小檗胺诱导Burkitt淋巴瘤Raji细胞凋亡的实验研究
摘要
非霍奇金淋巴瘤(NHL)是一组发生于淋巴结和(或)结外部位淋巴组织的恶性肿瘤,通常NHL患者对传统的化疗方案(如COP,CHOP等)表现出较好的反应,但多数往往在达到缓解后数月至数年的时间内出现复发。加大剂量的强化疗仅能使部分患者达到长期的缓解,但同时带来较大的药物毒性及较高致死率。因此寻求治疗NHL的新手段成了当前的一个紧迫问题。
祖国传统中药小檗胺提取于小檗属植物的根茎,是一种钙调素拮抗剂。体外和动物实验显示小檗胺对多种恶性肿瘤有抗癌活性,其作用机制为改变钙调素的分子构象,抑制钙调素调控的靶酶,从而影响细胞的代谢、周期生长及其他生物活性,发挥抗肿瘤效应,其毒副作用小,与常规化疗药物合用有效应相加作用。
本实验从细胞生长增殖、凋亡及凋亡相关蛋白表达等细胞生物学行为的角度,探讨小檗胺对淋巴瘤细胞株Raji细胞的影响。我们采用Wright-Giemsa染色法观察细胞凋亡的形态学变化；MTS法分析小檗胺对淋巴瘤细胞增殖抑制作用；采用流式细胞术分析小檗胺对细胞凋亡的影响；DNA片段化检测Raji细胞是否出现了"DNA梯子”的凋亡细胞表现；用Westernblot分析细胞内PARP(活化caspase3切割底物)的表达水平。结果显示,在0-32ug/ml小檗胺作用下,随着小檗胺浓度的升高,Raji细胞的存活率逐步降低,此外,小檗胺对Raji细胞的生长抑制也呈时间依赖性,同一浓度下,药物作用时间越长,细胞存活率也越低,小檗胺作用于Raji细胞24h,48h,72h的IC50分别为16.01μg/ml,11.74μg/ml,9.90μg/ml。形态学观察可见,小檗胺处理24h后的Raji细胞出现了凋亡形态的改变：细胞异型性明显,胞核深染,胞质浓缩,染色质成团块状。DNA片段化检测可见经小檗胺处理的Raji细胞出现了典型的"DNA梯子”,随着药物浓度的增加,"DNA梯子”也越明显,AV-PI法检测也发现,1,2,4,8,16,24,32μg/ml小檗胺处理Raji细胞24h后,早期凋亡细胞比例分别为38.89±2.11%,41.43±3.45%,44.54±3.02%,47.09±2.69%,54.20±5.67%,57.47±6.99%,60.87±5.38%,同时,小檗胺还可以诱导Raji细胞PARP剪切激活。
综上所述，小檗胺对Raji细胞有抑制增殖和诱导凋亡作用,可能通过caspase途径诱导Raji细胞凋亡。
Non-Hodgkin's lymphoma(NHL) is one of malignant tumors sourced from lymph nodes and(or) extra-nodal lymphoid tissue,NHL Patients usually showed good response on traditional chemotherapy(such as COP,CHOP,etc.),but most of them often easily recurred after relief for a few months even several years.The patients could achieve long- term relief time after stronger chemotherapy,but the stronger chemotherapy drugs followed the more toxicity and higher mortality rate,which made new therapy for the NHL become a hot spot in recent years.
The berbamine has shown obvious anti-tumor effect on a variety of malignant tumors.The mechanism are related to change the molecular conformation of calmodulin, and inhibit the target enzyme which are regulated by calmodulin,so as to affect the metabolism, cycle growth and other biological activity of tumor cell.It has tolerable side effect and no cross-resistant drug with other chemodrugs.
To provide theoretical proof for new combination chemotherapy programs in clinic, we treated human lymphoma cells Raji with berbamine,and observed the changes of the cells proliferation and cells apoptosis rate.Wright-Giemsa dyeing assay was used to observe apoptosis morphology of lymphoma cells.Detecting proliferation of the cells with berbamine by the method of MTS.Flow cytometry(FCM) and DNA ladder was used to detect apoptosis of lymphoma cells.western blot was used for detecting PARP(poly ADP-ribose polymerase).The result shows that the cell viabilites were significantly inhibited by BBM at the concentration of 0-32μg/ml,The cell survival rate was decreased with the increasing concentration of BBM.Furthermore,BBM inhibited cell growth in a time dependent manner. If treated at the same concentration of BBM,fewer cells would survive when the cells were treated for an increasing time.The 24h,48h,72h IC50 of BBM on Raji cell were 16.01μg/ml,11.74μg/ml,9.90μg/ml.After treated by BBM for 24h,Raji cells observed by Wright-Giemsa stain,showed a typical apoptotic morphology,nuclear fragmentation and apoptotic body.Typical DNA ladders were also seen in Raji cells treated by BBM for 24h.With the concentration of BBM increasing,The DNA ladder became more significant.BBM could induce apoptosis in Raji cells,as measured by AV-PI assay. After treated by 1,2,4,8,16,24,32μg/ml BBM for 24h,the Raji cells in early apoptosis were38.89±2.11%,41.43±3.45%,44.54±3.02%, 47.09±2.69%,54.20±5.67%,57.47±6.99%,60.87±5.38%,Furthermore,PAR P were cleaved and activated,as measured by western blot in Raji cells treated by BBM。
In conelusion,berbamine can inhibit Raji cells proliferation,induce cells apoptosis,which may through caspase way.
祖国传统中药小檗胺提取于小檗属植物的根茎,是一种钙调素拮抗剂。体外和动物实验显示小檗胺对多种恶性肿瘤有抗癌活性,其作用机制为改变钙调素的分子构象,抑制钙调素调控的靶酶,从而影响细胞的代谢、周期生长及其他生物活性,发挥抗肿瘤效应,其毒副作用小,与常规化疗药物合用有效应相加作用。
本实验从细胞生长增殖、凋亡及凋亡相关蛋白表达等细胞生物学行为的角度,探讨小檗胺对淋巴瘤细胞株Raji细胞的影响。我们采用Wright-Giemsa染色法观察细胞凋亡的形态学变化；MTS法分析小檗胺对淋巴瘤细胞增殖抑制作用；采用流式细胞术分析小檗胺对细胞凋亡的影响；DNA片段化检测Raji细胞是否出现了"DNA梯子”的凋亡细胞表现；用Westernblot分析细胞内PARP(活化caspase3切割底物)的表达水平。结果显示,在0-32ug/ml小檗胺作用下,随着小檗胺浓度的升高,Raji细胞的存活率逐步降低,此外,小檗胺对Raji细胞的生长抑制也呈时间依赖性,同一浓度下,药物作用时间越长,细胞存活率也越低,小檗胺作用于Raji细胞24h,48h,72h的IC50分别为16.01μg/ml,11.74μg/ml,9.90μg/ml。形态学观察可见,小檗胺处理24h后的Raji细胞出现了凋亡形态的改变：细胞异型性明显,胞核深染,胞质浓缩,染色质成团块状。DNA片段化检测可见经小檗胺处理的Raji细胞出现了典型的"DNA梯子”,随着药物浓度的增加,"DNA梯子”也越明显,AV-PI法检测也发现,1,2,4,8,16,24,32μg/ml小檗胺处理Raji细胞24h后,早期凋亡细胞比例分别为38.89±2.11%,41.43±3.45%,44.54±3.02%,47.09±2.69%,54.20±5.67%,57.47±6.99%,60.87±5.38%,同时,小檗胺还可以诱导Raji细胞PARP剪切激活。
综上所述，小檗胺对Raji细胞有抑制增殖和诱导凋亡作用,可能通过caspase途径诱导Raji细胞凋亡。
Non-Hodgkin's lymphoma(NHL) is one of malignant tumors sourced from lymph nodes and(or) extra-nodal lymphoid tissue,NHL Patients usually showed good response on traditional chemotherapy(such as COP,CHOP,etc.),but most of them often easily recurred after relief for a few months even several years.The patients could achieve long- term relief time after stronger chemotherapy,but the stronger chemotherapy drugs followed the more toxicity and higher mortality rate,which made new therapy for the NHL become a hot spot in recent years.
The berbamine has shown obvious anti-tumor effect on a variety of malignant tumors.The mechanism are related to change the molecular conformation of calmodulin, and inhibit the target enzyme which are regulated by calmodulin,so as to affect the metabolism, cycle growth and other biological activity of tumor cell.It has tolerable side effect and no cross-resistant drug with other chemodrugs.
To provide theoretical proof for new combination chemotherapy programs in clinic, we treated human lymphoma cells Raji with berbamine,and observed the changes of the cells proliferation and cells apoptosis rate.Wright-Giemsa dyeing assay was used to observe apoptosis morphology of lymphoma cells.Detecting proliferation of the cells with berbamine by the method of MTS.Flow cytometry(FCM) and DNA ladder was used to detect apoptosis of lymphoma cells.western blot was used for detecting PARP(poly ADP-ribose polymerase).The result shows that the cell viabilites were significantly inhibited by BBM at the concentration of 0-32μg/ml,The cell survival rate was decreased with the increasing concentration of BBM.Furthermore,BBM inhibited cell growth in a time dependent manner. If treated at the same concentration of BBM,fewer cells would survive when the cells were treated for an increasing time.The 24h,48h,72h IC50 of BBM on Raji cell were 16.01μg/ml,11.74μg/ml,9.90μg/ml.After treated by BBM for 24h,Raji cells observed by Wright-Giemsa stain,showed a typical apoptotic morphology,nuclear fragmentation and apoptotic body.Typical DNA ladders were also seen in Raji cells treated by BBM for 24h.With the concentration of BBM increasing,The DNA ladder became more significant.BBM could induce apoptosis in Raji cells,as measured by AV-PI assay. After treated by 1,2,4,8,16,24,32μg/ml BBM for 24h,the Raji cells in early apoptosis were38.89±2.11%,41.43±3.45%,44.54±3.02%, 47.09±2.69%,54.20±5.67%,57.47±6.99%,60.87±5.38%,Furthermore,PAR P were cleaved and activated,as measured by western blot in Raji cells treated by BBM。
In conelusion,berbamine can inhibit Raji cells proliferation,induce cells apoptosis,which may through caspase way.
引文
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