【作者】 胡修明；

【导师】 蒋纪恺；

【作者基本信息】 汕头大学 ， 肿瘤学， 2010， 硕士

【摘要】 背景简述中药苦参味苦性寒,始载于《神农本草经》,主要含苦参碱和氧化苦参碱等生物碱,常被中医临床组方于清热解毒方中用于肿瘤治疗。当前关于苦参碱抗白血病机制的研究主要集中在抑制增殖、诱导分化和凋亡,及逆转耐药等方面,而对细胞活动最基本要素—物质和能量代谢方面作用的研究则很少见于报道。细胞通过物质代谢获取能量支撑生命活动,肿瘤细胞增殖旺盛,需增强糖代谢以获取更多的能量,因此靶向抑制肿瘤细胞旺盛的物质和能量代谢是目前研究肿瘤治疗的选项之一。本研究以白血病K562细胞为研究模型,从抗癌药物靶向作用肿瘤细胞物质和能量代谢的角度,探讨苦参中主要生物碱抗白血病的作用机制。研究目的本研究旨在从糖代谢的角度选取糖酵解关键酶丙酮酸激酶(pyruvate kinase,PK)作为抑制肿瘤增殖的靶点,探讨苦参及所含主要生物碱苦参碱、氧化苦参碱,或苦参碱联合氧化苦参碱抑制白血病细胞增殖、诱导凋亡等可能机制。研究内容首先通过建立高效液相色谱方法,测定苦参碱作用白血病细胞后在细胞培养体系中的含量分布,以便为后续研究提供苦参碱作用浓度和时间的依据。将苦参液、苦参总碱、苦参碱、氧化苦参碱,或苦参碱联合氧化苦参碱作用白血病K562细胞,通过丙酮酸激酶活性测定实验,了解各药物组是否可抑制丙酮酸激酶的活性,以探明各实验组是否通过抑制K562细胞丙酮酸激酶的活性从而阻断糖酵解途径,达到抑制增殖的作用。最后通过测定糖酵解代谢终产物乳酸生成情况进一步验证实验结果。实验方法1.使用HPLC建立苦参碱浓度与检测峰面积对应的标准曲线,然后取对数期生长的K562细胞,加入苦参碱并调整其浓度分别为400μg/mL、200μg/mL作用培养细胞,按不同时间点分别收集培养液和细胞,并裂解细胞,用HPLC法测定培养基和细胞裂解液(即细胞内)的苦参碱含量。2.利用丙酮酸激酶活性测定试剂盒测定苦参碱、氧化苦参碱、苦参碱联合氧化苦参碱各实验组先作用K562细胞一定时间后,再裂解细胞,并测定细胞裂解液中丙酮酸激酶的活性。取对数生长期K562细胞分别加入苦参碱、氧化苦参碱,并调整其浓度分别为400μg/mL和200μg/mL,苦参碱联合氧化苦参碱实验组为苦参碱和氧化苦参碱各自按400μg/mL和200μg/mL等比例加入细胞培养体系,培养3天。然后换入新鲜培养基,调整培养体系中的药物浓度与上述一致,继续培养3天(简称3+3组),或继续培养4天(简称3+4组),取出细胞裂解。按试剂盒操作方法,用紫外分光光度计测定并计算各实验组对丙酮酸激酶的活性的影响。每组均设置阴性对照,且每组都重复培养、测定三次。3.利用丙酮酸激酶活性测定试剂盒直接测定细胞裂解液中丙酮酸激酶的活性。取对数生长期的K562细胞,先裂解细胞,在酶活性测定体系中先分别加入适当浓度的苦参提取液(简称苦参液)、苦参总碱、苦参碱、氧化苦参碱,及苦参碱等比例浓度联合氧化苦参碱等药物,再在各实验组加入含丙酮酸激酶的K562细胞裂解液,按前述同样的方法测定裂解液中丙酮酸激酶的活性。每组均设置阴性对照组,且每组测定三次。4.利用HPLC建立测定培养液中乳酸浓度。取对数生长期的K562细胞,加入苦参碱和氧化苦参碱,使其终浓度分别为400μg/mL、200μg/mL,然后培养3天。取培养基测定其中的乳酸浓度,培养瓶换入新鲜的培养基并重新调整相应的药物浓度与更换前相同,相同条件再次培养24、48h、72小时,取出培养基并利用HPLC测定其中乳酸浓度。每组均设置阴性对照,且每组都重复培养测定三次。实验结果1.培养体系中苦参碱浓度分布:苦参碱作用于K562细胞后,培养基中的苦参碱含量下降,200μg/mL苦参碱处理组下降至190μg/mL左右,400μg/mL苦参碱处理组下降至390μg/mL附近,大约24小时后趋于稳定。与对照组比较,200μg/mL或400μg/mL苦参碱处理的K562细胞裂解液(细胞内)的苦参碱有所升高。2.实验组药物先作用K562细胞,再裂解细胞测定其丙酮酸激酶活性:分别为200μg/mL、400μg/mL苦参碱、氧化苦参碱,及分别为200μg/mL、400μg/mL的苦参碱联合氧化苦参碱作用K562细胞,其丙酮酸激酶的活性抑制率分别为:(1)苦参碱(3+3组) 0.56±0.97%(200μg/mL)、2.66±2.31%(400μg/mL);(3+4组)2.73±2.74%(200μg/mL)、4.06±2.00%(400μg/mL);(2)氧化苦参碱(3+3组) 1.35±0.66%(200μg/mL)、3.11±1.30%(400μg/mL);(3+4组)1.56±0.73%(200μg/mL)、4.31±2.47%(400μg/mL);(3)苦参碱联合氧化苦参碱(3+3组)10.85±3.76%(200μg/mL)、39.96±2.28%(400μg/mL);(3+4组)17.00±2.87%(200μg/mL)、47.78±2.42%(400μg/mL)。实验结果显示与对照组比较,苦参碱联合氧化苦参碱组对K562细胞内丙酮酸激酶活性抑制明显,有统计学意义(P<0.01)。3.实验组药物直接作用细胞裂解液的丙酮酸激酶活性测定:苦参液和苦参总碱对K562细胞裂解后的裂解液中丙酮酸激酶活性抑制率随药物浓度的增加而增加,苦参液和苦参总碱浓度分别从0.4mg/mL增加到2.0mg/mL的过程中,抑制率分别从21.64%、43.57%上升到88.07%和94.10%,对K562细胞裂解液丙酮酸激酶活性有抑制作用(P<0.01),并呈浓度依赖性。而在上述浓度下,苦参碱、氧化苦参碱、等比例的苦参碱联合氧化苦参碱等实验组对K562细胞裂解液丙酮酸激酶活性的抑制并未随药物浓度的增加而增加,只在2.18%到17.56%这个很小的范围内波动,对K562细胞裂解液丙酮酸激酶活性无抑制作用。4.苦参碱联合氧化苦参碱组对K562细胞乳酸生成影响:无论是对照组还是苦参碱联合氧化苦参碱处理组,乳酸产量都在3.2μg/104个细胞/mL左右波动。与对照组比较,分别为200μg/mL和400μg/mL的苦参碱等比例联合氧化苦参碱对K562细胞乳酸生成的差异无统计学意义(P=0.74)。苦参液和苦参总碱实验组对K562细胞乳酸生成的影响,因仪器设备问题未做进一步验证。小结①一定浓度苦参碱作用于K562细胞后,培养基中的苦参碱含量下降,大约24小时后趋于平稳,苦参碱在细胞中的浓度有所升高,提示培养基中苦参碱作用K562细胞一定时间后可能进入细胞发挥作用。②无论200μg/mL、400μg/mL的苦参碱或氧化苦参碱分别单独先加入培养体系中,作用K562细胞一定时间再裂解细胞,测定丙酮酸激酶活性,还是先收集裂解K562细胞,再将上述浓度的苦参碱或氧化苦参碱直接作用裂解液中的丙酮酸激酶,测定丙酮酸激酶活性,结果显示二者均不能抑制丙酮酸激酶活性,提示单独使用苦参碱或氧化苦参碱对K562细胞丙酮酸激酶活性无间接或直接作用。③200μg/mL或400μg/mL苦参碱等比例联合氧化苦参碱加入培养基作用K562细胞一定时间后,收集并裂解细胞,再测定其丙酮酸激酶活性,发现可显著抑制丙酮酸激酶活性。但将苦参碱联合氧化苦参碱加入已经裂解含丙酮酸激酶的K562细胞裂解液中,测定丙酮酸激酶活性,发现其对裂解液丙酮酸激酶活性无抑制作用。提示苦参碱联合氧化苦参碱对丙酮酸激酶活性影响可能主要通过K562细胞间接发挥抑制作用,而对裂解液中丙酮酸激酶活性无直接的作用。④实验发现苦参液、苦参总碱在实验浓度下均可直接抑制K562细胞裂解液中丙酮酸激酶活性,提示苦参所含的其它生物碱可能对丙酮酸激酶活性有直接抑制作用。⑤在本课题建立的实验方法和条件下未发现200μg/mL或400μg/mL的苦参碱联合氧化苦参碱对K562细胞的乳酸生成有显著影响。更多还原

【Abstract】 Background: Matrine and oxymatrine, the major components in Traditional Chinese medicine kuh-seng, are often used clinically in oncotherapy. However, the exactly underlying mechanism remains elusive. Lines of evidence have indicated that the dysregulated glucose metabolism plays an important role for the vigorous proliferation of tumor cells and has also proved to be the potential target for cancer therapy. Previous studies show that matrine treatment inhibits proliferation and induces cell differentiation and apoptosis in leukemia cells. In this study, the effects of matrine on glucose metabolism in human leukemia K562 cells were investigated.Objectives: 1. To determine the content of matrine in the cell lysate and the cell culture supernate in matrine-treated K562 cells. 2. To investigate the changes of pyruvate kinase (PK) activity in K562 cells incubated alone in the presence of matrine(M), oxymatrine(OM), the combination of M with OM (M&OM), kuh-seng alkaloids and Sophora flavescens extracts, respectively. 3. To investigate the changes of the amount of lactic acid in K562 cells exposed to the combination of M and OM.Methods: High pressure liquid chromatography (HPLC) was estabalished to measure the content of matrine in the cell lysate and the cell culture supernate in K562 cells exposed to matrine. The Pyruvate Kinase Assay Kit was used to detect PK activity in K562 cells incubated with the agents and the lysate of K562 cells treated directly by agents. HPLC was adopted to test the amount of lactic acid in K562 cells incubated with the combination of M with OM.Results: 1.The content of matrine in the culture supernate was found to be decreased to mearly 190μg/mL and 390μg/mL respectively in K562 cells exposed to 200μg/mL and 400μg/mL of matrine for 24 h.On the contrary, the content of matrine in the cell lysate increased slightly.2. The inhibition rate of PK activity in K562 cells in the presence of 200μg/mL or 400μg/mL of M, the same dose of OM, and the combination of M and OM (M& OM) for 6 days or 7 days (medium containg agent was changed after 3 days incubation) are shown below: (1) M 0.56±0.97%, 2.66±2.31%(3d+3d); 2.73±2.74%, 4.06±2.00%(3d+4d); (2) OM 1.35±0.66%, 3.11±1.30%(3d+3d); 1.56±0.73%, 4.31±2.47%(3d+4d); (3) M & OM 10.85±3.76%, 39.96 ±2.28%(3d +3d); 17.00±2.87%, 47.78±2.42%(3d+4) . The statistically significant difference was found in the combination group (P<0.01).3. PK activity was noticed to be down-regulated in a dose-dependent fashion after the lysate of K562 cells treated directly with kuh-seng alkaloids or Sophora flavescens extracts. The rate of inhibition increased from 21.64% and 43.57% to 88.07% and 94.10% in cells incubated with kuh-seng alkaloids and Sophora flavescens extracts at the doses of 0.4 mg/mL and 2.0 mg/mL respectively. No significant change of PK activity was observed in the lysate exposed to the agents including M, OM, and M&OM.4. The amount of lactic acid in K562 cells incubated with the combination of M with OM was maintained at the level of approximately 3.2μg/mL per 104 cells, which had no statistic significant difference compared to the untreated K562 cells.Conclusion: The content of matrine in K562 cells was increased and maintained at a stabe level in cells incubated with matrine for 24h. The treatment with combination of M and OM decreased the activity of PK in K562 cells. Kuh-seng alkaloids and Sophora flavescens extracts alone inhibited the activity of PK in the lysate of K562 cells in a dose-dependent pattern. No significant effect of the combination of M and OM on the production of lactate was observed in K562 cells.更多还原