明显减轻(P值为0.017)。40mg／kg、80mg／kg汉防己甲素治疗组H22移植瘤微血管计数与NS对照组相比明显减少(P值分别为0.037、0.003)。20g／kg白头翁醇提物治疗组H22移植瘤重与生理盐水对照组相比明显减轻(P值为0.010)。10／kg、20g／kg白头翁醇提物治疗组H22移植瘤微血管计数与NS对照组相比明显减少(P值分别为0.037、0.010)。结论重楼醇提物、白头翁醇提物及汉防己甲素体内对KM小鼠H22移植瘤具有明显抗肿瘤作用；同时可明显抑制肿瘤组织微血管密度，推测其抗肿瘤作用可能与血管生成抑制有关。
第五部分重楼、白头翁醇提物和汉防己甲素对裸鼠Lovo细胞移植瘤的作用
目的研究重楼、白头翁醇提物和汉防己甲素体内对裸鼠Lovo细胞皮下移植瘤的作用。方法建立人大肠癌细胞Lovo移植瘤裸鼠模型，分别给予重楼、白头翁醇提物和汉防己甲素灌胃治疗14天，观察用药后肿瘤体积、肿瘤重量、肿瘤微血管密度(MVD)和裸鼠体重变化。结果4g／kg重楼醇提物治疗组Lovo移植瘤重与NS对照组相比明显减轻(P值为0.017)。2g／kg、4g／kg重楼醇提物治疗组Lovo移植瘤微血管计数与NS对照组相比明显减少(P值分别为0.035、0.003)。20g／kg白头翁醇提物治疗组Lovo移植瘤重与NS对照组相比明显减轻(P值为0.036)。10g／kg、20g／kg白头翁醇提物治疗组Lovo移植瘤的微血管计数与NS对照组相比明显减少(P值分别为0.043、0.033)。80mg／kg汉防己甲素治疗组Lovo移植瘤重与NS对照组相比明显减轻(P值为0.040)。80mg／kg汉防己甲素治疗组Lovo移植瘤的微血管计数与NS对照组相比明显减少(P值为0.035)。10g／kg、20g／kg白头翁醇提物与2g／kg、4g／kg重楼醇提物和80mg／kg汉防己甲素用药后Lovo荷瘤裸鼠体重无明显改变。结论重楼醇提物、白头翁醇提物及汉防己甲素体内对裸鼠Lovo移植瘤具有明显抗肿瘤作用；且可明显抑制肿瘤组织微血管密度，推测其抗肿瘤作用可能与血管生成抑制有关。
第六部分重楼、白头翁醇提物和汉防己甲素对裸鼠Lovo移植瘤血管生成基因表达的芯片分析
目的了解重楼、白头翁醇提物和汉防己甲素对裸鼠Lovo细胞移植瘤肿瘤组织血管生成相关的基因表达的变化。方法采用基因芯片技术，检测重楼、白头翁醇提物和汉防己甲素对裸鼠Lovo细胞移植瘤组织血管生成相关基因的影响，从基因水平进一步探讨其作用的靶点。结果在裸鼠Lovo细胞移植瘤肿瘤组织中，13种血管生成相关基因受到了调节，其中表达下调的基因有10种，表达上调的基因有3种。4g／kg的重楼醇提物作用后的裸鼠Lovo细胞移植瘤肿瘤组织上调的抑血管基因有3种：Ⅳ型胶原α3(COL4A3)上调3倍，趋化因子配体9(CXCL9)上调4倍，血小板反应蛋白1(THBS1)上调10倍；下调的促血管基因有3种：趋化因子配体3(CXCL3)下调2.7倍，肝素酶(HPSE)下调113倍，分化抑制因子1(ID1)下调12倍。80mg／kg的汉防己甲素作用后的裸鼠Lovo细胞移植瘤肿瘤组织下调的促血管基因有7种：血管生成素样蛋白4(ANGPTL4)下调3.8倍。碱性成纤维细胞生长因子2(FGF2)下调5.5倍，成纤维细胞生长因子6(FGF6)下调2.4倍，c-fos引导生长因子(FIGF)下调3.8倍，肝素酶(HPSE)下调4.8倍，层粘连蛋白α5(LAMA5)下调2.9倍，血管内皮生长因子B(VEGFB)下调7.9倍。20g／kg的白头翁醇提物作用后的裸鼠Lovo细胞移植瘤肿瘤组织下调的促血管基因有3种：血管生成素样蛋白4(ANGPTL4)下调3.6倍，趋化因子配体2(CCL2)下调4.5倍，血管肉皮生长因子B(VEGFB)下调7.7倍。结论重楼、白头翁醇提物和汉防己甲素可通过不同靶点调控裸鼠Lovo细胞移植瘤肿瘤组织血管生成相关基因的表达。
Part One Screen Traditional Chinese Medicine Which Having Anti-angiogenesis Effect
Objectives To screen Traditional Chinese Medicine composition of14 alcohol extracts of Traditional Chinese Medicine, paridis and pulsatillae, obviouslydifference cyotoxic on human hepatoma HepG2, SMC7721 cell strain; human colon carcinomaLovo, SW-116 cell strain; human gastric carcinoma AGS, BGC-823 cell strain; humanesophageal carcinoma CaEs-17 cell strain and endothelial cell (EC) of human umbilical veinin vitro. Methods The MTT assay had been developed for quantitative evaluation of theinhibited effect of 14 alcohol extracts of Traditional Chinese Medicine on proliferation ofhuman cell strains of hepatoma、colon carcinoma、gastric carcinoma、esophageal carcinomaand EC of human umbilical vein. Used growth curve assay to detect the variability cell toxicaction to tumour cell and EC which was cultured in vitro by 14 alcohol extracts of TraditionalChinese Medicine. Screened for the most sensitive cell strain to the Traditional ChineseMedicine and for the Traditional Chinese Medicine which had different cytotoxic to EC.Results The most sensitive cell to the medicine was human colon carcinoma Lovo cell.Alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae could obviously inhibitthe proliferation of EC in deuto-cytotoxic concentration (P=0.003、0.006、0.024). Effect wasmost obviously at 48h, and had fine dose-dependent relationship. Conclusion Alcoholextract of paridis in 15-60μg/ml, tetrandrine in 2-8μg/ml and alcohol extract of pulsatillae in2-8μg/ml had obviously variability cell toxic action to tumour cell and EC, the inhibitory effectto EC proliferation was most obviously at 48h, and had fine dose-dependent relationship.
Part Two Alcohol Extract of Paridis, Puisatillae and Tetrandrine Effect to The BiologicalBehaviour of Vascular Endothelial Cell in Vitro
Objectives To investigate the effect of migration, cell cycle andapoptosis mechanism of alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillaeto vascular endothelial cell. Methods Used transwell cabin test and out-body canaliculizationtest to observe the alcohol extract of paridis impact the migrational and vaso-formed ability ofHUVEC. And used Annexin V fluorescein stain and flow cytometry to detect the developmentof apoptosis and the variation of the cell cycle after drugs used to the EC. Results While thealcohol extract of paridis in 15-60μg/ml, tetrandrine in 2-8μg/ml and alcohol extract ofpulsatillae in 2-8μg/ml effected at 24 hours to the EC, the number of the tubule was reducedand lumina became unintegrated. There was significant difference compared with the controlgroup, and the inhibition was dose-dependent to canaliculization of human umbilical veinendothelial cell, R=-0.934,-0.982,-0.931; P=0.000,0.000,0.000. The number of cell migrationhad obviously different compared with the control group after medicine effected for 12 hours,and the capability of HUVEC migration was dose-dependent, R=-0.933,-0.929,-0.933;P=0.000,0.000,0.000. And when alcohol extract of paridis in 60μg/ml, tetrandrine in 8μg/mland alcohol extract of pulsatillae in 8μg/ml at 48h, it could obviously induce human umbilicalvein EC apoptosis. The alcohol extract of paridis and tetrandrine could block the cell cycle andarrest at G0-G1 after 24 hours, and the alcohol extract of pulsatillae arrest at G2-M after 24hours. Conclusion The alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillaecould actively suppress angiogenesis in vitro and the mechanism may related to block themigration, canaliculization of the EC and induce the EC apoptosis, suppress the endothelial cellcycle.
Part Three Research The Mechanism of Alcohol Extract of Paridis, Pulsatillae and Tetrandrine toAnti-angiogenesis of The Chick Embryo in Vivo
Objectives To further understand the effect of anti-angiogenesis of thealcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae in vivo. MethodsUsed chickchorio allantoic membrane (CAM) model to observe the effect of anti-angiogenesisof the alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae. ResultsSurrounding the carrying agent of CAM the blood vessel was suppressed after added medicinefor 72h, then there appeares avascular area that inequality of size. There was inhibitory actionto the CAM angiogenesis when the density of the alcohol extract of paridis is between15-60ng/ml, tetrandrine between 2-8ng/ml and alcohol extract of pulsatillae between2-8ng/ml.Conclusion The alcohol extract of paridis, tetrandrine and alcohol extract ofpulsatillae could inhibit CAM angiogenesis in vivo.
Part Four The Effect of Alcohol Extract of Paridis, Pulsatillae and Tetrandrine on Bearing H22Tumor Mice
Objectives To study the antiangiogenic effect of alcohol extract ofparidis, pulsatillae and tetrandrine in vivo on bearing tumor Mice. Methods We discoveredthat the drugs were more sensitive to H22 cell line than S180 cell line in vitro and selected H22cell line to establish H22 transplantation tumor mice models, then intragastric administrationthe alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae. To observe weightmagnitude and microvessel density of the tumor after management. Results The tumorweight of the alcohol extract of paridis in 2g/kg and 4g/kg were lighter than NS control group(P=0.012, 0.001). The microvessel density of the alcohol extract of paridis in 2g/kg and4g/kg were fewer than NS control group (P=0.015, 0.001). The tumor weight of tetrandrinein 80mg/kg were lighter than NS control group (P=0.017). The microvessel density oftetrandrine in 40mg/kg and 80mg/kg were fewer than NS control group(P=0.015,0.001). Thetumor weight of alcohol extract of pulsatillae in 20g/kg wre lighter than NS control group (P=0.01). The microvessel density of alcohol extract of pulsatillae in 10g/kg and 20g/kg werefewer than NS control group(P=0.037,0.010). Conclusion This experiment showed that thealcohol extract of paridis, alcohol extract of pulsatillae and tetrandrine had antitumor ability invivo on H22 transplantation tumor. They also inhibited tumor MVD. So we presume that theantitumor ability was correlate with the antiangiogenic effect.
Part Five The Effect of Alcohol Extract of Paridis, Pulsatillae and Tetrandrine on Nude MiceBearing Human Colon Carcinoma (Lovo)
Objectives To study the effect of alcohol extract of paridis,pulsatillae and tetrandrine in vivo on nude mice bearing Lovo transplantation tumor. MethodsEstablished Lovo transplantation tumor nude mouse models, then intragastric administrationthe alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae. To observe grosstumor volume, weight magnitude, microvessel density of the tumor and body weight aftermanagement. Results The tumor weight of the alcohol extract of paridis in 4g/kg was lighterthan NS control group (P=0.017). The microvessel density of the alcohol extract of paridis in2g/kg and 4g/kg were fewer than NS control group (P=0.035, 0.003). The tumor weight ofalcohol extract of pulsatillae in 20g/kg was lighter than NS control group (P=0.036). Themicrovessel density of alcohol extract of pulsatillae in 10g/kg and 20g/kg were fewer than NScontrol group (P=0.043,0.033). The tumor weight of tetrandrine in 80mg/kg was lighter thanNS control group (P=0.040). The microvessel density oftetrandrine in 80mg/kg was fewerthan NS control group (P=0.035).The body weight of bearing Lovo transplantation tumornude mouse post-treatment with alcohol extract of pulsatillae (10g/kg,20g/kg), alcohol extractof paridis (2g/kg,4g/kg) and tetrandrine (80mg/kg) had no significant change. ConclusionThis experiment showed that the alcohol extract of paridis, alcohol extract of pulsatillae andtetrandrine had antitumor ability in vivo on Lovo transplantation tumor. They also inhibitedtumor MVD. So we presume that the antitumor ability was correlate with the antiangiogenic effect.
Part Six Differential Angiogenesis Related Genes Expression of Alcohol Extract of Paridis,pulsatillae and Tetrandrine on nude mice bearing Lovo transplantation tumor
Objectives To study the differential angiogenesis related genesexpression of alcohol extract of paridis, pulsatillae and tetrandrine in vivo on nude micebearing Lovo transplantation tumor. Methods We used microarray analysis to detect thedifferential genes expression of alcohol extract of paridis, pulsatillae and tetrandrine in vivo onnude mice bearing Lovo transplantation tumor.Then we approached the effective target on thelevel of gene deeply. Results 13 angiogenesis related genes were found regulated above 2folds or more by the arrays on the tissue of nude mice bearing Lovo transplantation tumor, andamong them 3 up and 10 down. 3 genes were found up regulated effected by alcohol extract ofparidis (4g/kg): COL4A3 up regulated above 3 folds, CXCL9 up regulated above 4 folds,THBS1 up regulated above 10 folds. 3 genes were found down regulated effected by alcoholextract of pulsatillae (4g/kg): CXCL3 down regulated above 2.7 folds, HPSE down regulatedabove 11.3 folds, ID1 down regulated above 12 folds. 7 genes were found down regulatedeffected by tetrandrine (80mg/kg): ANGPTL4 down regulated above 3.8 folds, FGF2 downregulated above 5.5 folds, FGF6 down regulated above 2.4 folds, FIGF down regulated above3.8 folds, HPSE down regulated above 4.8 folds, LAMA5 down regulated above 2.9 folds,VEGFB down regulated above 7.9 folds. 3 genes were found down regulated effected byalcohol extract of pulsatillae (20g/kg): ANGPTL4 down regulated above 3.6 folds, CCL2down regulated above 4.5 folds, VEGFB down regulated above 7.7 folds. Conclusion Basedon our nude mice bearing Lovo transplantation tumor model, and by oligonucleotide arrays, wefound paridis, pulsatillae and tetrandrine can ragulate the expression of angiogenesis relatedgenes through different target in vivo on nude mice bearing Lovo transplantation tumor.
引文
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