吴茱萸碱诱导肿瘤细胞凋亡及与D-mitosis和M-slippage的关系研究
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摘要
一、研究目的及意义:吴茱萸碱(Evodiamine)是一种吲哚喹唑啉生物碱,是中药吴茱萸的主要活性物质。迄今报道,吴茱萸碱具有较强的抗肿瘤活性,可以诱导肿瘤细胞凋亡、抑制肿瘤组织的侵袭和转移、抑制肿瘤组织的血管生成、抑制多药耐药细胞株的活性等。本课题组前期研究亦证实,吴茱萸碱有显著的诱导肿瘤细胞凋亡的作用,在预实验中发现吴茱萸碱诱导肿瘤细胞凋亡的作用发生G2/M期阻滞,存在Delay-mitosis(D-mitosis,有丝分裂延缓)现象。有学者认为,只有在细胞周期调控中的有丝分裂纺锤体组装检验点(SAC)引起的D-mitosis和Mitotic-slippage(M-slippage,有丝分裂跳出)这两个事件都发生后,细胞凋亡才会发生。细胞凋亡与细胞周期是有着紧密联系的生命活动。不同因素诱导的凋亡,可发生在细胞的不同时相。那么,吴茱萸碱影响相应细胞周期的调控诱导肿瘤细胞凋亡的作用如何以及其诱导肿瘤细胞的凋亡是否与D-mitosis和M-slippage事件相关?这是本课题研究的目的。本研究对阐明吴茱萸碱诱导肿瘤细胞凋亡机制、凋亡与细胞周期调控的关系以及对吴茱萸碱的进一步开发应用都有很大的意义。同时阐明这一理论也将为进一步寻找更多针对细胞周期的有效中药成分和开拓以药物作用机制为靶点进行中药复方组方的理论打下基础,对传统中药的现代化具有深远的意义。
二、研究方法:
2.1吴茱萸碱对体外培养细胞的生长抑制作用。实验中选择人胃癌细胞SGC-7901、人肝癌细胞HepG2、大鼠肝癌细胞CBRH7919、Kidney、Liver小鼠原代细胞,MTT法观察吴茱萸碱对以上5种细胞生长的影响。通过流式细胞仪检测药物作用后对人胃癌细胞SGC-7901细胞周期、凋亡指数的影响,克隆集落形成实验观察克隆集落形成抑制率的影响,镜下观察细胞形态学的改变。
2.2建立细胞周期同步化模型。采用胸苷嘧啶核苷酸(TD)双阻断法及血清饥饿法建立人胃癌细胞SGC-7901细胞周期同步化的细胞模型,选择最佳的方法。
2.3探讨吴茱萸碱对SGC-7901细胞M期的影响。通过流式细胞仪对细胞周期、凋亡指数的检测寻找吴茱萸碱作用的可逆时间点。根据寻找到的可逆时间点,吴茱萸碱作用不同的时间后进行细胞凋亡的形态学、生化学检测;流式细胞仪对细胞周期、凋亡指数的检测;Western blotting分析CyclinB1、CyclinE的表达,彗星电泳检测,克隆集落形成实验观察。
2.4构建不可降解CyclinB1(ND-CyclinB1)及全长CyclinB1(FL-CyclinB1)载体,探讨吴茱萸碱诱导细胞凋亡与细胞周期调控的关系。构建目的基因ND-CyclinB1与GFP的融合基因表达系统以及目的基因FL-CyclinB1与GFP的融合基因表达系统,运用流式细胞仪对细胞凋亡指数检测分析及荧光定量分析研究。
2.5探讨吴茱萸碱影响细胞周期调控诱导凋亡的可能机制。通过JC-1染色观察线粒体膜电位的改变,微管蛋白α-tubulin的改变,Western blotting分析抗凋亡基因Survivin的表达。
2.6吴茱萸合用青黛体内抗瘤实验。建立移植性肝癌(H22)昆明小鼠模型,从整体抗肿瘤作用进行研究,观察吴茱萸、青黛联合作用对荷瘤小鼠的一般情况、抑瘤率、瘤体积、生存期的影响。
三、研究结果:
3.1吴茱萸碱对体外培养细胞的生长抑制作用结果显示。
①人胃癌细胞SGC-7901、人肝癌细胞HepG2及大鼠肝癌细胞CBRH7919三种肿瘤细胞在48hr内分别增长了0.9、1.13、1.39倍,Kidney及Liver原代细胞在48hr内分别增长了0.24和0.18倍;同时细胞的急性毒性实验显示,三种肿瘤细胞对1μM吴茱萸碱的敏感性也显著高于原代细胞。吴茱萸碱对分裂指数低的原代细胞无明显作用。
②MTT实验结果显示吴茱萸碱对SGC-7901细胞的生长抑制作用呈时间和剂量依赖关系,IC_(50)为1.36μM。吴茱萸碱对肿瘤细胞的起效作用浓度低,小剂量的吴茱萸碱对肿瘤细胞即有明显的抑制作用。
③流式细胞仪检测,经1μmol·L~(-1)吴茱萸碱作用12hr后G2/M期的细胞占到78.8%,但凋亡指数仅为4.1%与阴性对照组的4.4%无明显差别。作用24hr后细胞G2/M期的比例下降但凋亡指数增至了34.0%,36hr组G2/M期的比例为0.7%凋亡指数则是52.5%。
④克隆集落形成实验筛选得2×10~3cells/孔为6孔板中最佳实验用细胞密度。用0.25μM、0.5μM、1μM、2μM的吴茱萸碱作用24hr的集落形成抑制率分别为:32.37%、37.17%、48.21%、58.27%。作用36hr的集落形成抑制率分别为:31.3%、49.62%、56.23%、62.85%。
⑤细胞形态学实验观察到典型的凋亡细胞形态学改变,如细胞的体积变小、空泡化、染色质的凝聚、核固缩、凋亡小体的出现。
综上结果提示,吴茱萸碱具有显著抑制肿瘤细胞的生长和抑制肿瘤细胞分裂复制的能力,且这种抑制作用只对分裂指数高的细胞起作用,对低分裂指数细胞株和原代培养细胞生长无明显影响。吴茱萸碱通过直接诱导细胞凋亡而对肿瘤细胞起抑制作用。
3.2建立细胞周期同步化模型。
①经TD双阻断法诱导细胞周期同步化的方法,TD终浓度分别为2mM、4mM、8mM作用后,G0/G1期细胞数分别为77.3%、77.5%和77.0%,而G2/M期细胞数分别为2.0%、2.1%和2.1%。另一大组于上述TD双阻断后各释放12hr后分别收集细胞,TD终浓度为2mM、4mM、8mM的G0/G1期细胞数下降分别为44.0%、38.0%和36.6%,而G2/M期细胞数上升分别为39.9%、46.2%和49.1%,各期细胞数恢复正常范围。TD终浓度为2mM经双阻断法诱导SGC-7901细胞即可在短时间内获得大量可用于后续实验的G0/G1期细胞。
②采用血清饥饿法诱导细胞周期同步化的方法,流式细胞仪检测显示,经无血清培养基分别培养24hr、30hr、36hr后,G0/G1期细胞数分别为57.4%、59.9%和68.4%,而G2/M期细胞数分别为11.4%、5.0%和10.1%。另一大组于上述血清饥饿法诱导各释放12hr后分别收集细胞,经无血清培养基分别培养24hr、30hr、36hr的G0/G1期细胞数下降分别为50.3%、51.4%和51.4%,而G2/M期细胞数上升分别为13.8%、22.7%和22.7%,各期细胞数变化不明显。且无血清培养基培养24hr、30hr及36hr组均出现典型的亚二倍体凋亡峰。
综上结果提示,采用血清饥饿法诱导SGC-7901细胞周期化的方法不够理想。TD双阻断法诱导细胞周期同步化的方法是一种较理想的获得大量处于“基态”的细胞,即G0/G1期细胞的方法,且不存在TD浓度依赖。
3.3探讨吴茱萸碱对SGC-7901细胞M期的影响。
①寻找吴茱萸碱作用后可逆时间点实验中,1μmol·L~(-1)吴茱萸碱作用12hr后,凋亡指数为4.1%。作用16hr后,凋亡指数为4.0%,释放药物维持培养后凋亡指数为8.5%。作用20hr后,两类不同的处理方法的凋亡指数开始存在明显的差别,释放组的凋亡指数为40.4%要显著高于不释放组的凋亡指数16.0%。作用24hr、28hr、32hr和36hr释放组的凋亡指数均显著高于不释放组。因此,吴茱萸碱诱导SGC-7901细胞凋亡的可逆时间点在16-20hr之间。后续实验采用1μmol·L~(-1)吴茱萸碱分别作用16hr、20hr、24hr和36hr后予以研究。
②Western Blot分析CyclinB1的水平,吴茱萸碱作用16hr后CyclinB1蛋白水平显著高于阴性对照。作用20hr后CyclinB1蛋白水平较之作用16hr发生显著下调,作用24hr及作用36hr组CyclinB1蛋白无明显表达。Western Blot分析CyclinE的表达,阴性对照组、1μmol·L~(-1)吴茱萸碱作用16hr、20hr及24hr组均未见表达,而作用36hr组出现特异性条带。结果CyclinB1随吴茱萸碱作用时间的增加表达下降,作用36hr后不表达,而CyclinE则在吴茱萸碱作用36hr后开始表达,提示吴茱萸碱作用36hr后经历了M期阻滞后跳出M期进入了G1期。
③SGC-7901细胞为贴壁生长类型,呈多角形或菱形。PI染色,荧光显微镜下观察,吴茱萸碱处理16hr可看到染色体固缩呈三叉样;处理24hr后并不能看到明显的凋亡小体产生,大部分细胞仍处于染色体固缩的状态;处理36hr后出现大量的凋亡小体,同时多核细胞增加。
④彗星电泳实验,受损伤的DNA泳出,在细胞后面形成长的拖尾。吴茱萸碱作用后细胞平均光密度值较阴性对照均降低,具有统计学意义。彗星尾距较阴性对照均增加,具有统计学意义,二者的改变与作用时间相关。
⑤流式细胞仪检测,7.5μM CDK1I作用SGC-7901细胞凋亡指数为7.6%与阴性对照组的3.5%比较无显著差异。吴茱萸碱作用18hr后再加入CDK1I二者共同作用6hr后较单独吴茱萸碱作用24hr的凋亡指数均显著升高。0.5μM吴茱萸碱组的凋亡指数为8.9%,加入CDK1I共同作用组的凋亡指数则升至28.2%。1μM吴茱萸碱组的凋亡指数为20.0%,加入CDK1I共同作用组的凋亡指数则升至32.0%。抑制作用呈剂量依赖关系。
⑥克隆集落形成实验以阴性对照组的集落形成抑制率0%为参照,7.5μM CDK1I作用SGC-7901细胞的集落形成抑制率为24.84%。吴茱萸碱作用18hr后再加入CDK1I二者共同作用6hr后较单独吴茱萸碱作用的集落形成抑制率均显著升高。0.5μM吴茱萸碱组的集落形成抑制率为38.01%,加入CDK1I共同作用组的凋亡指数则升至65.34%。1μM吴茱萸碱组的凋亡指数为49.81%,加入CDK1I共同作用组的凋亡指数则升至70.32%。
⑦Dapi染色,光学显微镜下观察,阴性对照的细胞贴壁生长牢固,细胞明亮清晰,折光性好,充分伸展,细胞膜完整,细胞质分布均匀,核较大而圆,发出均匀的荧光。SGC-7901细胞经CDK1I作用后体积变大,贴壁能力增强。0.5μM及1μM吴茱萸碱组可见细胞变圆,染色质出现浓缩状态,出现凋亡小体。加入CDK1I 3hr左右即可见变圆细胞重新贴壁,同时至共同作用6hr后多核形态的细胞增加与PI染色细胞核改变现象类似。
综上结果提示,吴茱萸碱影响细胞周期调控诱导SGC-7901细胞凋亡而发生肿瘤细胞的死亡,而凋亡的发生与D-mitosis事件的产生和维持4-8hr相关,协同发生M-slippage事件可增强凋亡作用。
3.4构建不可降解CyclinB1(ND-CyclinB1)及全长CyclinB1(FL-CyclinB1)载体,探讨吴茱萸碱诱导细胞凋亡与细胞周期调控的关系。
①经限制性酶切鉴定和DNA序列测定分析,目的基因ND-CyclinB1、FL-CyclinB1成功与GFP基因连接。插入处的密码阅读框与GFP的密码阅读框吻合,无移码突变产生。重组质粒pEGFP-N1-ND CyclinB1、pEGFP-N1-FL CyclinB1转染SGC-7901细胞,ND-CyclinB1、FL-CyclinB1基因在SGC-7901中都获得表达。
②重组质粒转染SGC-7901细胞,经吴茱萸碱作用后流式细胞仪检测显示,阴性对照组的凋亡指数为4.1%。重组pEGFP-N1-ND与pEGFP-N1-FL经吴茱萸碱作用后二者的凋亡指数分别为38.2%和41.3%,与不经吴茱萸碱作用的凋亡指数14.5%和10.7%比较均差别显著。转染细胞经吴茱萸碱作用组的凋亡指数远不及非转染细胞经吴茱萸碱作用36hr的56.8%。
③重组质粒转染SGC-7901细胞,经吴茱萸碱作用36hr后盲法摄取图像,采用盲法评分,评分结果可得重组pEGFP-N1-ND CyclinB1经吴茱萸碱作用组分值为3.60,均高于pEGFP-N1-FL CyclinB1及空载pEGFP-N1经吴茱萸碱作用的2.50与1.40。
综上结果提示,重组质粒pEGFP-N1-ND CyclinB1与pEGFP-N1-FL CyclinB1的构建是成功的。采用让细胞长时间阻滞于M期无法进入下一细胞进程的方法可得出:吴茱萸碱诱导SGC-7901细胞凋亡而发生肿瘤细胞的死亡与其引起D-mitosis事件相关,阻断M-slippage事件可减弱凋亡作用。
3.5探讨吴茱萸碱影响细胞周期调控诱导凋亡的可能机制。
①应用JC-1观察细胞线粒体膜电位的改变,结果显示,阴性对照组红、绿色荧光均匀,红色荧光散布于细胞中。吴茱萸碱作用16hr后,红色荧光强度减弱,而绿色荧光强度增强,表明线粒体膜电位受到破坏。作用20hr,红色荧光强度显著减弱,而绿色荧光强度显著增强。作用24hr只见散在红色荧光,36hr后则视野中几乎无红色荧光。
②荧光显微镜下观察α-tubulin微管蛋白的改变,阴性对照组的微管蛋白纤维清晰可见,呈细丝状,沿着细胞的长轴分布。作用16hr后可见微管蛋白纤维排列开始紊乱,断裂变短。作用20hr可见微管蛋白纤维发生弯曲,聚合状态发生改变,随着作用时间的增加,改变进一步加剧。至作用36hr微管蛋白聚合在一侧,荧光强度增加,纤维不再清晰可见,与紫杉醇作用36hr的微管蛋白改变类似。吴茱萸碱作用后使得细胞微管蛋白的聚合状态发生了改变。
③Western Blot分析Survivin凋亡抑制基因的表达,实验中阴性对照组Survivin是高表达的,吴茱萸碱作用16hr后,Survivin的表达较阴性对照组即以开始下降,作用20hr、24hr及36hr则均已无Survivin的特异性条带出现。
综上结果提示,吴茱萸碱通过影响细胞周期调控诱导SGC-7901细胞凋亡的作用机制可能与线粒体跨膜电位的丢失、影响微管蛋白聚合以及抑制Survivin基因的表达相关,是一个多靶点作用的过程。
3.6吴茱萸合用青黛体内抗瘤实验。
①动物整体水平研究选用皮下移植性小鼠(正常鼠)肝癌模型的方法,成瘤率100%。
②实验一中,最终存活下来的小鼠一般情况以环磷酰胺组最佳,但环磷酰胺组仅存活5只。环磷酰胺组、吴茱萸组、青黛组以及联合组的抑瘤率分别为43.9%、35.6%、27.7%和37.2%。瘤体积比较结果表明:各治疗组瘤体积均较模型对照组小,各组间两两比较仅联合组与模型对照组间有统计学意义(p=0.048)。
③实验二中,存活小鼠一般情况类似实验一部分。环磷酰胺组、吴茱萸组、青黛组以及联合组的抑瘤率分别为50.1%、44.9%、45.9%和51.9%。瘤体积比较结果表明:各治疗组瘤体积均较模型对照组小,且各治疗组与模型比较均有统计学意义。联合组与环磷酰胺组之间虽无统计学差异,但联合组的瘤体积平均值小于环磷酰胺组。同时,吴茱萸组、青黛组也均小于环磷酰胺组。进行生存期观察小鼠,当药物干预进行到第19d时环磷酰胺组陆续出现恶病质表现,体重明显下降,目光晦暗呆滞,弓背,张口呼吸,肉眼可见肿块虽较其他治疗组小但小鼠的一般情况远不及其他治疗组小鼠。各中药组改善移植瘤小鼠生存质量的优势显现了出来。药物作用30d后,不治疗继续观察至小鼠全部死亡,计算各治疗组的生命延长率。各治疗组较模型对照组均能延长存活时间,环磷酰胺组、联合组与模型对照组的平均存活时间之间具有统计学意义。环磷酰胺组、吴茱萸组、青黛组以及联合组的生命延长率分别为40.1%、36.04%、18.78%、70.05%,联合组要显著高于环磷酰胺组,但二者间差别无统计学意义。
综上结果提示,吴茱萸合用青黛对移植肿瘤小鼠具有抗肿瘤作用,提示吴茱萸碱与靛玉红合用可以增加荷瘤小鼠瘤体生长抑制作用和延长荷瘤小鼠生存时间作用。
四、结论:
吴茱萸碱具有显著抑制肿瘤细胞的生长和抑制肿瘤细胞分裂复制的能力,且这种抑制作用可能具有细胞分裂指数选择性。吴茱萸碱对SGC-7901细胞持续作用16-20hr后即启动了细胞的凋亡程序。吴茱萸碱诱导SGC-7901细胞凋亡而发生肿瘤细胞的死亡,凋亡的发生既与D-mitosis事件的产生和维持4-8hr相关又与M-slippage事件的发生相关。吴茱萸碱通过影响细胞周期调控诱导SGC-7901细胞凋亡的作用机制可能与线粒体跨膜电位的丢失、影响微管蛋白聚合以及抑制Survivin基因的表达相关,是一个多靶点作用的过程。吴茱萸与青黛合用可以增加荷瘤小鼠瘤体生长抑制作用和延长荷瘤小鼠生存时间作用。吴茱萸碱作为抗肿瘤药具有极大的开发潜力。
Objective:
Evodiamine is an indole quinazoline alkaloid,the main active substance of evodiae.Cell experiments show that evodiamine with antineoplastic activity, directly induce tumor cell apoptosis,inhibit tumor invasion and metastasis, inhibit tumor angiogenesis,inhibit activity of multidrug resistance cell line and so on.Preliminary studies have confirmed that evodiamine could induce tumor cells apoptosis and induce tumor cells arresting in G2/M phase,there is the phenomenon of D-mitosis.D-mitosis and M-slippage,these two events have taken place,apoptosis would be induced only.The current view consider that apoptosis is closely linked to the cell cycle as activities of life.Apoptosis induced by different factors,can occur in different cell cycle.The study was to elucidate the mechanisms of apoptosis induced by evodiamine and the relationship between apoptosis and cell cycle regulation,as well as the further development and application of evodiamine.At the same time,it was provided foundation of more effective Chinese herbal against the cell cycle and developed complex prescription according to the mechanism of Chinese herbal.
Methods:
2.1 Evodiamine could inhibit the growth of tumor cells in vitro.We selected SGC-7901、HepG2、CBRH7919、primary Kidney,primary Liver cells,used MTT assay to test above 5 cells antipoliferative effect after being treated with evodiamine.The cell cycle and apoptosis index of SGC-7901 were examined using FACScan flow cytometry,the colone assay was used to detect the colone inhibition ratio,microscope was used to observe the changes in cell morphology.
2.2 In order to reproduct the best method of SGC-7901 cell cycle synchronization,we used two methods,one was double TD blocking,the other was serum starvation.
2.3 We used FACScan flow cytometry to look for reversible drug detection time after being treated with evodiamine.Accordance with irreversible point in time,after being treated with evodiamine the morphology of apoptosis and chemical detection were done;The cell cycle and apoptosis index of SGC-7901 were examined using FACScan flow cytometry;Expression of CyclinB1 and CyclinE were examined using Western blotting analysis;Comet assay and colone assay were used to prove hypothesis.
2.4Construction of gene ND-cyclinB1 with GFP fusion gene expression systems, as well as FL-cyclinB1 gene fusion with the GFP gene expression system to confirm hypothesis by FACScan flow cytometry and Quantitative analysis of fluorescence.
2.5 The initial mechanisms of Evodiamine induced apoptosis requires both activation of D-mitosis and M-slippage:through the JC-1 staining of mitochondrial membrane potential change,α-tubulin changes,Western blotting analysis of anti-apoptosis gene Survivin expression.
2.6 Produce the transplanted liver cancer(H22) in Kunming mice,the general situation、tumor inhibition rate、tumor volume、survival period were determed after being treated with Evodiae and Indigo naturalis.
Results:
3.1Evodiamine in vitro cell growth inhibition results show:
①SGC-7901,HepG2,CBRH7919 three tumor cells respectively increased 0.9,1.13,1.39 times after 48hr,Kidney and Liver cells respectively increased 0.24,0.18 times after 48hr;Simultaneously cell acute toxicity experiments reveaIed that the evodiamine sensitivity of three types of tumor cells were significantly higher than the primary cells.
②MTT assay confirmed growth inhibition of evodiamine was time and dose-dependent,IC_(50) was1.36μmol.
③FACScan flow cytometry showed 1μmol·L~(-1) evodiamine for 12hr the G2/M phase cells account for 78.8%,but the apoptotic index was only 4.1%and negative was 4.4%in the control group no significant difference,1μmol·L~(-1) evodiamine for 24hr the G2/M phase declineed in the proportion,the apoptotic index increased to 34.0%,evodiamine for 36hr the G2/M phase ceils accounted for 0.7%,the apoptotic index was 52.5%.
④Colone assay we found 2×10~3cells/hole was the best experiment cell density in 6-well plates,evodiamine had significant inhibition of colony formation.
⑤Experimental observation of cell morphology,we saw typical morphological changes of apoptotic cells,such as smaller cell size,vacuolization, chromatin cohesion,karyopyknosis,the emergence of apoptotic bodies.
The results suggested that Evodiamine could inhibit the growth and division of tumor cells,and this inhibition was selective,that was,the faster growth cells were stronger sensitive to evodiamine.
3.2 To product SGC-7901 cell cycle synchronization.
①we used double TD blocking method.TD final concentration of 2mM,4mM,8mM after,G0/G1 phase cells were77.3%,77.5%and 77.0%,while G2/M phase cells were 2.0%,2.1%and 2.1%.Another large group in the above mentioned double TD blocking then released 12hr,TD final concentration of 2mM,4mM,8mM of G0/G1 phase cells decreased to 44.0%,38.0%and 36.6%,and G2/M phase cells increased to39.9%,46.2%and 49.1%.
②The other method-serum starvation synchronization,flow cytometry indicated that the serum-free culture medium,respectively,24hr,30hr,36hr,G0/G1 phase cells were 57.4%,59.9%and 68.4%,and G2/M phase cells were11.4%,5.0% and 10.1%.Another large group in the above mentioned serum starvation then released 12hr,respectively,24hr,30hr,36hr of G0/G1 phase cells decreased to 50.3%,51.4%and 51.4%,while G2/M phase cells increased to 13.8%,22.7% and 22.7%,the period of cell number was not change significantly.Serum-free culture medium for 24hr,30hr and 36hr,both appeared typical hypodiploid apoptotic peak.
The results suggested that Double TD blocking cell cycle synchronization method was an ideal access to gain a large number of ground state cells,and didn't rely on the TD concentration.Treatment by evodiamine for 16-20hr, SGC-7901 switched on apoptotic process,resulting in irreversible death.
3.3 Explore the impaction in M phase of SGC-7901 by evodiamine.
①About the reversible point in time after experiment we found, 1μmol·L~(-1)evodiamine for12hr the apoptotic index was 4.1%.For16hr,the apoptotic index was 4.0%,then released the drug apoptotic index was 8.5%. For 20hr two different types had significant difference,the released group apoptotic index increased to40.4%,was significantly higher than non-released group-16.0%.For 24hr,28hr,32hr and 36hr the apoptotic index of the released group were significantly higher than non-released group.According to evodiamine-induced apoptosis irreversible point in time between 16-20hr,we used 1μmol·L~(-1) evodiamine respectively for 16hr,20hr,24hr and 36hr.
②Western Blot analysis of the level of CyclinB1:Treated by evodiamine for 16hr the level was significantly higher than negative cells,but the level of evodiamine for 20hr was lower than 16hr,while 24hr and 36hr with no protein expression.Western Blot analysis of the expression of CyclinE,negative cells, 1μmol·L~(-1) evodiamine for16hr,20hr and 24hr were both no specific bands,and evodiamine for 36hr appeared band.The expression of CyclinE tangled to CyclinB1,suggesting that after treating by evodiamine for 36hr cells arrested in M phase,then went into G1 phase.
③The growth type of SGC-7901 was polygonal or diamond.PI staining was observed under fluorescence microscope,chromosomes can be seen shrinkaged assuming trigeminal after treating evodiamine for 16hr;After treatment for 24hr,could not see the obvious apoptotic bodies,the majority of cells wore still in a state of chromosome condensation;After treatment for 36hr,we could see the obvious apoptotic bodies,at the same time could see the multinucleated cells.
④The comet assay,we found the damaged DNA swam,a long tail appeared behind the cell.The average optic density was lower than the negative cells after treating with statistical significance.The comet tail was longer than the negative cells after treating with statistical significance,both two changes were related to time.
⑤FACScan flow cytometry showed:apoptosis index of 1μmol·L~(-1) CDK1I was 7.6%, the negative cell was 3.5%,no significant difference.After treating by evodiamine forl8hr added CDK1I for 6hr the apoptotic index significantly increased.0.5μmol·L~(-1) evodiamine for 24hr the apoptotic index was 8.9%,then another group added CDK1I the apoptotic index increased to 28.2%.1μmol·L~(-1)evodiamine for 24hr the apoptotic index was 20.0%,then another group added CDK1I the apoptotic index increased to 32.0%.
⑥The colone assay,we found the colone inhibition index was 24.84%after treating by 7.5μM CDK1I,and added CDK1I combinding to evodiamine the colone inhibition index significantly increased.0.5μmol·L~(-1) evodiamine for 24hr the colone inhibition index was 38.01%,then another group added CDK1I the colone inhibition index increased to 65.34%.1μmol·L~(-1) evodiamine for 24hr the colone inhibition index was 49.81%,then another group added CDK1I the colone inhibition index increased to 70.32%.The colone Inhibition was dose-dependent.
⑦Dapi staining was observed under fluorescence microscope,negative cells were firmly adherent,bright,extended,uniform.While SGC-7901 after treating by CDK1I were bigger and stronger.0.5μmol·L~(-1)and 1μmol·L~(-1)evodiamine group could be seen larger sizes,condensed chromatin status,apoptotic bodies.Added CDK1I for 3hr cells re-adherent and at the same time the combined effect for 6hr,multinucleated cells appeared concording with PI staining.
The results suggested that Evodiamine induced SGC-7901 apoptosis,and signal apoptosis related on the generation and maintenance of D-mitosis and Mitotic-slippage event.
3.4 Gene ND-CyclinB1,FL-CyclinB1 have successfully connected with the GFP gene.
①Recombinant plasmid pEGFP-N1-ND CyclinB1,pEGFP-N1-FL CyclinB1 transfected SGC-7901,ND-CyclinB1,FL-CyclinB1 gene were expressed in SGC-7901.
②Recombinant plasmid transfected SGC-7901,after treating by evodiamine, FACScan flow cytometry showed that the apoptotic index of negative cell was 4.1%,pEGFP-N1-ND CyclinB1,pEGFP-N1-FL CyclinB1 respectively was 38.2%,41.3%, and without the role of evodiamine the apoptotic index respectively was14.5%,10.7%,with significant difference.
③Recombinant plasmid transfected SGC-7901 cells,1μmol·L~(-1)evodiamine for 36hr the blind score of pEGFP-N1-ND CyclinB1was 3.60,were higher than pEGFP-N1-FL CyclinB1 and empty pEGFP-N1 as the role of evodiamine 2.50 and 1.40.
The results suggested that Recombinant plasmid of pEGFP-N1-ND and pEGFP-N1-FL were success,they could applicated to research cell cycle-related study in the future.Method of making cells arrest in M phase,we can get the same conclusion,that is evodiamine induced SGC-7901 apoptosis relatde on D-mitosis event.
3.5 Explore the possible mechanisms of cell cycle regulation inducting apoptosis
①Application JC-1 to observe mitochondrial membrane potential changes,result showed:red,green fluorescence dispersed uniformly in the negative cells, red fluorescent spread in cells.After treatment for 16hr,the red fluorescence intensity decreased,while the green fluorescence intensity enhanced,indicating that mitochondrial membrane potential damaged.After treatment for 20hr,the red fluorescence intensity decreased significantly, while the green fluorescence intensity increased significantly.After treatment for 24hr,red fluorescence scattered,while for 36hr almost no red fluorescence.
②Then we Observedα-tubulin,the negative cells with clearly visible fibers, showing fine filaments.After treatment for 16hr,tubulin fibers could be seen beginning with disorders,shorter fracture.After treatment for 20hr,tubulin fibers could be seen bending happened,changing the status polymerization.Treatment lasted 36hr,tubulin polymerized in the side, fluorescence intensity increased,the changes similar to taxol-tubulin.
③Western Blot analysis Survivin expression,negative cell was in a high expression,after treatment for 16hr evodiamine,Survivin begen to decline.After treatment for 20hr,24hr and 36hr then had no Survivin specific bands appeare.
The results suggested that Evodiamine induced SGC-7901 apoptosis might be related to the loss of mitochondrial transmembrane potential,the impact of tubulin polymerization and inhibit the expression of Survivin gene,was a multi-target process.
3.6 Rutaecarpa combined Qingdai anti-tumor experiments in vivo.
①Transplanted subcutaneously mice of liver cancer model could formed tumor 100%.
②Experiment I,we found that Cyclophosphamide group mice was in best general state of health,but only five mice survived.The tumor inhibition rate of cyclophosphamide group,evodiamine group,qingdai group and combinded group respectively was 43.9%,35.6%27.7%and 37.2%.Tumor size results showed that the treatment groups' were smaller than control group and combinded group compared with cyclophosphamide group had statistical significace(p=0.048).
③ExperimentⅡ,general state of health was similar to the experimentⅠ.The tumor inhibition rate of cyclophosphamide group,evodiamine group,qingdai group and combinded group respectively was 50.1%,44.9%,45.9%and 51.9%.Tumor size results showed that the treatment groups' were smaller than control group. The life extension rate of cyclophosphamide group,evodiamine group,qingdai group and combinded group respectively was 40.1%,36.04%,18.78%,70.05%,the combinded group was significant higher than cyclophosphamide group.
Conclusion:
Evodiamine could inhibit the growth and division of tumor cells,and this inhibition is selective,that is,the faster growth cells are stronger sensitive to evodiamine.Treatment by evodiamine for 16-20hr,SGC-7901 switches on apoptotic process,resulting in irreversible death.Evodiamine induces SGC-7901 apoptosis,and signal apoptosis relats to the generation and maintenance of D-mitosis and Mitotic-slippage event,Evodiamine induces SGC-7901 apoptosis may be related to the loss of mitochondrial transmembrane potential,the impact of tubulin polymerization and inhibit the expression of Survivin gene,is a multi-target process.
二、研究方法:
2.1吴茱萸碱对体外培养细胞的生长抑制作用。实验中选择人胃癌细胞SGC-7901、人肝癌细胞HepG2、大鼠肝癌细胞CBRH7919、Kidney、Liver小鼠原代细胞,MTT法观察吴茱萸碱对以上5种细胞生长的影响。通过流式细胞仪检测药物作用后对人胃癌细胞SGC-7901细胞周期、凋亡指数的影响,克隆集落形成实验观察克隆集落形成抑制率的影响,镜下观察细胞形态学的改变。
2.2建立细胞周期同步化模型。采用胸苷嘧啶核苷酸(TD)双阻断法及血清饥饿法建立人胃癌细胞SGC-7901细胞周期同步化的细胞模型,选择最佳的方法。
2.3探讨吴茱萸碱对SGC-7901细胞M期的影响。通过流式细胞仪对细胞周期、凋亡指数的检测寻找吴茱萸碱作用的可逆时间点。根据寻找到的可逆时间点,吴茱萸碱作用不同的时间后进行细胞凋亡的形态学、生化学检测;流式细胞仪对细胞周期、凋亡指数的检测;Western blotting分析CyclinB1、CyclinE的表达,彗星电泳检测,克隆集落形成实验观察。
2.4构建不可降解CyclinB1(ND-CyclinB1)及全长CyclinB1(FL-CyclinB1)载体,探讨吴茱萸碱诱导细胞凋亡与细胞周期调控的关系。构建目的基因ND-CyclinB1与GFP的融合基因表达系统以及目的基因FL-CyclinB1与GFP的融合基因表达系统,运用流式细胞仪对细胞凋亡指数检测分析及荧光定量分析研究。
2.5探讨吴茱萸碱影响细胞周期调控诱导凋亡的可能机制。通过JC-1染色观察线粒体膜电位的改变,微管蛋白α-tubulin的改变,Western blotting分析抗凋亡基因Survivin的表达。
2.6吴茱萸合用青黛体内抗瘤实验。建立移植性肝癌(H22)昆明小鼠模型,从整体抗肿瘤作用进行研究,观察吴茱萸、青黛联合作用对荷瘤小鼠的一般情况、抑瘤率、瘤体积、生存期的影响。
三、研究结果:
3.1吴茱萸碱对体外培养细胞的生长抑制作用结果显示。
①人胃癌细胞SGC-7901、人肝癌细胞HepG2及大鼠肝癌细胞CBRH7919三种肿瘤细胞在48hr内分别增长了0.9、1.13、1.39倍,Kidney及Liver原代细胞在48hr内分别增长了0.24和0.18倍;同时细胞的急性毒性实验显示,三种肿瘤细胞对1μM吴茱萸碱的敏感性也显著高于原代细胞。吴茱萸碱对分裂指数低的原代细胞无明显作用。
②MTT实验结果显示吴茱萸碱对SGC-7901细胞的生长抑制作用呈时间和剂量依赖关系,IC_(50)为1.36μM。吴茱萸碱对肿瘤细胞的起效作用浓度低,小剂量的吴茱萸碱对肿瘤细胞即有明显的抑制作用。
③流式细胞仪检测,经1μmol·L~(-1)吴茱萸碱作用12hr后G2/M期的细胞占到78.8%,但凋亡指数仅为4.1%与阴性对照组的4.4%无明显差别。作用24hr后细胞G2/M期的比例下降但凋亡指数增至了34.0%,36hr组G2/M期的比例为0.7%凋亡指数则是52.5%。
④克隆集落形成实验筛选得2×10~3cells/孔为6孔板中最佳实验用细胞密度。用0.25μM、0.5μM、1μM、2μM的吴茱萸碱作用24hr的集落形成抑制率分别为:32.37%、37.17%、48.21%、58.27%。作用36hr的集落形成抑制率分别为:31.3%、49.62%、56.23%、62.85%。
⑤细胞形态学实验观察到典型的凋亡细胞形态学改变,如细胞的体积变小、空泡化、染色质的凝聚、核固缩、凋亡小体的出现。
综上结果提示,吴茱萸碱具有显著抑制肿瘤细胞的生长和抑制肿瘤细胞分裂复制的能力,且这种抑制作用只对分裂指数高的细胞起作用,对低分裂指数细胞株和原代培养细胞生长无明显影响。吴茱萸碱通过直接诱导细胞凋亡而对肿瘤细胞起抑制作用。
3.2建立细胞周期同步化模型。
①经TD双阻断法诱导细胞周期同步化的方法,TD终浓度分别为2mM、4mM、8mM作用后,G0/G1期细胞数分别为77.3%、77.5%和77.0%,而G2/M期细胞数分别为2.0%、2.1%和2.1%。另一大组于上述TD双阻断后各释放12hr后分别收集细胞,TD终浓度为2mM、4mM、8mM的G0/G1期细胞数下降分别为44.0%、38.0%和36.6%,而G2/M期细胞数上升分别为39.9%、46.2%和49.1%,各期细胞数恢复正常范围。TD终浓度为2mM经双阻断法诱导SGC-7901细胞即可在短时间内获得大量可用于后续实验的G0/G1期细胞。
②采用血清饥饿法诱导细胞周期同步化的方法,流式细胞仪检测显示,经无血清培养基分别培养24hr、30hr、36hr后,G0/G1期细胞数分别为57.4%、59.9%和68.4%,而G2/M期细胞数分别为11.4%、5.0%和10.1%。另一大组于上述血清饥饿法诱导各释放12hr后分别收集细胞,经无血清培养基分别培养24hr、30hr、36hr的G0/G1期细胞数下降分别为50.3%、51.4%和51.4%,而G2/M期细胞数上升分别为13.8%、22.7%和22.7%,各期细胞数变化不明显。且无血清培养基培养24hr、30hr及36hr组均出现典型的亚二倍体凋亡峰。
综上结果提示,采用血清饥饿法诱导SGC-7901细胞周期化的方法不够理想。TD双阻断法诱导细胞周期同步化的方法是一种较理想的获得大量处于“基态”的细胞,即G0/G1期细胞的方法,且不存在TD浓度依赖。
3.3探讨吴茱萸碱对SGC-7901细胞M期的影响。
①寻找吴茱萸碱作用后可逆时间点实验中,1μmol·L~(-1)吴茱萸碱作用12hr后,凋亡指数为4.1%。作用16hr后,凋亡指数为4.0%,释放药物维持培养后凋亡指数为8.5%。作用20hr后,两类不同的处理方法的凋亡指数开始存在明显的差别,释放组的凋亡指数为40.4%要显著高于不释放组的凋亡指数16.0%。作用24hr、28hr、32hr和36hr释放组的凋亡指数均显著高于不释放组。因此,吴茱萸碱诱导SGC-7901细胞凋亡的可逆时间点在16-20hr之间。后续实验采用1μmol·L~(-1)吴茱萸碱分别作用16hr、20hr、24hr和36hr后予以研究。
②Western Blot分析CyclinB1的水平,吴茱萸碱作用16hr后CyclinB1蛋白水平显著高于阴性对照。作用20hr后CyclinB1蛋白水平较之作用16hr发生显著下调,作用24hr及作用36hr组CyclinB1蛋白无明显表达。Western Blot分析CyclinE的表达,阴性对照组、1μmol·L~(-1)吴茱萸碱作用16hr、20hr及24hr组均未见表达,而作用36hr组出现特异性条带。结果CyclinB1随吴茱萸碱作用时间的增加表达下降,作用36hr后不表达,而CyclinE则在吴茱萸碱作用36hr后开始表达,提示吴茱萸碱作用36hr后经历了M期阻滞后跳出M期进入了G1期。
③SGC-7901细胞为贴壁生长类型,呈多角形或菱形。PI染色,荧光显微镜下观察,吴茱萸碱处理16hr可看到染色体固缩呈三叉样;处理24hr后并不能看到明显的凋亡小体产生,大部分细胞仍处于染色体固缩的状态;处理36hr后出现大量的凋亡小体,同时多核细胞增加。
④彗星电泳实验,受损伤的DNA泳出,在细胞后面形成长的拖尾。吴茱萸碱作用后细胞平均光密度值较阴性对照均降低,具有统计学意义。彗星尾距较阴性对照均增加,具有统计学意义,二者的改变与作用时间相关。
⑤流式细胞仪检测,7.5μM CDK1I作用SGC-7901细胞凋亡指数为7.6%与阴性对照组的3.5%比较无显著差异。吴茱萸碱作用18hr后再加入CDK1I二者共同作用6hr后较单独吴茱萸碱作用24hr的凋亡指数均显著升高。0.5μM吴茱萸碱组的凋亡指数为8.9%,加入CDK1I共同作用组的凋亡指数则升至28.2%。1μM吴茱萸碱组的凋亡指数为20.0%,加入CDK1I共同作用组的凋亡指数则升至32.0%。抑制作用呈剂量依赖关系。
⑥克隆集落形成实验以阴性对照组的集落形成抑制率0%为参照,7.5μM CDK1I作用SGC-7901细胞的集落形成抑制率为24.84%。吴茱萸碱作用18hr后再加入CDK1I二者共同作用6hr后较单独吴茱萸碱作用的集落形成抑制率均显著升高。0.5μM吴茱萸碱组的集落形成抑制率为38.01%,加入CDK1I共同作用组的凋亡指数则升至65.34%。1μM吴茱萸碱组的凋亡指数为49.81%,加入CDK1I共同作用组的凋亡指数则升至70.32%。
⑦Dapi染色,光学显微镜下观察,阴性对照的细胞贴壁生长牢固,细胞明亮清晰,折光性好,充分伸展,细胞膜完整,细胞质分布均匀,核较大而圆,发出均匀的荧光。SGC-7901细胞经CDK1I作用后体积变大,贴壁能力增强。0.5μM及1μM吴茱萸碱组可见细胞变圆,染色质出现浓缩状态,出现凋亡小体。加入CDK1I 3hr左右即可见变圆细胞重新贴壁,同时至共同作用6hr后多核形态的细胞增加与PI染色细胞核改变现象类似。
综上结果提示,吴茱萸碱影响细胞周期调控诱导SGC-7901细胞凋亡而发生肿瘤细胞的死亡,而凋亡的发生与D-mitosis事件的产生和维持4-8hr相关,协同发生M-slippage事件可增强凋亡作用。
3.4构建不可降解CyclinB1(ND-CyclinB1)及全长CyclinB1(FL-CyclinB1)载体,探讨吴茱萸碱诱导细胞凋亡与细胞周期调控的关系。
①经限制性酶切鉴定和DNA序列测定分析,目的基因ND-CyclinB1、FL-CyclinB1成功与GFP基因连接。插入处的密码阅读框与GFP的密码阅读框吻合,无移码突变产生。重组质粒pEGFP-N1-ND CyclinB1、pEGFP-N1-FL CyclinB1转染SGC-7901细胞,ND-CyclinB1、FL-CyclinB1基因在SGC-7901中都获得表达。
②重组质粒转染SGC-7901细胞,经吴茱萸碱作用后流式细胞仪检测显示,阴性对照组的凋亡指数为4.1%。重组pEGFP-N1-ND与pEGFP-N1-FL经吴茱萸碱作用后二者的凋亡指数分别为38.2%和41.3%,与不经吴茱萸碱作用的凋亡指数14.5%和10.7%比较均差别显著。转染细胞经吴茱萸碱作用组的凋亡指数远不及非转染细胞经吴茱萸碱作用36hr的56.8%。
③重组质粒转染SGC-7901细胞,经吴茱萸碱作用36hr后盲法摄取图像,采用盲法评分,评分结果可得重组pEGFP-N1-ND CyclinB1经吴茱萸碱作用组分值为3.60,均高于pEGFP-N1-FL CyclinB1及空载pEGFP-N1经吴茱萸碱作用的2.50与1.40。
综上结果提示,重组质粒pEGFP-N1-ND CyclinB1与pEGFP-N1-FL CyclinB1的构建是成功的。采用让细胞长时间阻滞于M期无法进入下一细胞进程的方法可得出:吴茱萸碱诱导SGC-7901细胞凋亡而发生肿瘤细胞的死亡与其引起D-mitosis事件相关,阻断M-slippage事件可减弱凋亡作用。
3.5探讨吴茱萸碱影响细胞周期调控诱导凋亡的可能机制。
①应用JC-1观察细胞线粒体膜电位的改变,结果显示,阴性对照组红、绿色荧光均匀,红色荧光散布于细胞中。吴茱萸碱作用16hr后,红色荧光强度减弱,而绿色荧光强度增强,表明线粒体膜电位受到破坏。作用20hr,红色荧光强度显著减弱,而绿色荧光强度显著增强。作用24hr只见散在红色荧光,36hr后则视野中几乎无红色荧光。
②荧光显微镜下观察α-tubulin微管蛋白的改变,阴性对照组的微管蛋白纤维清晰可见,呈细丝状,沿着细胞的长轴分布。作用16hr后可见微管蛋白纤维排列开始紊乱,断裂变短。作用20hr可见微管蛋白纤维发生弯曲,聚合状态发生改变,随着作用时间的增加,改变进一步加剧。至作用36hr微管蛋白聚合在一侧,荧光强度增加,纤维不再清晰可见,与紫杉醇作用36hr的微管蛋白改变类似。吴茱萸碱作用后使得细胞微管蛋白的聚合状态发生了改变。
③Western Blot分析Survivin凋亡抑制基因的表达,实验中阴性对照组Survivin是高表达的,吴茱萸碱作用16hr后,Survivin的表达较阴性对照组即以开始下降,作用20hr、24hr及36hr则均已无Survivin的特异性条带出现。
综上结果提示,吴茱萸碱通过影响细胞周期调控诱导SGC-7901细胞凋亡的作用机制可能与线粒体跨膜电位的丢失、影响微管蛋白聚合以及抑制Survivin基因的表达相关,是一个多靶点作用的过程。
3.6吴茱萸合用青黛体内抗瘤实验。
①动物整体水平研究选用皮下移植性小鼠(正常鼠)肝癌模型的方法,成瘤率100%。
②实验一中,最终存活下来的小鼠一般情况以环磷酰胺组最佳,但环磷酰胺组仅存活5只。环磷酰胺组、吴茱萸组、青黛组以及联合组的抑瘤率分别为43.9%、35.6%、27.7%和37.2%。瘤体积比较结果表明:各治疗组瘤体积均较模型对照组小,各组间两两比较仅联合组与模型对照组间有统计学意义(p=0.048)。
③实验二中,存活小鼠一般情况类似实验一部分。环磷酰胺组、吴茱萸组、青黛组以及联合组的抑瘤率分别为50.1%、44.9%、45.9%和51.9%。瘤体积比较结果表明:各治疗组瘤体积均较模型对照组小,且各治疗组与模型比较均有统计学意义。联合组与环磷酰胺组之间虽无统计学差异,但联合组的瘤体积平均值小于环磷酰胺组。同时,吴茱萸组、青黛组也均小于环磷酰胺组。进行生存期观察小鼠,当药物干预进行到第19d时环磷酰胺组陆续出现恶病质表现,体重明显下降,目光晦暗呆滞,弓背,张口呼吸,肉眼可见肿块虽较其他治疗组小但小鼠的一般情况远不及其他治疗组小鼠。各中药组改善移植瘤小鼠生存质量的优势显现了出来。药物作用30d后,不治疗继续观察至小鼠全部死亡,计算各治疗组的生命延长率。各治疗组较模型对照组均能延长存活时间,环磷酰胺组、联合组与模型对照组的平均存活时间之间具有统计学意义。环磷酰胺组、吴茱萸组、青黛组以及联合组的生命延长率分别为40.1%、36.04%、18.78%、70.05%,联合组要显著高于环磷酰胺组,但二者间差别无统计学意义。
综上结果提示,吴茱萸合用青黛对移植肿瘤小鼠具有抗肿瘤作用,提示吴茱萸碱与靛玉红合用可以增加荷瘤小鼠瘤体生长抑制作用和延长荷瘤小鼠生存时间作用。
四、结论:
吴茱萸碱具有显著抑制肿瘤细胞的生长和抑制肿瘤细胞分裂复制的能力,且这种抑制作用可能具有细胞分裂指数选择性。吴茱萸碱对SGC-7901细胞持续作用16-20hr后即启动了细胞的凋亡程序。吴茱萸碱诱导SGC-7901细胞凋亡而发生肿瘤细胞的死亡,凋亡的发生既与D-mitosis事件的产生和维持4-8hr相关又与M-slippage事件的发生相关。吴茱萸碱通过影响细胞周期调控诱导SGC-7901细胞凋亡的作用机制可能与线粒体跨膜电位的丢失、影响微管蛋白聚合以及抑制Survivin基因的表达相关,是一个多靶点作用的过程。吴茱萸与青黛合用可以增加荷瘤小鼠瘤体生长抑制作用和延长荷瘤小鼠生存时间作用。吴茱萸碱作为抗肿瘤药具有极大的开发潜力。
Objective:
Evodiamine is an indole quinazoline alkaloid,the main active substance of evodiae.Cell experiments show that evodiamine with antineoplastic activity, directly induce tumor cell apoptosis,inhibit tumor invasion and metastasis, inhibit tumor angiogenesis,inhibit activity of multidrug resistance cell line and so on.Preliminary studies have confirmed that evodiamine could induce tumor cells apoptosis and induce tumor cells arresting in G2/M phase,there is the phenomenon of D-mitosis.D-mitosis and M-slippage,these two events have taken place,apoptosis would be induced only.The current view consider that apoptosis is closely linked to the cell cycle as activities of life.Apoptosis induced by different factors,can occur in different cell cycle.The study was to elucidate the mechanisms of apoptosis induced by evodiamine and the relationship between apoptosis and cell cycle regulation,as well as the further development and application of evodiamine.At the same time,it was provided foundation of more effective Chinese herbal against the cell cycle and developed complex prescription according to the mechanism of Chinese herbal.
Methods:
2.1 Evodiamine could inhibit the growth of tumor cells in vitro.We selected SGC-7901、HepG2、CBRH7919、primary Kidney,primary Liver cells,used MTT assay to test above 5 cells antipoliferative effect after being treated with evodiamine.The cell cycle and apoptosis index of SGC-7901 were examined using FACScan flow cytometry,the colone assay was used to detect the colone inhibition ratio,microscope was used to observe the changes in cell morphology.
2.2 In order to reproduct the best method of SGC-7901 cell cycle synchronization,we used two methods,one was double TD blocking,the other was serum starvation.
2.3 We used FACScan flow cytometry to look for reversible drug detection time after being treated with evodiamine.Accordance with irreversible point in time,after being treated with evodiamine the morphology of apoptosis and chemical detection were done;The cell cycle and apoptosis index of SGC-7901 were examined using FACScan flow cytometry;Expression of CyclinB1 and CyclinE were examined using Western blotting analysis;Comet assay and colone assay were used to prove hypothesis.
2.4Construction of gene ND-cyclinB1 with GFP fusion gene expression systems, as well as FL-cyclinB1 gene fusion with the GFP gene expression system to confirm hypothesis by FACScan flow cytometry and Quantitative analysis of fluorescence.
2.5 The initial mechanisms of Evodiamine induced apoptosis requires both activation of D-mitosis and M-slippage:through the JC-1 staining of mitochondrial membrane potential change,α-tubulin changes,Western blotting analysis of anti-apoptosis gene Survivin expression.
2.6 Produce the transplanted liver cancer(H22) in Kunming mice,the general situation、tumor inhibition rate、tumor volume、survival period were determed after being treated with Evodiae and Indigo naturalis.
Results:
3.1Evodiamine in vitro cell growth inhibition results show:
①SGC-7901,HepG2,CBRH7919 three tumor cells respectively increased 0.9,1.13,1.39 times after 48hr,Kidney and Liver cells respectively increased 0.24,0.18 times after 48hr;Simultaneously cell acute toxicity experiments reveaIed that the evodiamine sensitivity of three types of tumor cells were significantly higher than the primary cells.
②MTT assay confirmed growth inhibition of evodiamine was time and dose-dependent,IC_(50) was1.36μmol.
③FACScan flow cytometry showed 1μmol·L~(-1) evodiamine for 12hr the G2/M phase cells account for 78.8%,but the apoptotic index was only 4.1%and negative was 4.4%in the control group no significant difference,1μmol·L~(-1) evodiamine for 24hr the G2/M phase declineed in the proportion,the apoptotic index increased to 34.0%,evodiamine for 36hr the G2/M phase ceils accounted for 0.7%,the apoptotic index was 52.5%.
④Colone assay we found 2×10~3cells/hole was the best experiment cell density in 6-well plates,evodiamine had significant inhibition of colony formation.
⑤Experimental observation of cell morphology,we saw typical morphological changes of apoptotic cells,such as smaller cell size,vacuolization, chromatin cohesion,karyopyknosis,the emergence of apoptotic bodies.
The results suggested that Evodiamine could inhibit the growth and division of tumor cells,and this inhibition was selective,that was,the faster growth cells were stronger sensitive to evodiamine.
3.2 To product SGC-7901 cell cycle synchronization.
①we used double TD blocking method.TD final concentration of 2mM,4mM,8mM after,G0/G1 phase cells were77.3%,77.5%and 77.0%,while G2/M phase cells were 2.0%,2.1%and 2.1%.Another large group in the above mentioned double TD blocking then released 12hr,TD final concentration of 2mM,4mM,8mM of G0/G1 phase cells decreased to 44.0%,38.0%and 36.6%,and G2/M phase cells increased to39.9%,46.2%and 49.1%.
②The other method-serum starvation synchronization,flow cytometry indicated that the serum-free culture medium,respectively,24hr,30hr,36hr,G0/G1 phase cells were 57.4%,59.9%and 68.4%,and G2/M phase cells were11.4%,5.0% and 10.1%.Another large group in the above mentioned serum starvation then released 12hr,respectively,24hr,30hr,36hr of G0/G1 phase cells decreased to 50.3%,51.4%and 51.4%,while G2/M phase cells increased to 13.8%,22.7% and 22.7%,the period of cell number was not change significantly.Serum-free culture medium for 24hr,30hr and 36hr,both appeared typical hypodiploid apoptotic peak.
The results suggested that Double TD blocking cell cycle synchronization method was an ideal access to gain a large number of ground state cells,and didn't rely on the TD concentration.Treatment by evodiamine for 16-20hr, SGC-7901 switched on apoptotic process,resulting in irreversible death.
3.3 Explore the impaction in M phase of SGC-7901 by evodiamine.
①About the reversible point in time after experiment we found, 1μmol·L~(-1)evodiamine for12hr the apoptotic index was 4.1%.For16hr,the apoptotic index was 4.0%,then released the drug apoptotic index was 8.5%. For 20hr two different types had significant difference,the released group apoptotic index increased to40.4%,was significantly higher than non-released group-16.0%.For 24hr,28hr,32hr and 36hr the apoptotic index of the released group were significantly higher than non-released group.According to evodiamine-induced apoptosis irreversible point in time between 16-20hr,we used 1μmol·L~(-1) evodiamine respectively for 16hr,20hr,24hr and 36hr.
②Western Blot analysis of the level of CyclinB1:Treated by evodiamine for 16hr the level was significantly higher than negative cells,but the level of evodiamine for 20hr was lower than 16hr,while 24hr and 36hr with no protein expression.Western Blot analysis of the expression of CyclinE,negative cells, 1μmol·L~(-1) evodiamine for16hr,20hr and 24hr were both no specific bands,and evodiamine for 36hr appeared band.The expression of CyclinE tangled to CyclinB1,suggesting that after treating by evodiamine for 36hr cells arrested in M phase,then went into G1 phase.
③The growth type of SGC-7901 was polygonal or diamond.PI staining was observed under fluorescence microscope,chromosomes can be seen shrinkaged assuming trigeminal after treating evodiamine for 16hr;After treatment for 24hr,could not see the obvious apoptotic bodies,the majority of cells wore still in a state of chromosome condensation;After treatment for 36hr,we could see the obvious apoptotic bodies,at the same time could see the multinucleated cells.
④The comet assay,we found the damaged DNA swam,a long tail appeared behind the cell.The average optic density was lower than the negative cells after treating with statistical significance.The comet tail was longer than the negative cells after treating with statistical significance,both two changes were related to time.
⑤FACScan flow cytometry showed:apoptosis index of 1μmol·L~(-1) CDK1I was 7.6%, the negative cell was 3.5%,no significant difference.After treating by evodiamine forl8hr added CDK1I for 6hr the apoptotic index significantly increased.0.5μmol·L~(-1) evodiamine for 24hr the apoptotic index was 8.9%,then another group added CDK1I the apoptotic index increased to 28.2%.1μmol·L~(-1)evodiamine for 24hr the apoptotic index was 20.0%,then another group added CDK1I the apoptotic index increased to 32.0%.
⑥The colone assay,we found the colone inhibition index was 24.84%after treating by 7.5μM CDK1I,and added CDK1I combinding to evodiamine the colone inhibition index significantly increased.0.5μmol·L~(-1) evodiamine for 24hr the colone inhibition index was 38.01%,then another group added CDK1I the colone inhibition index increased to 65.34%.1μmol·L~(-1) evodiamine for 24hr the colone inhibition index was 49.81%,then another group added CDK1I the colone inhibition index increased to 70.32%.The colone Inhibition was dose-dependent.
⑦Dapi staining was observed under fluorescence microscope,negative cells were firmly adherent,bright,extended,uniform.While SGC-7901 after treating by CDK1I were bigger and stronger.0.5μmol·L~(-1)and 1μmol·L~(-1)evodiamine group could be seen larger sizes,condensed chromatin status,apoptotic bodies.Added CDK1I for 3hr cells re-adherent and at the same time the combined effect for 6hr,multinucleated cells appeared concording with PI staining.
The results suggested that Evodiamine induced SGC-7901 apoptosis,and signal apoptosis related on the generation and maintenance of D-mitosis and Mitotic-slippage event.
3.4 Gene ND-CyclinB1,FL-CyclinB1 have successfully connected with the GFP gene.
①Recombinant plasmid pEGFP-N1-ND CyclinB1,pEGFP-N1-FL CyclinB1 transfected SGC-7901,ND-CyclinB1,FL-CyclinB1 gene were expressed in SGC-7901.
②Recombinant plasmid transfected SGC-7901,after treating by evodiamine, FACScan flow cytometry showed that the apoptotic index of negative cell was 4.1%,pEGFP-N1-ND CyclinB1,pEGFP-N1-FL CyclinB1 respectively was 38.2%,41.3%, and without the role of evodiamine the apoptotic index respectively was14.5%,10.7%,with significant difference.
③Recombinant plasmid transfected SGC-7901 cells,1μmol·L~(-1)evodiamine for 36hr the blind score of pEGFP-N1-ND CyclinB1was 3.60,were higher than pEGFP-N1-FL CyclinB1 and empty pEGFP-N1 as the role of evodiamine 2.50 and 1.40.
The results suggested that Recombinant plasmid of pEGFP-N1-ND and pEGFP-N1-FL were success,they could applicated to research cell cycle-related study in the future.Method of making cells arrest in M phase,we can get the same conclusion,that is evodiamine induced SGC-7901 apoptosis relatde on D-mitosis event.
3.5 Explore the possible mechanisms of cell cycle regulation inducting apoptosis
①Application JC-1 to observe mitochondrial membrane potential changes,result showed:red,green fluorescence dispersed uniformly in the negative cells, red fluorescent spread in cells.After treatment for 16hr,the red fluorescence intensity decreased,while the green fluorescence intensity enhanced,indicating that mitochondrial membrane potential damaged.After treatment for 20hr,the red fluorescence intensity decreased significantly, while the green fluorescence intensity increased significantly.After treatment for 24hr,red fluorescence scattered,while for 36hr almost no red fluorescence.
②Then we Observedα-tubulin,the negative cells with clearly visible fibers, showing fine filaments.After treatment for 16hr,tubulin fibers could be seen beginning with disorders,shorter fracture.After treatment for 20hr,tubulin fibers could be seen bending happened,changing the status polymerization.Treatment lasted 36hr,tubulin polymerized in the side, fluorescence intensity increased,the changes similar to taxol-tubulin.
③Western Blot analysis Survivin expression,negative cell was in a high expression,after treatment for 16hr evodiamine,Survivin begen to decline.After treatment for 20hr,24hr and 36hr then had no Survivin specific bands appeare.
The results suggested that Evodiamine induced SGC-7901 apoptosis might be related to the loss of mitochondrial transmembrane potential,the impact of tubulin polymerization and inhibit the expression of Survivin gene,was a multi-target process.
3.6 Rutaecarpa combined Qingdai anti-tumor experiments in vivo.
①Transplanted subcutaneously mice of liver cancer model could formed tumor 100%.
②Experiment I,we found that Cyclophosphamide group mice was in best general state of health,but only five mice survived.The tumor inhibition rate of cyclophosphamide group,evodiamine group,qingdai group and combinded group respectively was 43.9%,35.6%27.7%and 37.2%.Tumor size results showed that the treatment groups' were smaller than control group and combinded group compared with cyclophosphamide group had statistical significace(p=0.048).
③ExperimentⅡ,general state of health was similar to the experimentⅠ.The tumor inhibition rate of cyclophosphamide group,evodiamine group,qingdai group and combinded group respectively was 50.1%,44.9%,45.9%and 51.9%.Tumor size results showed that the treatment groups' were smaller than control group. The life extension rate of cyclophosphamide group,evodiamine group,qingdai group and combinded group respectively was 40.1%,36.04%,18.78%,70.05%,the combinded group was significant higher than cyclophosphamide group.
Conclusion:
Evodiamine could inhibit the growth and division of tumor cells,and this inhibition is selective,that is,the faster growth cells are stronger sensitive to evodiamine.Treatment by evodiamine for 16-20hr,SGC-7901 switches on apoptotic process,resulting in irreversible death.Evodiamine induces SGC-7901 apoptosis,and signal apoptosis relats to the generation and maintenance of D-mitosis and Mitotic-slippage event,Evodiamine induces SGC-7901 apoptosis may be related to the loss of mitochondrial transmembrane potential,the impact of tubulin polymerization and inhibit the expression of Survivin gene,is a multi-target process.
引文
[1]陈意生,史景泉.肿瘤分子细胞生物学[M].北京:人民军医出版社,2002,291-308.
[2]Kin KL,Cidlowaki-JA.Cell cycle and apoptosis:common pathways to life and death[J].Cell Biochem,1995,58(2):175-180.
[3]Tao,WK,V.J.South,Y.Zhang,et al,Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage[J].Cancer Cell,2005,8:49-59.
[4]Jie Guang Chen,Chia Ping Huang Yang,Michael Cammer,et al.Gene expression and mitotic exitinducedby microtubule-stabilizing drugs[J].Cancer research,2003,15,7891-7899
[5]Weikang Tao,Victoria J.South,Ronald E.Diehl,et al.An inhibitor of the Kinesin Spindle Protein activates the intrinsic apoptotic pathway independently of p53 and de novo protein synthesis[J].Molecular and cellular biology,2007,27,689-698.
[6]郁仁存,姜廷良,于尔辛.肿瘤研究[M].上海:上海科学技术出版社,1991.
[7]杨柱,陈学习.肿瘤的中医病因病机初探[J].辽宁中医杂志,2002,29(4):197-198.
[8]李庆玲.谈中医药治疗肿瘤的特色及其作用[J].中医药学报,2001,29(5):4-6.
[9]沈珝琁,方福德.真核基因表达调控[M].高等教育出版社-斯普林格出版社,1999,151.
[10]Catania A,Urban S,Yan E,et al.Expression and localization of cyclin-dependent kinase 5 in apoptotic human glioma cell[J].Neuro-onco,2001,3:89-98.
[11]Sun Y,Tang J.Expression of CyclinE and CDK5 in lung cancer[J].J Dalian Med Uni,2003,25:4-6.
[12]Garber K,Beyond the Nobel Price:cell cycle researcs new view on cancer[J].Natl Can-Cer Inst,2001,93:1766-1768.
[13]Jiang M,Shao ZM,Wu J,et al.p21/walf/cipl and mdm22 expression in breast carcinoms patients as related to prognosis[J].Int J Cancer,1997,74:529-534.
[14]Liang B,Wang S,Yang X,et al.Expression of cyclin E,cyclin dependent kinase 2 and p57(KIP2)in human gastric cancer[J].Chin Med,2003,116:20-23.
[I5]ItoY,Takeda T,Sakon M,et al,Expression of p57/kip2 protein in hepatocellular carcinoma[J].Oncology,2001,61:221-225.
[16]Rosenberg E,Demopoulos RI,Zeleniuch-Jacquotte A,et al,Expression of cell cycle regulators p57(KIP2),cyclin D1 and cyclin E in epithelial ovarian tumors and survival[J].Hum Pathal,2001,32:808-813.
[17]Yu HY,Shi CG,Zhu JJ,et al,CyclinA expression in non-small cell lung carcinoma as related to proliferative activity and prognosis[J].Chin J Cancer,2001,20:38-40.
[18]Zhang Y,Peng ZH,Qiu GQ,et al,Overexpression of cyclinA in hepatocellular carcinoma and its relationship with HBx gene integration[J].Chin J Oncol,2002,24:353-355.
[19]申漫里,冯永东,高纯等.细胞周期蛋白B1在肿瘤细胞中的非时相性表达[J].中华肿瘤杂志,2002,24(3):215-219.
[20]Barrett K L,Demiranda D,Katula K S.Cyclin B1 promoter activity and functional CDK1 complex formation in G1 phase of human breast cancer cells[J].Cell Biol Int,2002,26(1):19-28.
[21]Mishina T,Dosaka-Akita H,Hmmura F,et al.CyclinE expression,a potential prognostic marker for non-small cell lung cancer.Cancer Res,2000,6:11-16.
[22]Resnitzky D,Reed SI.Different roles for cyclinD1 and E in regulation of the G1-to-S transition.Mol Cell Biol,1995,15:3463-3469.
[23]Mikhailov A,Cole RW,Rieder CL.DNA damage during mitosis in human cells delays the metaphase/anaphase transition via the spindle-assembly checkpoint[J].Curr Biol,2002,12(21):1797-1806.
[24]Zhou J,Yao J,Joshi HC.Attachment and tension in the spindle assembly checkpoint[J].J Cell Sci,2002,115(18):3547-3555.
[25]Vigneron S,Prieto S,Bernis C,et al,Kinetochore localization of spindle checkpoint proteins:who controls whom[J].Mol Biol Cell,2004,15(10):4584-4596.
[26]Agarwal ML,Ramana CV,Hamilton M,et al,Regulation of p53 expression by the RAS-MAP kinase pathway[J].Oncogene.,2001,20(20):2527-36.
[27]Innocente SA,Abrahamson JL,Cogswell JP,et al,p53 regulates a G2 checkpoint through cyclin BI[J].Proc Natl Acad Sci U S A.,1999,9 6(5):2147-52.
[28]Gajate C,Barasoain I,Andreu JM,et al,Induction of apoptosis in leukemic cells by the reversible microtubule-disrupting agent 2-methoxy-5-2,4,6-cyclonheptatrien-1-one:protection by Bcl-2 and BcI-X(L) and cell cycle arrest.Cancer Res.2000,60(10):2651-2659.
[29]Panvichian R,Orth K,Day ML,et al,Paclitaxel-associated multimininucleation is permitted by the inhibition of caspase activation:a potential early step in drug resistance[J].Cancer Res,1998,58(20):4667-4672.
[30]Heide LF,Robert AS,James D.Cell cycle and growth control.2nd[J].NJ:Wiley-Liss,2004,95-128.
[31]Woods CM,Zhu J,McQueney PA,et al,Taxol-induced mitotic block trigger rapid onset of a p53-independent apoptotic pathway[J].Mol Med,1995,1(5):506-526.
[32]Sherwood SW,Sheridan JP,Schirnke RT.Induction of apoptosis by the anti-tubulin drug colcemid:relationship of mitotic checkpoint control to the induction of apoptosis in Hela S3cells[J].Exp Cell Res,1994,215(2):373-379.
[33]Michalak,E.,A.Villunger,M.Erlacher,and A.Strasser.Death squadsenlisted by the tumour suppressor p53[J].Biochem.Biophys.Res.Commun.2005,331:786-798.
[34]Maria Castedo,Arnaud Coquelle,Sonia Vivet,et al,Apoptosis regulation in tetraploid cancer cells[J].The EMBO Journal,2006,25,2584-2596.
[35]Huang YC,Guh JH,Teng CM.Induction of mitotic arrest and apoptosis by evodiamine in humanleukemic T-lymphocytes[J].Life sci,2004,75(1):35.
[36]吴琦玮,葛忠良,高月等.靛玉红对肿瘤细胞抑制作用的研究及相关机制探讨[J].天津中医药,2008,25(1):55-58.
[37]Knowles LM,Milner JA.Diallyl disulfide induces ERK phosphorylation and ahem gene expression profiles in human colon tumor cels[J].J Nutr,2003,133(9):2901-2906.
[38]Panvichian R,Orth K,Day ML,et al,Paclitaxel-associated multimininucleation is permitted by the inhibition of caspase activation:a potential early step in drug resistance[J].Cancer Res,1998,58(20):4667-4672.
[39]王宗伟,杨安平,吴庆光.中华芦荟多糖对辐射小鼠胸腺细胞凋亡及细胞周期的影响[J].中药新药与临床药理,2005,16(4):240-243.
[40]刘宇炜,文若剑,易卉玲.高温和当归联合作用对Hela细胞周期的影响[J].中华中西医杂志,2005,6(21).
[41]赵连梅,单保恩,艾军等.香加皮杠柳苷对人食管癌细胞TE-13生长抑制作用[J].肿瘤,2008,28(3):203-206.
[42]赵丕文,牛建昭,王继峰等.6种中药活性成分植物雌激素作用的比较研究[J].中国药学杂志,2007,42(24):1852-1855.
[43]刘展华,曹洋,林丽珠等.斑蝥酸钠对人肺癌LAC细胞周期的影响及其分子机制的研究[J].广州中医药大学学报,2007,24(6):486-489.
[44]王鹂,吴军,李敏等.华蟾毒精抑制HeLa细胞增殖作用机制的探讨[J].中华肿瘤杂志,2005,27(12):717-720.
[45]周曦,陈忠东.三氧化二砷对宫颈癌Hela细胞体外生长的影响[J].南华大学学报医学版,2007,35(4):525-528.
[46]Janos H,Fendler.Membrane mimetic chemistry:characterizations and applications of micells,microemulsions,monolayers,bilayers,vesicles,host-guest systems,and polyions[J].New York:Wiley,1982:35-36.
[47]傅炜昕,梁再赋,李铁英等.槲寄生凝集素对肿瘤细胞细胞周期的影响[J].沈阳药科大学学报,2005,22(1):59-62.
[48]王菁鹏,林青.姜黄素对子宫颈癌HeLa细胞增殖抑制作用及其机制的研究[J].现代肿瘤医学,2006,14(8):1001-1003.
[49]张大鹏,孔棣.中药对体外人结肠癌THC8908细胞的生长抑制作用[J].中国中西医 结合外科杂志,2007,13(3):270-272.
[50]肖宝红,张春玲,武晓.猫眼草水提物对小鼠Lewis肺癌的抑制作用及机制探讨[J].中国中药杂志,2006,31(12):1002-1005.
[51]陈晓莉,王骊骊.葛根提取物对肝癌细胞增殖及细胞周期的影响[J].广东药学院学报,2001,17(3):183-184.
[52]李飞.方剂学[M].北京:人民卫生出版社,2002;125-128.
[53]Chiou WF,Chou CJ,Shum AY.The vasorelaxant effect of evodiamine in rat isolated mesenteric arteries mode of action[J].Eur J Pharmacol,1992,215(3):277-283.
[54]Wu CL,Hung CR,Chang FY,et al,Effects of evodiamine on gastrointestinal motility in male rats[J].Eur J Pharmacol,2002,457(2-3):169-176.
[55]Rang WQ,Du YH,Hu CP,et al,Protective effects of evodiamine on myocardial ischemia- reperfusion injury in rats[J].Planta Med,2004,70(12):1140-1143.
[56]Lin H,Tsai SC,Chen JJ,et al,Effects of evodiamine on the secretion of testosterone in rat testicular interstitial cells[J].Metabolism,1999,48(12):1532-1535.
[57]Chiou WF,Sung YJ,Liao JF,et al,Inhibitory effect of dehydroevodiamine and evodiamine on nitric oxide production in cultured murine macrophages[J].J Nat Prod,1997,60(7):708-711.
[58]Chou WF,Chen CF.Pharmacological profile of evodiamine in isolated rabbit corpus cavernosum[J].Eur J Pharmacol,2002,446(1-3):151-159.
[59]Kobayashi Y,Nakano Y,Hoshikuma K.The bronchoconstricttive action of evodiamine,an indoloquinazoline alkaloidisolated from the fruits of Evodia rutaecarpa,on guineapig isolated bronchus:possible involvement on vanilloid receptors[J].Planta Med,2000,66(6):526-530.
[60]张莹,吴立军,田代真一等.吴茱萸碱诱导人黑色素瘤A375-S2细胞的两种死亡机制[J].药学学报,2003,38(9):650-3.
[61]Zhang Y,wu L J,Tashiro S,et al,Evodiamine induces tumor cel ideath through different pathways:apoptosis and necrosis[J].Acta Pharmacol Sin,2004,25(1):83-8.
[62]Huang DM,Guh JH,Huang YT,et al,Induction of mitotic arrest and apoptosis in human prostate cancer pc-3 cells by evodiamine[J].J Urol,2005 Jan,173(1):256-61.
[63]Kan SF,Huang WJ,Lin LC,et al,Inhibitory effects of evodiamine on the growth of human prostate cancer cell line LNCaP[J].Int J Cancer,2004 Jul 10,110(5):641-51.
[64]Huang YC,Guh JH,Teng CM.Induction of mitotic arrest and apoptosis by evodiamine in human leukemic T-lymphocytes[J].Life sci,2004,75(1):35.
[65]Zhang V,Zhang Q H,Wu L J,et al,Atypical apoptosis in L929 cells induced by evodiamine isolated from Evodia rutaecarpa[J].J Asian Nat Prod Res,2004,6(1):19.
[66]Yu-Hui Tan,Wei-Wen Chen,Ying-Ya Wu,et al,Effects of berberine,evodiamine and indirubin on gastric cancer cell[J].World Chin J Digestol,2005 February 15,13(4):472-476.
[67]ChoHwa Liao,ShiowLin Pan,Jih-Hwa Guh,et al,Antitumor mechanism of evodiamine,aconstituent from Chinese herb Evodiae fructus,in human multiple-drug resistant breast cancer NCI/ADR-RES cells in in vitro and in vivo Carcinogenesis vol[J].26 no.5 2005pp,968-975.
[68]赞晓方,王本祥,池岛乔.吴茱萸碱诱导人子宫颈癌HeLa细胞凋亡的机制研究[J].药学学报,2002,37(9):673-5.
[69]Wang C,Wang MW,Tashiro S,et al,Roles of SIRT1 and phosphoinositide 3-OH kinase/protein kinase C pathways in evodiamine-induced human melanoma A375-S2 cell death[J].J Pharmacol Sci.2005 Apr,97(4):494-500.
[70]Wang C,Wang MW,Tashiro S,et al,Effect of protein kinase C on human melanoma A375-S2cell death induced by evodiamine[J].Yao Xue Xue Bao.2005 Nov,40(11):1033-6.
[71]Wang C,Wang MW,Tashiro S,et al,Evodiamine induced human melanoma A375-S2cell death partially through interleukin 1 mediated pathway[J].Biol Pharm Bull.2005Jun,28(6):984-9.
[72]Zhang Y,Wu L J,Tashiro S,et al,Intracellular regulation of evodiamine-induced A375-S2 cell death[J].Biol Pharm Bul 1,2003,26(11):1543-6.
[73]Ogasawara M,Matsubara T,Suzuki H.Inhibitory effects of evodiamine on in vitro invasion and experimental lung metastasis of murine colon cancer cells[J].Biol Pharm Bull.2001 Aug,24(8):917-20.
[74]Ogasawara M,Suzuki H.Inhibition by evodiamine of hepatocyte growth factor-induced invasion and migration of tumor cells[J].Biol Pharm Bull.2004 Apr,27(4):578-82.
[75]KouGiShyu,ShankungLin,ChunChungLee,et al,Evodiamine inhibits in vitro angiogenesis:Implication for antitumorgenicity[J].Life Sciences.78(2006),2234-2243.
[76]Liao CH,Pan SL,Gu LH,et al,Antitumor mechanism of evodiamine,a constituent from Chinese herb Evodiae fructus,in human multiple-drug resistant breast cancer NCI/ADR-RES cells in vitro and in vivo[J].Carcinogenesis,2005,26(5):968-975.
[77]Shimomura 0,Johnson F H,Saiga Y.Extraction,purification and properties of aequorin,a bioluminescent protein from the luminous hydromedusan,Aequorea[J].J Cell Comp Physiol,1962,59:223-229.
[78]Prasher D C,Eckenrode V K,Ward W W,et al,Primary structure of the Aequorea victorea green fluorescent protein[J].Gene,1992,111(2):229-233.
[79]Chalfle M,Tu Y,Euskirchen G,et al,Green fluorescent protein as a marker for gene expression[J].Science,1994,263(5148):802-805.
[80]Heim R,Prasher D C,Tsien R Y.Wavelength mutations and posttranslational autoxidati -on of green fluorescent protein[J].Proc Natl Acad Sci USA,1994,91(26):12501-12504.
[81]Helm R,Tsien R Y.Engineering green fluorescent protein for improved brightness,long -er wavelengths and fluorescence energy transfer[J].Curr Biol,1996,6(2):178-182.
[82]Ormo M,Cubitt AB,Kallio K et al,Crystal structure of the Aequorea victoria green fluorescent protein[J].Science,1996,273(5280):1392-1395.
[83]Muller-Taubenberger A,Anderson K I.Recent advances using green and red fluorescent protein variants[J].Appl Microbiol Biotechnol,2007,77(1):1-12.
[84]Yang F,Moss L G,Phillips G N.The molecular structure of green fluoresent protein[J].Nat Biotech,1996,14(10):1246-1251.
[85]储军,施华,杨杰等.分子与细胞事件的光学可视化[J].生物化学与生物物理进展.2008,35(10):1104-1111.
[86]刘艳丽,鲁澄宇.绿色荧光蛋白在药学研究中的应用进展[J].药学服务与研究,2008,8(5):326-328.
[87]Andreas Jacobs,Michael Dubrovin,et al,Functional Coexpression of HSV-1 Thymidine Kinase and Green Fluorescent Protein:Implications for Noninvasive Imaging of Transgene Expression[J].Neoplasia,1999,1(2):154-161.
[88]杨洁,聂青和.绿色荧光蛋白标记技术及其在基础临床研究中的应用[J].实用肝脏病杂志,2007,12(6):420-422.
[89]潘启超,管彬主编.肿瘤药理学与化学治疗学[M].河南医科大学出版社,第二版,2000,2-9.
[90]Cooper S.Reappraisal of serum starvation,the restriction point,G0 and G1 phase arrest points[J].FASEB J,2003,17(3):333-340.
[91]斯佩克特DL.细胞实验指南[M].黄培堂译.北京:科学出版社,2001,93-98.
[92]Collin AR,Dusinska M,Franklin M,et al,Comet assay in human biomonitoring studies:reliability,validation,and applications[J].Environ Mol mutagen,1997,30:139-146.
[93]Wolf F,Wandke C,Isenberg N,et al,Dose-dependent effects of stable cyclin B1 on progression through mitosis in human cells[J].EMBO J.,2006,25(12):2802-13.
[94]Salvioli S,Ardizzoni A,Franeoschi C,et al.JC-1,but not DiOC6(3)or Rho123,is a reliable fluorescent probe to assess changes in intact cells:implications for studies on mitoehondflal functionality during apoptosis[J].FEBS Lett,1997,411(1):77-82.
[95]翟中和,王喜忠,丁明孝.细胞生物学[M].北京:高等教育出版社,2000.
[96]Hammond JW,Cai D,Verhey KJ.Tubulin modifications and their cellular functions[J].Curr Opin Cell Biol,2008,20(1):71-76.
[97]徐忠东,吴琴.微管蛋白研究进展[J].安徽教育学院学报,1999,(2):73-74.
[98]吴平平,李苏宜.紫杉醇耐药与微管蛋白的研究进展[J].临床肿瘤学杂志,2008,1(8):755-757.
[99]Red R,Oakley.An abundance of tubulins[J].Trends in Cell Biology,2000,(10):537-542.
[100]Schmidt M,Bastian H.Mitotic drug targets and the development of novel anti mitotic anticancer drugs[J].Drug Resist Updat,2007,10(4-5):162-181.
[101]Ambrosini G.,Adida C,Ahieri D C.A novel anti-apoptosisgene survivin expressed in cancer and lymphoma[J].Nat Med,Ⅰ997,3(8):917-921.
[102]Javle M M,Tan D,Yu J,et al,Nuclear survivin expression predicts poor outcome in cholan-giocarcinoma[J].Hepatogastroenterology,2004,51(60):1653-1657.
[103]Span P N,Sweep FC,Wiegerinck E T,et al,Survivin is an independent prognostic marker for risk stratification of breast cancer patients[J].Clin Chem,2004,50(11):1986-1
[104]Huber BE,Austin EA,Good SS.In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase[J].CancerRes,1993;53(19):4619
[105]李翠云,段小娴.肝癌动物模型研究进展[J].医学研究杂志,2006,35(10):71-73.
[106]胡卫,陈涛.鼠移植性肝癌模型研究进展[J].河南肿瘤学杂志,2003,16(6):463-466.
[107]徐静,李旭.肝癌动物模型的建立[J].实用肝脏病杂志,2005,8(2):116-118.
[108]王海宁,熊慧林,黄作苏.HPLC测定吴茱萸中吴茱萸碱的含量[J].贵州师范大学学报(自然科学版),2001,19(1):74-75.
[109]于澍,王楚盈,魏中元等.吴茱萸碱抗肿瘤作用研究[J].吉林中医药,2008,28(3):223-223.
[110]王通,曾耀英,肇静娴等.靛蓝和靛玉红对小鼠T细胞活化与增殖的影响[J].中国医科大学学报,2005,36(5):444-447.
[111]Xiao Z,Hao Y,Liu B,et al.Indirubin and meisoindigo in the treatment of chronic myelogenous leukemia in China[J].Leuk Lymphoma,2002,43:1763-1768.
[112].Knockaert M,Blondel M,Bach S,et al,IndeDendent actions on cyclin-dependent kinases and aryl hydrocarbon receptor mediate the antiDroliferative effects of indirubins[J].Oncogene,2004,23:4400-4412.
[113].沈映君.中药药理学[M].北京:人民卫生出版社.2000.165.
[114]Eisenbrand G,Hippe F,Jakobs S,et al,Molecular mechanisms of indirubin and its derivatives:novel anticancer molecules with their origin in traditional Chinese phytomedicine[J].J Cancer Res Clin Oncol,2004,130(11):627-635.
[2]Kin KL,Cidlowaki-JA.Cell cycle and apoptosis:common pathways to life and death[J].Cell Biochem,1995,58(2):175-180.
[3]Tao,WK,V.J.South,Y.Zhang,et al,Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage[J].Cancer Cell,2005,8:49-59.
[4]Jie Guang Chen,Chia Ping Huang Yang,Michael Cammer,et al.Gene expression and mitotic exitinducedby microtubule-stabilizing drugs[J].Cancer research,2003,15,7891-7899
[5]Weikang Tao,Victoria J.South,Ronald E.Diehl,et al.An inhibitor of the Kinesin Spindle Protein activates the intrinsic apoptotic pathway independently of p53 and de novo protein synthesis[J].Molecular and cellular biology,2007,27,689-698.
[6]郁仁存,姜廷良,于尔辛.肿瘤研究[M].上海:上海科学技术出版社,1991.
[7]杨柱,陈学习.肿瘤的中医病因病机初探[J].辽宁中医杂志,2002,29(4):197-198.
[8]李庆玲.谈中医药治疗肿瘤的特色及其作用[J].中医药学报,2001,29(5):4-6.
[9]沈珝琁,方福德.真核基因表达调控[M].高等教育出版社-斯普林格出版社,1999,151.
[10]Catania A,Urban S,Yan E,et al.Expression and localization of cyclin-dependent kinase 5 in apoptotic human glioma cell[J].Neuro-onco,2001,3:89-98.
[11]Sun Y,Tang J.Expression of CyclinE and CDK5 in lung cancer[J].J Dalian Med Uni,2003,25:4-6.
[12]Garber K,Beyond the Nobel Price:cell cycle researcs new view on cancer[J].Natl Can-Cer Inst,2001,93:1766-1768.
[13]Jiang M,Shao ZM,Wu J,et al.p21/walf/cipl and mdm22 expression in breast carcinoms patients as related to prognosis[J].Int J Cancer,1997,74:529-534.
[14]Liang B,Wang S,Yang X,et al.Expression of cyclin E,cyclin dependent kinase 2 and p57(KIP2)in human gastric cancer[J].Chin Med,2003,116:20-23.
[I5]ItoY,Takeda T,Sakon M,et al,Expression of p57/kip2 protein in hepatocellular carcinoma[J].Oncology,2001,61:221-225.
[16]Rosenberg E,Demopoulos RI,Zeleniuch-Jacquotte A,et al,Expression of cell cycle regulators p57(KIP2),cyclin D1 and cyclin E in epithelial ovarian tumors and survival[J].Hum Pathal,2001,32:808-813.
[17]Yu HY,Shi CG,Zhu JJ,et al,CyclinA expression in non-small cell lung carcinoma as related to proliferative activity and prognosis[J].Chin J Cancer,2001,20:38-40.
[18]Zhang Y,Peng ZH,Qiu GQ,et al,Overexpression of cyclinA in hepatocellular carcinoma and its relationship with HBx gene integration[J].Chin J Oncol,2002,24:353-355.
[19]申漫里,冯永东,高纯等.细胞周期蛋白B1在肿瘤细胞中的非时相性表达[J].中华肿瘤杂志,2002,24(3):215-219.
[20]Barrett K L,Demiranda D,Katula K S.Cyclin B1 promoter activity and functional CDK1 complex formation in G1 phase of human breast cancer cells[J].Cell Biol Int,2002,26(1):19-28.
[21]Mishina T,Dosaka-Akita H,Hmmura F,et al.CyclinE expression,a potential prognostic marker for non-small cell lung cancer.Cancer Res,2000,6:11-16.
[22]Resnitzky D,Reed SI.Different roles for cyclinD1 and E in regulation of the G1-to-S transition.Mol Cell Biol,1995,15:3463-3469.
[23]Mikhailov A,Cole RW,Rieder CL.DNA damage during mitosis in human cells delays the metaphase/anaphase transition via the spindle-assembly checkpoint[J].Curr Biol,2002,12(21):1797-1806.
[24]Zhou J,Yao J,Joshi HC.Attachment and tension in the spindle assembly checkpoint[J].J Cell Sci,2002,115(18):3547-3555.
[25]Vigneron S,Prieto S,Bernis C,et al,Kinetochore localization of spindle checkpoint proteins:who controls whom[J].Mol Biol Cell,2004,15(10):4584-4596.
[26]Agarwal ML,Ramana CV,Hamilton M,et al,Regulation of p53 expression by the RAS-MAP kinase pathway[J].Oncogene.,2001,20(20):2527-36.
[27]Innocente SA,Abrahamson JL,Cogswell JP,et al,p53 regulates a G2 checkpoint through cyclin BI[J].Proc Natl Acad Sci U S A.,1999,9 6(5):2147-52.
[28]Gajate C,Barasoain I,Andreu JM,et al,Induction of apoptosis in leukemic cells by the reversible microtubule-disrupting agent 2-methoxy-5-2,4,6-cyclonheptatrien-1-one:protection by Bcl-2 and BcI-X(L) and cell cycle arrest.Cancer Res.2000,60(10):2651-2659.
[29]Panvichian R,Orth K,Day ML,et al,Paclitaxel-associated multimininucleation is permitted by the inhibition of caspase activation:a potential early step in drug resistance[J].Cancer Res,1998,58(20):4667-4672.
[30]Heide LF,Robert AS,James D.Cell cycle and growth control.2nd[J].NJ:Wiley-Liss,2004,95-128.
[31]Woods CM,Zhu J,McQueney PA,et al,Taxol-induced mitotic block trigger rapid onset of a p53-independent apoptotic pathway[J].Mol Med,1995,1(5):506-526.
[32]Sherwood SW,Sheridan JP,Schirnke RT.Induction of apoptosis by the anti-tubulin drug colcemid:relationship of mitotic checkpoint control to the induction of apoptosis in Hela S3cells[J].Exp Cell Res,1994,215(2):373-379.
[33]Michalak,E.,A.Villunger,M.Erlacher,and A.Strasser.Death squadsenlisted by the tumour suppressor p53[J].Biochem.Biophys.Res.Commun.2005,331:786-798.
[34]Maria Castedo,Arnaud Coquelle,Sonia Vivet,et al,Apoptosis regulation in tetraploid cancer cells[J].The EMBO Journal,2006,25,2584-2596.
[35]Huang YC,Guh JH,Teng CM.Induction of mitotic arrest and apoptosis by evodiamine in humanleukemic T-lymphocytes[J].Life sci,2004,75(1):35.
[36]吴琦玮,葛忠良,高月等.靛玉红对肿瘤细胞抑制作用的研究及相关机制探讨[J].天津中医药,2008,25(1):55-58.
[37]Knowles LM,Milner JA.Diallyl disulfide induces ERK phosphorylation and ahem gene expression profiles in human colon tumor cels[J].J Nutr,2003,133(9):2901-2906.
[38]Panvichian R,Orth K,Day ML,et al,Paclitaxel-associated multimininucleation is permitted by the inhibition of caspase activation:a potential early step in drug resistance[J].Cancer Res,1998,58(20):4667-4672.
[39]王宗伟,杨安平,吴庆光.中华芦荟多糖对辐射小鼠胸腺细胞凋亡及细胞周期的影响[J].中药新药与临床药理,2005,16(4):240-243.
[40]刘宇炜,文若剑,易卉玲.高温和当归联合作用对Hela细胞周期的影响[J].中华中西医杂志,2005,6(21).
[41]赵连梅,单保恩,艾军等.香加皮杠柳苷对人食管癌细胞TE-13生长抑制作用[J].肿瘤,2008,28(3):203-206.
[42]赵丕文,牛建昭,王继峰等.6种中药活性成分植物雌激素作用的比较研究[J].中国药学杂志,2007,42(24):1852-1855.
[43]刘展华,曹洋,林丽珠等.斑蝥酸钠对人肺癌LAC细胞周期的影响及其分子机制的研究[J].广州中医药大学学报,2007,24(6):486-489.
[44]王鹂,吴军,李敏等.华蟾毒精抑制HeLa细胞增殖作用机制的探讨[J].中华肿瘤杂志,2005,27(12):717-720.
[45]周曦,陈忠东.三氧化二砷对宫颈癌Hela细胞体外生长的影响[J].南华大学学报医学版,2007,35(4):525-528.
[46]Janos H,Fendler.Membrane mimetic chemistry:characterizations and applications of micells,microemulsions,monolayers,bilayers,vesicles,host-guest systems,and polyions[J].New York:Wiley,1982:35-36.
[47]傅炜昕,梁再赋,李铁英等.槲寄生凝集素对肿瘤细胞细胞周期的影响[J].沈阳药科大学学报,2005,22(1):59-62.
[48]王菁鹏,林青.姜黄素对子宫颈癌HeLa细胞增殖抑制作用及其机制的研究[J].现代肿瘤医学,2006,14(8):1001-1003.
[49]张大鹏,孔棣.中药对体外人结肠癌THC8908细胞的生长抑制作用[J].中国中西医 结合外科杂志,2007,13(3):270-272.
[50]肖宝红,张春玲,武晓.猫眼草水提物对小鼠Lewis肺癌的抑制作用及机制探讨[J].中国中药杂志,2006,31(12):1002-1005.
[51]陈晓莉,王骊骊.葛根提取物对肝癌细胞增殖及细胞周期的影响[J].广东药学院学报,2001,17(3):183-184.
[52]李飞.方剂学[M].北京:人民卫生出版社,2002;125-128.
[53]Chiou WF,Chou CJ,Shum AY.The vasorelaxant effect of evodiamine in rat isolated mesenteric arteries mode of action[J].Eur J Pharmacol,1992,215(3):277-283.
[54]Wu CL,Hung CR,Chang FY,et al,Effects of evodiamine on gastrointestinal motility in male rats[J].Eur J Pharmacol,2002,457(2-3):169-176.
[55]Rang WQ,Du YH,Hu CP,et al,Protective effects of evodiamine on myocardial ischemia- reperfusion injury in rats[J].Planta Med,2004,70(12):1140-1143.
[56]Lin H,Tsai SC,Chen JJ,et al,Effects of evodiamine on the secretion of testosterone in rat testicular interstitial cells[J].Metabolism,1999,48(12):1532-1535.
[57]Chiou WF,Sung YJ,Liao JF,et al,Inhibitory effect of dehydroevodiamine and evodiamine on nitric oxide production in cultured murine macrophages[J].J Nat Prod,1997,60(7):708-711.
[58]Chou WF,Chen CF.Pharmacological profile of evodiamine in isolated rabbit corpus cavernosum[J].Eur J Pharmacol,2002,446(1-3):151-159.
[59]Kobayashi Y,Nakano Y,Hoshikuma K.The bronchoconstricttive action of evodiamine,an indoloquinazoline alkaloidisolated from the fruits of Evodia rutaecarpa,on guineapig isolated bronchus:possible involvement on vanilloid receptors[J].Planta Med,2000,66(6):526-530.
[60]张莹,吴立军,田代真一等.吴茱萸碱诱导人黑色素瘤A375-S2细胞的两种死亡机制[J].药学学报,2003,38(9):650-3.
[61]Zhang Y,wu L J,Tashiro S,et al,Evodiamine induces tumor cel ideath through different pathways:apoptosis and necrosis[J].Acta Pharmacol Sin,2004,25(1):83-8.
[62]Huang DM,Guh JH,Huang YT,et al,Induction of mitotic arrest and apoptosis in human prostate cancer pc-3 cells by evodiamine[J].J Urol,2005 Jan,173(1):256-61.
[63]Kan SF,Huang WJ,Lin LC,et al,Inhibitory effects of evodiamine on the growth of human prostate cancer cell line LNCaP[J].Int J Cancer,2004 Jul 10,110(5):641-51.
[64]Huang YC,Guh JH,Teng CM.Induction of mitotic arrest and apoptosis by evodiamine in human leukemic T-lymphocytes[J].Life sci,2004,75(1):35.
[65]Zhang V,Zhang Q H,Wu L J,et al,Atypical apoptosis in L929 cells induced by evodiamine isolated from Evodia rutaecarpa[J].J Asian Nat Prod Res,2004,6(1):19.
[66]Yu-Hui Tan,Wei-Wen Chen,Ying-Ya Wu,et al,Effects of berberine,evodiamine and indirubin on gastric cancer cell[J].World Chin J Digestol,2005 February 15,13(4):472-476.
[67]ChoHwa Liao,ShiowLin Pan,Jih-Hwa Guh,et al,Antitumor mechanism of evodiamine,aconstituent from Chinese herb Evodiae fructus,in human multiple-drug resistant breast cancer NCI/ADR-RES cells in in vitro and in vivo Carcinogenesis vol[J].26 no.5 2005pp,968-975.
[68]赞晓方,王本祥,池岛乔.吴茱萸碱诱导人子宫颈癌HeLa细胞凋亡的机制研究[J].药学学报,2002,37(9):673-5.
[69]Wang C,Wang MW,Tashiro S,et al,Roles of SIRT1 and phosphoinositide 3-OH kinase/protein kinase C pathways in evodiamine-induced human melanoma A375-S2 cell death[J].J Pharmacol Sci.2005 Apr,97(4):494-500.
[70]Wang C,Wang MW,Tashiro S,et al,Effect of protein kinase C on human melanoma A375-S2cell death induced by evodiamine[J].Yao Xue Xue Bao.2005 Nov,40(11):1033-6.
[71]Wang C,Wang MW,Tashiro S,et al,Evodiamine induced human melanoma A375-S2cell death partially through interleukin 1 mediated pathway[J].Biol Pharm Bull.2005Jun,28(6):984-9.
[72]Zhang Y,Wu L J,Tashiro S,et al,Intracellular regulation of evodiamine-induced A375-S2 cell death[J].Biol Pharm Bul 1,2003,26(11):1543-6.
[73]Ogasawara M,Matsubara T,Suzuki H.Inhibitory effects of evodiamine on in vitro invasion and experimental lung metastasis of murine colon cancer cells[J].Biol Pharm Bull.2001 Aug,24(8):917-20.
[74]Ogasawara M,Suzuki H.Inhibition by evodiamine of hepatocyte growth factor-induced invasion and migration of tumor cells[J].Biol Pharm Bull.2004 Apr,27(4):578-82.
[75]KouGiShyu,ShankungLin,ChunChungLee,et al,Evodiamine inhibits in vitro angiogenesis:Implication for antitumorgenicity[J].Life Sciences.78(2006),2234-2243.
[76]Liao CH,Pan SL,Gu LH,et al,Antitumor mechanism of evodiamine,a constituent from Chinese herb Evodiae fructus,in human multiple-drug resistant breast cancer NCI/ADR-RES cells in vitro and in vivo[J].Carcinogenesis,2005,26(5):968-975.
[77]Shimomura 0,Johnson F H,Saiga Y.Extraction,purification and properties of aequorin,a bioluminescent protein from the luminous hydromedusan,Aequorea[J].J Cell Comp Physiol,1962,59:223-229.
[78]Prasher D C,Eckenrode V K,Ward W W,et al,Primary structure of the Aequorea victorea green fluorescent protein[J].Gene,1992,111(2):229-233.
[79]Chalfle M,Tu Y,Euskirchen G,et al,Green fluorescent protein as a marker for gene expression[J].Science,1994,263(5148):802-805.
[80]Heim R,Prasher D C,Tsien R Y.Wavelength mutations and posttranslational autoxidati -on of green fluorescent protein[J].Proc Natl Acad Sci USA,1994,91(26):12501-12504.
[81]Helm R,Tsien R Y.Engineering green fluorescent protein for improved brightness,long -er wavelengths and fluorescence energy transfer[J].Curr Biol,1996,6(2):178-182.
[82]Ormo M,Cubitt AB,Kallio K et al,Crystal structure of the Aequorea victoria green fluorescent protein[J].Science,1996,273(5280):1392-1395.
[83]Muller-Taubenberger A,Anderson K I.Recent advances using green and red fluorescent protein variants[J].Appl Microbiol Biotechnol,2007,77(1):1-12.
[84]Yang F,Moss L G,Phillips G N.The molecular structure of green fluoresent protein[J].Nat Biotech,1996,14(10):1246-1251.
[85]储军,施华,杨杰等.分子与细胞事件的光学可视化[J].生物化学与生物物理进展.2008,35(10):1104-1111.
[86]刘艳丽,鲁澄宇.绿色荧光蛋白在药学研究中的应用进展[J].药学服务与研究,2008,8(5):326-328.
[87]Andreas Jacobs,Michael Dubrovin,et al,Functional Coexpression of HSV-1 Thymidine Kinase and Green Fluorescent Protein:Implications for Noninvasive Imaging of Transgene Expression[J].Neoplasia,1999,1(2):154-161.
[88]杨洁,聂青和.绿色荧光蛋白标记技术及其在基础临床研究中的应用[J].实用肝脏病杂志,2007,12(6):420-422.
[89]潘启超,管彬主编.肿瘤药理学与化学治疗学[M].河南医科大学出版社,第二版,2000,2-9.
[90]Cooper S.Reappraisal of serum starvation,the restriction point,G0 and G1 phase arrest points[J].FASEB J,2003,17(3):333-340.
[91]斯佩克特DL.细胞实验指南[M].黄培堂译.北京:科学出版社,2001,93-98.
[92]Collin AR,Dusinska M,Franklin M,et al,Comet assay in human biomonitoring studies:reliability,validation,and applications[J].Environ Mol mutagen,1997,30:139-146.
[93]Wolf F,Wandke C,Isenberg N,et al,Dose-dependent effects of stable cyclin B1 on progression through mitosis in human cells[J].EMBO J.,2006,25(12):2802-13.
[94]Salvioli S,Ardizzoni A,Franeoschi C,et al.JC-1,but not DiOC6(3)or Rho123,is a reliable fluorescent probe to assess changes in intact cells:implications for studies on mitoehondflal functionality during apoptosis[J].FEBS Lett,1997,411(1):77-82.
[95]翟中和,王喜忠,丁明孝.细胞生物学[M].北京:高等教育出版社,2000.
[96]Hammond JW,Cai D,Verhey KJ.Tubulin modifications and their cellular functions[J].Curr Opin Cell Biol,2008,20(1):71-76.
[97]徐忠东,吴琴.微管蛋白研究进展[J].安徽教育学院学报,1999,(2):73-74.
[98]吴平平,李苏宜.紫杉醇耐药与微管蛋白的研究进展[J].临床肿瘤学杂志,2008,1(8):755-757.
[99]Red R,Oakley.An abundance of tubulins[J].Trends in Cell Biology,2000,(10):537-542.
[100]Schmidt M,Bastian H.Mitotic drug targets and the development of novel anti mitotic anticancer drugs[J].Drug Resist Updat,2007,10(4-5):162-181.
[101]Ambrosini G.,Adida C,Ahieri D C.A novel anti-apoptosisgene survivin expressed in cancer and lymphoma[J].Nat Med,Ⅰ997,3(8):917-921.
[102]Javle M M,Tan D,Yu J,et al,Nuclear survivin expression predicts poor outcome in cholan-giocarcinoma[J].Hepatogastroenterology,2004,51(60):1653-1657.
[103]Span P N,Sweep FC,Wiegerinck E T,et al,Survivin is an independent prognostic marker for risk stratification of breast cancer patients[J].Clin Chem,2004,50(11):1986-1
[104]Huber BE,Austin EA,Good SS.In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase[J].CancerRes,1993;53(19):4619
[105]李翠云,段小娴.肝癌动物模型研究进展[J].医学研究杂志,2006,35(10):71-73.
[106]胡卫,陈涛.鼠移植性肝癌模型研究进展[J].河南肿瘤学杂志,2003,16(6):463-466.
[107]徐静,李旭.肝癌动物模型的建立[J].实用肝脏病杂志,2005,8(2):116-118.
[108]王海宁,熊慧林,黄作苏.HPLC测定吴茱萸中吴茱萸碱的含量[J].贵州师范大学学报(自然科学版),2001,19(1):74-75.
[109]于澍,王楚盈,魏中元等.吴茱萸碱抗肿瘤作用研究[J].吉林中医药,2008,28(3):223-223.
[110]王通,曾耀英,肇静娴等.靛蓝和靛玉红对小鼠T细胞活化与增殖的影响[J].中国医科大学学报,2005,36(5):444-447.
[111]Xiao Z,Hao Y,Liu B,et al.Indirubin and meisoindigo in the treatment of chronic myelogenous leukemia in China[J].Leuk Lymphoma,2002,43:1763-1768.
[112].Knockaert M,Blondel M,Bach S,et al,IndeDendent actions on cyclin-dependent kinases and aryl hydrocarbon receptor mediate the antiDroliferative effects of indirubins[J].Oncogene,2004,23:4400-4412.
[113].沈映君.中药药理学[M].北京:人民卫生出版社.2000.165.
[114]Eisenbrand G,Hippe F,Jakobs S,et al,Molecular mechanisms of indirubin and its derivatives:novel anticancer molecules with their origin in traditional Chinese phytomedicine[J].J Cancer Res Clin Oncol,2004,130(11):627-635.