【作者】 吴达；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2010， 博士

【摘要】 肝症是我国常见癌症之一,每年死于肝癌的患者约11万人,占全世界肝癌死亡人数的45%。病因目前尚未完全明了,可能是环境因素、病毒感染、食源性污染、肝炎癌变及免疫功能下降等综合作用的结果。由于发病机理多种多样,防治很难一蹴而就。目前治疗肝癌大多采用化学合成药物,在治疗过程中易产生毒副作用和多药耐药性。天然药物以其毒副作用低和不产生耐药性而引起研究者的高度关注。因此,筛选天然来源的肝癌药物成为药理学领域的研究热点。甘肃棘豆是棘豆属有毒植物品种之一,其分布面积广,营养价值高,但毒性也很强,严重威胁着草原畜牧业的发展。目前,棘豆属植物的防除和综合利用研究已取得了显著的成效。但其药用研究不深入也不系统,本试验以甘肃棘豆生物碱部位为研究对象,选择了较为常见的小鼠移植性肝癌H22和人原发性肝癌QGY-7703两种细胞株,分别进行体内外抗肿瘤作用及其机制方面的研究,获得以下结果:(1)生物碱分别用阳离子交换树脂和碱性环境溶剂提取法得到,采用碘化铋钾、硅钨酸和磷钼酸试剂进行生物碱定性检测,结果显示,所提生物碱部位沉淀反应显强阳性;出碱率碱性环境溶剂提取法(1.75%)高于阳离子交换树脂提取法(1.56%)。(2)研究甘肃棘豆生物碱对小鼠移植性H22肝癌的影响。结果表明,甘肃棘豆生物碱低、中、高剂量组(0.25、0.5、1.0 g/kg体重)均可抑制小鼠实体型与腹水型肝癌的生长,抑瘤率和生命延长率分别为38.6%、43.2%、53.8%和45.1%、55.1%、59.4%。表明甘肃棘豆生物碱具有抑制小鼠移植性肿瘤H22生长的作用。(3)对实体瘤小鼠瘤块进行肉眼观察发现,甘肃棘豆生物碱各剂量组小鼠肿瘤易于剥离,体积较小,紧实,血管不丰富。用高剂量组瘤块制备肿瘤病理切片镜检发现,甘肃棘豆生物碱抑制了肿瘤细胞的生长,部分细胞呈现坏死表现如核固缩、碎裂和溶解。(4)免疫组化SP染色结果表明,甘肃棘豆生物碱干预了增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和抑制肿瘤基因p53蛋白的表达,说明其抗肿瘤作用机理之一在于干预了肿瘤细胞的增殖能力和增殖周期。(5)甘肃棘豆生物碱高剂量组实体瘤小鼠肝、肾眼观色泽质地均正常,无病理学改变。病理切片镜检发现肝、肾细胞排列紧凑,结构清楚。表明甘肃棘豆生物碱剂量达到1.0 g/kg体重时不会引起小鼠毒性反应。(6)对实体瘤小鼠免疫器官胸腺和脾脏指数进行分析,并用放免法测定肿瘤坏死因子(tumor necrosis factor,TNF)和白细胞介素-2(interleukin-2,IL-2)的浓度。结果发现甘肃棘豆生物碱各剂量组均可提高小鼠胸腺指数和脾脏指数以及血清中TNF和IL-2的含量。表明甘肃棘豆生物碱抗肝癌作用与其改善免疫功能有关。(7)细胞毒性试验表明甘肃棘豆生物碱作用48h可显著抑制体外培养的人肝癌QGY-7703细胞的生长,剂量-效应呈正相关,相关系数为0.9886,半数抑制浓度(50% inhibiting concentration,IC50)为18.53μg/mL。但其对人外周血单核细胞抑制作用较弱,表明甘肃棘豆生物碱对细胞具有选择性,能特异性杀死肿瘤细胞。集落形成试验表明甘肃棘豆生物碱能显著降低QGY-7703细胞的增殖能力,且呈剂量-效应正相关性,相关系数是0.9929,IC50值为19.85μg/mL。(8)细胞形态学观察和DNA片段化分析证实甘肃棘豆生物碱诱导QGY-7703细胞发生了凋亡,细胞出现皱缩、变圆,核染色质固缩、碎裂、边集,形成凋亡小体和DNA梯形带等明显的凋亡特征。乳酸脱氢酶活力测定表明甘肃棘豆生物碱作用QGY-7703细胞48h内,细胞主要经历了凋亡性的死亡方式。流式细胞仪检测结果证实了甘肃棘豆生物碱可诱导QGY-7703细胞凋亡。(9)黏附抑制试验结果显示:甘肃棘豆生物碱部位不同剂量的处理组中,以高剂量组(100μg/mL)对细胞与胶原的黏附抑制作用最强,各组与对照组相比,差异具有统计学意义(P<0.05,P<0.01)。Boyden小室体外QGY-7703细胞的侵袭、迁移试验发现,不同浓度的甘肃棘豆生物碱处理细胞后,对QGY-7703肿瘤细胞株的迁移和侵袭能力与对照组相比均有不同程度的降低,呈一定的剂量依赖性,其中,以100μg/mL组对肿瘤细胞的侵袭能力、迁移能力的抑制作用最强,侵袭抑制率为37.83%,迁移抑制率为33.97%,提示甘肃棘豆生物碱对肿瘤的抑制作用不仅表现在抑制肿瘤细胞的生长和诱导细胞调亡,还能影响肿瘤细胞与基质的相互作用,从而抑制肿瘤的浸润转移。(10)Western免疫印迹实验发现甘肃棘豆生物碱下调了B细胞淋巴瘤-2(b-cell lymphoma gene 2,bcl-2)和PCNA的表达量,而凋亡活化基因bax与肿瘤抑制基因p53的表达量增高。说明甘肃棘豆生物碱诱导的凋亡与bcl-2家族、p53和PCNA基因调控有关。本试验多角度较系统地研究了甘肃棘豆生物碱抗肝癌的作用,并从甘肃棘豆生物碱改善机体免疫功能、诱导肿瘤细胞凋亡和抑制肿瘤的浸润转移等多方面探讨了其抗肿瘤的作用机制,为开发甘肃棘豆为新型天然抗癌药物奠定了理论基础。更多还原

【Abstract】 Liver cancer is one of common malignant tumors threatening people health and life. In China every year there are 110 thousand people died from it. The number about occupys 45% of entire world. It may be resulted from environment factor, virus infection, food pollution, pathological changes of liver inflammation and reduction of immune function, and so on. The prevention and cure are difficult because of various pathogenesis. The therapy of liver cancer mostly uses chemosynthesis drugs at present, but side effects and multidrug resistance of chemosynthesis drugs become frequent in treatment. Naturally occurring drugs have been paid close attention by researcher for their low side-effect and no-drug-resistance. So screening naturally occurring drugs of anti-liver cancer becomes the study hotspot of pharmacology field.Oxytropis Kansuensis Bunge is one of toxic whin category plants, which distributes abroad, has high nutrient value and toxicity, and seriously threatens the local stockbreading and causes massive economic losing. Now, the research of whin category plants in prevention or killing off and utilization has made good progress, but its officinal research is not in-depth and not systemic.In our study, we investigated anti-liver cancer activity and its action mechanism of alkaloid from Oxytropis Kansuensis by animal model of transplanted tumor H22 in vivo and QGY- 7703 cells in vitro. The results were as follows.(1) The alkaloid from Oxytropis Kansuensis was extracted by cation resin and basic solvent two methods, which qualitative detect by bismuth potassium iodide, silicotungstic acid and phosphomolybdic acid. The results showed that it is strongly positive of alkaloid precipitation reaction and cation resin and basic solvent two methods compared extraction yield the latter is higher(1.75%)than the former(1.56%).(2) The study on anti-liver cancer activity of alkaloid from Oxytropis Kansuensis showed that alkaloid (0.25, 0.5, 1.0 g/kg body weight) inhibited the growth of liver cancer H22 harboring mice. The tumor inhibition rate of solid tumor harboring mice and the life prolong rate of ascites tumor harboring mice were 38.6%,43.2%,53.8% and 45.1%,55.1%,59.4% separately. It indicated that alkaloid from Oxytropis kansuensis significantly inhibited the growth of liver cancer-transplanted of mice.(3) The observation with eyes on solid tumors showed that tumors of alkaloid groups were stripped easily, tumor volume was small, tumors were compact, and blood vessels were not abundant. Tumor slice in high dose alkaloids group showed that growth of tumor cells was inhibited and some tumor cells appeared necrosis such as pycnosis, breaking to pieces and dissolving of nuclear.(4) The research discussed the anti-tumor mechanism of alkaloid from Oxytropis kansuensis with immunohistochemistry method. The result showed that alkaloid of Oxytropis kansuensis had direct effect on H22 cell, this role was associated with the express of proliferating cell nuclear antigen(PCNA) and p53 protein.(5) The color and texture of liver and kidney in high dose alkaloid group were normality and no pathology change observed with eyes. The slices of liver and kidney indicated that cells of liver and kidney disposed compactly and cells structure was distinct. The results suggested that alkaloid dose up to 1.0 g/kg was no signs of toxicity to tumor harboring mice.(6) The immune organ assay and radioimmunoassay suggested that alkaloid significantly increased the index number of thymus and spleen and the concentration of tumor necrosis factor and interleukin-2 in serum of solid tumor harboring mice. It showed that anti-liver cancer activity of alkaloid from Oxytropis kansuensis was related with improvement of immune function.(7) The growth of cells was inhibited when QGY- 7703 cells cultured in vitro were treated with alkaloid of Oxytropis kansuensis for 48 h. The dose and effect appeared positive correlation. 50% inhibiting concentration (IC50) was 18.53μg/ml. But the cytotoxicity of alkaloid to human peripheral blood mononuclear cells was low. It suggested that cytotoxicity of alkaloid had selectivity and it could kill tumor cells specifically. The experiment of colony formation indicated that alkaloid reduced proliferation activity of QGY- 7703 cells and IC50 was 19.85μg/ml. The relation between dose and effect was positive.(8) The observation of cell morphology and DNA fragmentation analysis verified that alkaloid induced apoptosis of QGY- 7703 cells. The cells appeared apoptosis characteristic such as cells shrinkage and rounding, nucleus chromatin pyknosis, break and gather to nuclear membrane, and apoptotic bodies and DNA ladder-shaped band formation. LDH activity assay demonstrated that apoptosis pathway was mainly responsible for QGY- 7703 cells death treated with alkaloid in 48 h. Flow cytometry analysis confirmed that alkaloid could induce QGY- 7703 cell apoptosis.(9) Compared with the control group the adhesion ability of the QGY-7703 cell line was decreased significantly (P<0.01, P<0.05) after treatment with the alkaloid from Oxytropis kansuensis, the 100μg/ml group on the adhesion inhibiting ability was the most obvious. The Boyden chamber test showed that compared with the control group both the invasion and the motility abilities of the QGY-7703 cell line were decreased significantly(P<0.01)after treatment with the different alkaloid does. The relation between dose and effect was positive. Among the 100μg/ml group on the invasion and the motility inhibiting abilities was the most obvious. The invasion inhibiting rate was 37.83% and the motility inhibiting rate was 33.97%. It make clear that the inhibiting tumor effect not only is the inhibiting effect to the tumor cell itself but also is due to the interrelation between tumor cell and the periplast, which has significant effect to tumor infiltration and migration.(10) Western blot showed that alkaloid up-regulated the expression of bax and p53 protein and down-regulated the expression of b-cell lymphoma gene 2(bcl-2) and PCNA protein. The results above suggested that apoptosis induced by alkaloid of Oxytropis kansuensis was modulated by bcl-2 family, p53 and PCNA together.The study investigated anti-liver cancer effect of alkaloid of Oxytropis kansuensis systematically and discussed the action mechanism from the point of view of organism immunologic function, apoptosis and tumor infiltration and migration. It provided rationale to develop Oxytropis kansuensis as naturally occurring drug of anti-tumor.更多还原