【作者】 陈伟海；

【导师】 徐晓玉；

【作者基本信息】 重庆医科大学 ， 药理学， 2005， 硕士

【摘要】 研究背景 血管生成是指从业已存在的血管中产生新生血管的一个多步骤的复杂过程,可发生在许多生理和病理的过程中,如伤口修复、月经周期、肿瘤生长、糖尿病视网膜病变等。现在研究已经证实肿瘤的生长和转移离不开血管生成,抑制肿瘤血管生成已成为治疗肿瘤的有效途径之一,已有多种化合物及天然产物成份证实有抑制肿瘤血管血管生长的作用。但对于阿魏酸钠及白芍总苷的研究尚未见报道。 肿瘤血管生成的机制现已基本阐明,近年来的研究表明肿瘤微环境中有正负两大类调控因子调节肿瘤血管的生成。最重要的即是血管内皮生长因子(Vascular endothelial growth factor ,VEGF),具有诱导内皮细胞的增殖等多种生物学特性,VEGF 表达与肿瘤微血管计数、肿瘤分级以及生存率有明显相关,抑制 VEGF 的表达可抑制肿瘤血管的生成从而抑制肿瘤的生长。因此我们从 VEGF 入手研究阿魏酸钠抑制血管生成的机制。 目的 研究活血化瘀中药提取物阿魏酸钠及白芍总苷对 H22 小鼠肝癌模型肿瘤生长的抑制作用及对血管生成的抑制作用,并探讨其可能机制。为更加全面准确地掌握活血化瘀药物及其成份对于肿瘤的影响提供实验依据。 重庆医科大学硕士研究生学位论文31. 阿魏酸钠、白芍总苷对小鼠肝癌血管生成的抑制作用将腹水型H22肝癌小鼠的腹水稀释成2×106/ml接种于昆明小鼠的右侧腋下,随机将小鼠分为为阿魏酸钠 200mg/kg/d 组、100mg/kg/d 组、50mg/kg/d 组,白芍总苷 200 mg/kg/d 组、100 mg/kg/d 组、50 mg/kg/d组及生理盐水阴性对照组,自接种后第二天按不同的分组腹腔注射上述药物,连续用药 14d 后处死动物,测定瘤块大小及重量。用免疫组化染色标记肿瘤微血管,记数微血管密度、检测血管内皮生长因子VEGF、增殖细胞核抗原(PCNA)的表达。采用 RT-PCR 法检测 VEGF的转录。2.体外检测阿魏酸钠对内皮细胞及肿瘤细胞的作用采用 MTT 法观察阿魏酸钠对内皮细胞(ECV304)和 H22 小鼠肝癌细胞增殖的影响。在 96 孔板内按每孔接种 1×104 个内皮细胞或 H22小鼠肝癌,并按 10μg/ml、20μg/ml、40μg/ml、80μg/ml、160μg/ml 的浓度梯度加入阿魏酸钠,按 MTT 法常规操作,在全自动酶标仪上检测光密度(OD),并计算半数致死浓度。观察阿魏酸钠对 H22 肿瘤细胞诱导内皮细胞增殖的影响。首先制备内皮细胞的条件培养液,将体外培养的 H22 细胞培养在含阿魏酸钠的培养液中,各组培养液的药物浓度分别为:10μg/ml、20μg/ml、40μg/ml、80μg/ml、160μg/ml 的,对照组不含阿魏酸钠,继续孵育 48h;然后各组无菌收集培养液,3000 转离心 30min,除去细胞及残渣,收集上清液,0.22μm 滤膜无菌过滤。用条件培养液在 96 孔板内培养内皮细胞 24h,并按上述 MTT 法检测条件培养液对内皮细胞增殖的影响 。3.RT-PCR 检测阿魏酸钠各剂量组及对照组小鼠肿瘤组织 VEGFmRNA 表达的差异。结果重庆医科大学硕士研究生学位论文41.阿魏酸钠各剂量组小鼠肿瘤生长较对照组慢。各剂量组均能抑制肿瘤生长,200mg/kg/d 的剂量对小鼠肿瘤的抑瘤率为 55.61%(p<0.05),白芍总苷各剂量组小鼠肿瘤生长与对照组之间没有统计意义。2.阿魏酸钠各剂量组的小鼠肿瘤组织微血管密度和对照组相比较均显著降低(p<0.05)。其中,阿魏酸 200mg/kg/d 剂量组对血管的抑制作用最强,明显强于阿魏酸钠 100mg/kg/d 和 50mg/kg/d(p<0.05)。白芍总苷各剂量组之间及与对照组之间没有统计意义。3.免疫组织化学分析显示,阿魏酸各组小鼠肿瘤组织 VEGF 阳性细胞明显少于对照组(p<0.05),白芍总苷各剂量组与对照组之间无显著差异。阿魏酸各剂量组小鼠肿瘤 PCNA 阳性细胞率明显低于对照组(p<0.05),白芍总苷各剂量组与对照组相比无显著性差异。4.体外试验表明,阿魏酸钠对 H22 肿瘤细胞及内皮细胞(ECV304)没有抑制作用,但阿魏酸钠能抑制 H22 肿瘤细胞诱导的内皮细胞增殖(p<0.05),抑制率呈剂量依赖性增加,半数抑制浓度为 18.34μg/ml 。5.RT-PCR 显示阿魏酸钠能抑制小鼠肿瘤组织 VEGF mRNA 表达 。结论阿魏酸钠能抑制 H22 小鼠肿瘤的生长及肿瘤细胞的增殖,并能抑制肿瘤血管生长。阿魏酸钠对体外培养的内皮细胞(ECV304)的增殖没有抑制作用,对体外培养的 H22 肿瘤细胞也没有直接的细胞毒作用。但体外实验显示阿魏酸钠能抑制 H22 肿瘤细胞诱导的内皮细胞增殖。进一步实验表明,阿魏酸钠具有抑制 VEGF mRNA 的转录,降低了VEGF 蛋白的表达的药理作用。因些,阿魏酸钠抑制小鼠 H22 肿瘤生长及其血管生成的机制可能是通过抑制 VEGF mRNA 的转录,降低了VEGF 蛋白的表达,阻碍了内皮细胞的增殖,从而抑制了肿瘤血管生成 。更多还原

【Abstract】 Background Angiogenesis,the development of new blood vessels from the endothelium of a preexisting vasculature,is an important component of wound healing,embryonic vascular development as well as pathological processes such as tumor growth,diabetic retinopathies.It has been evidenced that angiogenesis is essential for tumor growth,invasion and metastasis.Antiangiogiogensis has been the one of the effective methods of antitumor. As we know,many compounds have proved effective to inhibit the growth of tumor. The mechanization have been mainly realized.Although a wide range of angiogenesis stimulators and inhibitors have been identified,the most of importance is vascular endothelial growth factor(VEGF),which has many biological characters such as inducing endothelial cell proliferation.VEGF expression is associated with tumor blood growth,and tumor can be inhibited by suppressing VEGF expression. Objectives This study was initiated to determined whether sodium ferulate and total glucosides of pacony inhibit angiogenesis of H22 tumor growing in the mice and to explore its action mechanism,which could give a preliminary experimental basis for antiangiogenesis of Traditional Chinese Drug for promoting Blood circulation to remove blood stasis. Methods Chapter 1 Animal Studies Cells of the tumor H22 were jnjected s.c. into the right flanks(2×106/ml ). Animals were randomized for therapy on the second day and treated with sodium ferulate 200,100,50mg/kg/d , total glucosides of pacony 200,100,50mg/kg/d as well as sodium as control group for 14 days.Then,the volume,weight,tumor microvessel density were determinated. Chapter 2 Endothelial and Tumor Cell Proliferation Assay Vascular endothelial cells(ECV304) and H22 cells. Cells were incubated with sodium ferulate in various concentrations, the number of metabolically active cells was determined by MTT assay and calculate IC50,and then it was investigated whether the tumor cells deduced vascular endothelial cells proliferating can be inhibited by SF . Chapter 3 RT-PCR RT-PCR was employed to assay the expression of VEGF mRNA in mice tumor treated with various concentrations of sodium ferulate. Results Treatments with total glucosides of pacony in any concentrations were ineffective on mice tumor growth.In contrast,injections of sodium ferulate reduced tumor volume significantly(p<0.05). Immunohistochemical analysis revealed that treatment with sodium ferulate inhibited expression of VEGF(p<0.05),leading to a decrease in microvessel density,which also decreased the staining of proliferating cell nuclear antigen within tumor.In addition,it can not be proved that the groups treated with total glucosides of pacony are different significantly compared with control group. In vitro,sodium ferulate did not inhibt the proliferation of vascular endothelial cells and H22 tumor cells.However, the ability of H22 tumor cells deducing the proliferation of vascular endothelial cells can be reduced by sodium ferulate(p<0.05).And the IC50 is 18.34μg/ml. The expression of VEGF gene was inhibited obviously by sodiumferulate(p<0.05). Conclusions Total glucosides of pacony can not inhibit the growth of H22 tumor,angiogenesis as well as the expression of VEGF. Sodium ferulate can inhibited the expression of VEGF mRNA and angiogenesis in tumor,which leading to suppressing the growth of tumor in mice. Sodium ferulate can not inhibit the proliferation of vascular endothelial cell and H22 tumor cell directly,its ability of inhibiting of tumor growth depend on reducing the expression of VEGF. Sodium ferulate can decrease the ability of tumor cell deducing the proliferation of vascular endothelail cell. Innovation and significance We first report sodium ferulate can inhibit the growth of tumor,and give the preliminary mechanism that Sodium Ferulate take effect by the means of antiangiogenesis caused by expression of VEGF being suppressed.This study enlarges the pharmacological effect of sodium ferulate and gives the expremental basis for its clinical applications of a更多还原