【作者】 刘永峰；

【导师】 陈虹；

【作者基本信息】 河北医科大学 ， 药理学， 2011， 硕士

【摘要】 目的:本文研究鬼臼毒素新型衍生物GMZ-1对肿瘤细胞的体外杀伤和抑制生长作用,并对其发挥作用的机制进行初步探索。方法:1 MTT法检测GMZ-1的体外抗肿瘤活性采用MTT法测定GMZ-1对多种肿瘤细胞（Hela、A549、MCF-7、HepG-2、SKOV3、BGC-823、MGC-803、KB、KBV200、K562、K562/A02）及正常细胞（FB）抑制作用的IC₅₀值。2 GMZ-1诱导肿瘤细胞凋亡的检测吉姆萨染色（Giemsa staining）和荧光染色（Heochst 33342 staining）观察GMZ-1 K562/A02细胞作用的形态学变化;琼脂糖凝胶电泳观察GMZ-1对KBV200细胞染色体DNA的影响;流式细胞术（FCM）测定经GMZ-1处理后K562/A02细胞凋亡率的变化。3流式细胞术（FCM）分析GMZ-1对K562/A02细胞周期的影响4 RT-PCR检测GMZ-1对K562/A02细胞p21、p53、Bax、Caspase-3、PCNA、CyclinA、CyclinB1、Mdr-1基因mRNA表达的影响5 Western-blot检测GMZ-1对K562/A02细胞Caspase-3、Mdr蛋白表达的影响6 GMZ-1对HepG-2细胞微管聚合的影响免疫荧光（Immunofluorescence）观察经GMZ-1处理后HepG-2细胞微管形态变化;荧光半定量测定GMZ-1对HepG-2细胞微管聚合影响的抑制率及IC₅₀值。7小鼠急性毒性试验初步考察GMZ-1体内毒性结果:1 GMZ-1的体外抗肿瘤活性经MTT法测定发现GMZ-1对多种肿瘤细胞均有较强杀伤作用和抑制生长活性,IC₅₀值范围为0.066μmol/L～0.27μmol/L,明显强于同类已上市抗肿瘤药物依托泊苷(IC₅₀值范围为1.06μmol/L～14.1μmol/L),且对于同一种细胞的耐药株与非耐药株敏感性差别明显弱于依托泊苷,有较好的抗多药耐药作用。同时对人正常成纤维细胞（FB）具有较低毒性,提示GMZ-1对肿瘤细胞有很好选择性。通过绘制K562/A02细胞生长曲线,发现低浓度GMZ-1作用下细胞存活数量高于高浓度作用下的细胞存活数量,提示GMZ-1的作用具有一定的剂量依赖性。2 GMZ-1诱导肿瘤细胞凋亡GMZ-1能够诱导K562/A02细胞凋亡,对经过GMZ-1处理后的K562/A02细胞进行染色（所用染料为Giemsa和Hoechst 33342）,并利用光学和荧光显微镜观察。光学显微镜下发现细胞核染色质发生聚集固缩,细胞核碎裂成多个染色浓密的凋亡小体;荧光显微镜下发现同正常细胞微弱蓝色荧光相比,GMZ-1处理组细胞出现强亮的蓝色荧光,细胞核呈致密浓染。这些都是细胞发生凋亡的典型形态,且高浓度GMZ-1处理后出现凋亡细胞的数量多于低浓度。提取经GMZ-1处理后KBV200细胞的总DNA,进行琼脂糖凝胶电泳,发现其泳道呈一条一条的梯子状,为细胞发生凋亡所出现的典型的“DNA梯子”（DNA ladder）。通过流式细胞仪对GMZ-1处理后的K562/A02细胞凋亡率的测定,发现在一定浓度和时间范围内,GMZ-1处理后的细胞凋亡率高于正常细胞凋亡率,且凋亡率随着浓度的增大和时间的增多而变大。3 GMZ-1对K562/A02细胞增值周期的影响采用流式细胞仪分析了经GMZ-1处理后K562/A02细胞的周期分布情况,结果显示,在一定浓度范围内,GMZ-1作用12h、24h、36h,细胞都发生G₂+M期阻滞。4 GMZ-1对K562/A02细胞凋亡相关基因p21、p53、Bax、Caspase-3,增殖细胞核抗原PCNA,细胞周期素CyclinA、CyclinB1,多药耐药基因Mdr-1等mRNA表达的影响RT-PCR分析结果显示,GMZ-1通过上调抑癌基因p21、p53、Bax mRNA的表达而发挥诱导肿瘤细胞凋亡的作用;通过上调CyclinA,下调CyclinB1、PCNA而发挥抑制肿瘤细胞分裂增殖的作用;通过下调Mdr-1基因mRNA的表达而产生抗多药耐药作用。5 GMZ-1对K562/A02细胞凋亡执行蛋白Caspase-3和耐药蛋白Mdr表达的影响通过Western-blot分析结果发现, GMZ-1能下调Mdr蛋白的表达,进而发挥抗多药耐药的作用。而对K562/A02细胞Caspase-3的表达影响不明显。6 GMZ-1对HepG-2细胞微管聚合的影响通过免疫荧光方法观察到正常HepG-2细胞微管呈致密的网状分布,荧光强度高,细胞立体感强,而经过GMZ-1处理后的微管,网状结构稀疏或消失,呈星点状分布,荧光强度明显减弱,说明HepG-2细胞微管发生了解聚。通过免疫荧光半定量法对微管聚合解聚动态平衡进行测定,结果显示GMZ-1对微管具有很强的抑制作用,其抑制作用的IC50值为0.066±0.007μmol/L,同鬼臼毒素的0.071±0.007μmol/L相差不大,同时GMZ-1各浓度对应抑制率正负值同长春新碱相似,说明GMZ-1可能和长春新碱一样发挥抑制微管聚合的作用。7 GMZ-1急性毒性试验昆明小鼠腹腔注射GMZ-1后观察到小鼠出现反应迟钝、蜷缩不活动、相继出现死亡现象,解剖死亡小鼠后发现大肠或小肠有阻塞,通过各组小鼠最后死亡数量计算得GMZ-1腹腔注射的LD50值为97.90±19.21mg/kg （95% confidence interval）。结论: GMZ-1在体外对多种肿瘤细胞均具有较强的杀伤作用,其对多数肿瘤细胞的IC50值在0.10μmol/L左右,活性强于依托泊苷10-100倍。多项试验证明GMZ-1对肿瘤细胞的杀伤作用可能是通过诱导肿瘤细胞凋亡的方式实现的。GMZ-1能影响肿瘤细胞正常分裂周期,进而抑制肿瘤细胞增殖。GMZ-1发挥这些药理学作用可能与调节多种凋亡相关基因和蛋白、核增殖基因、细胞周期素、多药耐药基因和蛋白的表达有关。腹腔注射GMZ-1显示出一定的胃肠道反应,其它不良反应有待于进一步研究。总之,GMZ-1是一个新的抗肿瘤化合物,其特点为活性高、选择性好、机制明确,有一定的开发前景,有必要进行进一步试验研究,以获得更加全面的评价指标。更多还原

【Abstract】 Objective: To study the anti-tumor effect in vitro of GMZ-1 which is a novel podophyllotoxin derivative, and the mechanism of its anti-tumor effect. Methods:1 The anti-tumor activity in vitro of GMZ-1 was measured by MTT assayMTT assay was used to measure the IC50 values of GMZ-1 on several tumor cell lines, including human cervical cancer cells (Hela), non-small-cell lung carcinoma cells(A549), human breast carcinoma cells (MCF-7), human liver cancer cells(HepG-2) , human ovarian carcinoma cells (SKOV3), human stomach carcinoma cells (BGC-823、MGC-803), human oral squamous carcinoma cells (KB), human oral squamous carcinoma cells resistance to VCR(KBV200), human erythroleukaemia cells (K562), human erythro- leukaemia cells resistance to ADM, and one normal human cell line, human hypertrophic scar fibroblast (FB).2 Detection of apoptosis of tumor cells induced by GMZ-1Giemsa staining and Heochst 33342 staining were used to observe the morphological changes of K562/A02 cells after treated with GMZ-1; agarose gel electrophoresis was used to observe the effect of GMZ-1 on DNA of KBV200 cells; the apoptotic rate of K562/A02 cells after treated with GMZ-1 was measured by flow cytometry(FCM).3 The effect of cell cycle of K562/A02 cells after treated with GMZ-1 was analyzed by FCM.4 The mRNA expressions of p21、p53、Bax、Caspase-3、PCNA、CyclinA、CyclinB1、Mdr-1 in K562/A02 cells after treated with GMZ-1 were semi-quantified by reverse transcription PCR (RT-PCR).5 The protein expressions of Caspase-3 and Mdr in K562/A02 after treated with GMZ-1 were detected by Western-blot. 6 Effect of GMZ-1 on microtubule of HepG-2 cellsImmunofluorescence was used to observe the morphological changes of microtubule in HepG-2 cells after treated with GMZ-1 and the inhibition ratio of GMZ-1 on microtubule was measured by fluorescent semi-quantitation.7 Toxicity indexs in vivo of GMZ-1 was detected initially by Acute Toxicity Tests.Results:1 The anti-tumor activity in vitro of GMZ-1 The results of MTT assay showed that GMZ-1 revealed a very intense cytotoxic activity towards many carcinoma cell lines, the range of IC50 values was 0.066 to 0.27μmol/L, which was much bertter than VP-16(range of IC50 values: 1.06μmol/L～14.1μmol/L). GMZ-1 was also more sensitive to multidrug resistant tumor cells than VP-16. What is more, GMZ-1 had low cytotoxic towards normal human cells like FB. According to the growth curve of K562/A02 cells, we found that GMZ-1 inhibited growth of tumor cells in a dose-dependent manner.2 GMZ-1 induced apoptosis of tumor cells.After treated with GMZ-1, the typical features of apoptosis such as nucleus shrinkage and splitting, chromatin condensation and deeply dyeing were observed by Giemsa and Heochst 33342 staining in K562/A02 cells. Typical DNA fragmentation pattern (DNA ladder) of apoptosis was observed by Agarose gel electrophoresis in KBV200 cells. FCM analysis indicated that the apoptotic rate of K562/A02 cells accreted consequently with the working concentration of GMZ-1 uprised.3 Effect of GMZ-1 on cell cycle of K562/A02 cellsAfter treated with GMZ-1 for 12h, 24h and 36h, the cell cycles of K562/A02 cells were all arrested at G2+M phase.4 Regulating expressions of p21, p53, Bax, Caspase-3, PCNA, CyclinA, CyclinB1, Mdr-1 in K562/A02 cellsThe results of RT-PCR showed that GMZ-1 could up-regulate the mRNA expressions of p21, p53, Bax, CyclinA, and down-regulate the mRNA expressions of CyclinB1, PCNA, Mdr-1, then induced apoptosis of tumor cells, inhibited proliferations of tumor cells and reversed multidrug resistance.5 Effect on protein expressions of Caspase-3 and Mdr in K562/A02 cellsThe Western blotting experiments showed that GMZ-1 could decrease protein expression of Mdr.6 Immunofluorescence was used to study the effect of GMZ-1 on microtubule of HepG-2 cellsThe microtubule was bushy, reticulodromous, fluorescent brightly and spatial in normal HepG-2 cells, while in the cells after treated with GMZ-1 it was discrete, astroid and fluorescent darkly. Like vincristine, the effect of GMZ-1 on microtubule polymerizing was inhibition, not promoting. The IC50 value of inhibitting microtubule polymerizing was 0.066±0.007μmol/L,a little difference compared with podophyllotoxin(0.071±0.007μmol/L).7 Toxicity indexs of GMZ-1The reactions of peritoneal injection with GMZ-1 were slow response, scrunch, quiescence, diarrhea and blockage in intestine. The LD50 value was 97.90±19.21mg/kg (95% confidence interval).Conclusions: GMZ-1 showed a powerful anti-tumor activity against many human tumor cell lines in vitro. The IC50 values were all about at 0.10μmol/L , 10~100 times more than that of VP-16. Inducing apoptosis might be a main mode of actions of GMZ-1, meanwhile, GMZ-1 could interfere cell cycles of tumor cells, and then inhibit proliferation of tumor cells. The mechanisms of these pharmacological effects were regulating apoptotic gene and protein expressions, regulating PCNA and cyclins, regulating expressions of multidrug resistance related gene and protein. GMZ-1 showed a little adverse reaction in intestine or stomach, other adverse effects were planed to study later. In a word, GMZ-1 was a new anti-tumor compound with high activity, high selectivity, definite mechanism. It was worth to study further more to get an overall estimated index.更多还原