蟾酥逆转白血病多药耐药的机制研究
摘要
目的
慢性粒细胞白血病是造血系统的原发恶性疾病,是35岁以下发病率、死亡率最高的恶性肿瘤。K562细胞是慢性粒细胞白血病细胞系中的一种急变细胞株,是人的造血干细胞向红系分化过程被Friends病毒感染,停止分化而形成的。经过多年研究,传统祖国医学对白血病的治疗具有很强的临床优势,很多中药制剂治疗白血病具有明显的疗效,本实验初步研究中药蟾酥对白血病耐药细胞株K562/ADR细胞在抑制生长、逆转耐药、诱导凋亡等方面的作用和影响,为蟾酥治疗难治/复发性急性白血病提供有益的细胞学、分子生物学依据。
方法
本研究以体外细胞培养实验为主,研究内容包括:
(1)中药蟾酥血清药理学的实验方法;
中药血清药理学是研究中药的一种较好的方法,但应区分不同的药物而采取不同的血清制备方案。在研究药理的同时,最好能和中药血清药化学结合起来,对有效成分能定性或定量分析,这对于中药的开发更具有实际意义。
(2)MTT比色法观察蟾酥对细胞的抑制:
取对数生长期细胞,调整细胞数为1×10~5/ml,将调整后的细胞悬液100ul加入96孔的酶标板中,每组设8个平行孔,每一板次测定,均设一列空白对照组,将酶标板放置于37℃、5%CO2浓度,饱和湿度条件下的培养箱内培养后,分别加入不同浓度的实验药物(实验药物包括:①生理盐水组②蟾酥高剂量组③蟾酥中剂量组④蟾酥低剂量组⑤蟾酥高剂量+柔红霉素组⑥蟾酥中剂量+柔红霉素组⑦蟾酥小剂量+柔红霉素组)分别培养不同时间后,于自动酶标仪检测各标本吸光度(OD)值。根据公式计算各实验组的药物对白血病细胞的细胞毒作用。
(3)流式细胞术检测细胞凋亡率;
收集以上分组作用后的细胞,用Annexin V和PI染色双染法对细胞进行一系列处理后,送流式细胞仪检测其各组的凋亡率。
(4)用RT—PCR检测Survivin mRNA的表达。
提取以上加入药物作用后的白血病细胞的RNA,逆转录反应cDNA,然后进行RT—PCR反应,获得数据。
通过这些观察一般的项目和较新的、水平较高、较为公认的稳定的分子生物学指标,力求客观的反映中药蟾酥抗难治/复发性急性白血病的分子作用机制。
结果
(1)蟾酥可抑制K562/ADR耐药细胞的生长,药物浓度、作用时间与K562/ADR细胞生长抑制率之间均呈时效关系;
(2)蟾酥作用48h后显微镜下观察到细胞形态出现凋亡特征;
(3)随着药物浓度的增加和时间的推移,K562/ADR细胞中Survivin mRNA表达下降;
(4)蟾酥随着药物浓度的增加,K562/ADR细胞凋亡率也增加。
结论
蟾酥可抑制K562/ADR细胞生长,逆转其耐药,促进细胞凋亡,呈剂量依赖性,其机制可能与Survivin mRNA表达下降有关。
Objective:
Chronic myeloid leukemia is the Primary malignant disease of Hematopoietic system,35 years of age the incidence of the highest mortality rate of malignant. K562 cells in chronic myeloid leukemia cell lines in a CML cell line,human hematopoietic stem cells to erythroid differentiation by Friends virus infection,to stop the formation of differentiation.After years of research, traditional Chinese medicine has a strong clinical advantage on the Treatment of leukemia.A lot of Chinese medicine treatment of leukemia has obvious effects.To study the effect of chansu on growing,the reverse and the apoptosis of the K562/ADR cells,in order to provide useful based of cytology,molecular biology for multidrug resistant leukemia.
Methods:
The main experiments is in vitro cell culture in this study,the study includes:
(1) Chansu experimental serum pharmacological method
Serum pharmacology of Chinese medicine is a better way to study Chinese medicine,however,must distinguish between the different drugs and different levels of preparation program.At the same time in the study of pharmacological, The Chinese medicine can be combined with the Chemical serum,the active ingredients can be qualitative or quantitative analysis.This is the development of Chinese medicine is more practical significance.
(2) Analyse the growth inhibition of the cells by MTT assay;
From logarithmic phase cells,Adjust the cell number is 1×105/ml, Adding 100ul to 96' ELISA plate,Each set of parallel 8-hole,ELISA plate will be placed in 37℃,5%CO2,adding different concentrations of experimental drugs,training the different times,detection by the Automatic ELISA' OD. Calculated in accordance with the experimental group of drugs on the leukemia cell cytotoxicity.
(3) Detect the cell apoptosis by flow cytometry;
Collecting more than the role of the above of division,With Annex in V and PI to treat a series of cells,send their to FCM apoptosis rate in each group.
(4) The molecular assay of RT-PCR was used to examine the mRNA level of gene Survivin mRNA.
Extract the above division of cells' RNA.Reverse transcription reaction, Followed by RT-PCR reaction,to get the data.
Through these observations,in order to reflect the molecular mechanism of the reverse of resistance using chansu in multidrug resistant leukemia.
Results:
(1)Treatment of K562/ADR cells with chansu inhibited the cell growth in a dose-dependent manner.
(2)Apoptotic K562/ADR Cells were found after incubation with drugs.
(3)Positive findings of Survivin mRNA decreased after incubation with drugs.
(4)The percent of apoptotic K562/ADR cells increased after incubation with drugs.
Conclusion:
Chansu can inhibit the K562/ADR cell growth,reverse the resistance and induce the apoptosis in a dose-and time-depentent manner,while positive findings of Survivin mRNA decreased.
慢性粒细胞白血病是造血系统的原发恶性疾病,是35岁以下发病率、死亡率最高的恶性肿瘤。K562细胞是慢性粒细胞白血病细胞系中的一种急变细胞株,是人的造血干细胞向红系分化过程被Friends病毒感染,停止分化而形成的。经过多年研究,传统祖国医学对白血病的治疗具有很强的临床优势,很多中药制剂治疗白血病具有明显的疗效,本实验初步研究中药蟾酥对白血病耐药细胞株K562/ADR细胞在抑制生长、逆转耐药、诱导凋亡等方面的作用和影响,为蟾酥治疗难治/复发性急性白血病提供有益的细胞学、分子生物学依据。
方法
本研究以体外细胞培养实验为主,研究内容包括:
(1)中药蟾酥血清药理学的实验方法;
中药血清药理学是研究中药的一种较好的方法,但应区分不同的药物而采取不同的血清制备方案。在研究药理的同时,最好能和中药血清药化学结合起来,对有效成分能定性或定量分析,这对于中药的开发更具有实际意义。
(2)MTT比色法观察蟾酥对细胞的抑制:
取对数生长期细胞,调整细胞数为1×10~5/ml,将调整后的细胞悬液100ul加入96孔的酶标板中,每组设8个平行孔,每一板次测定,均设一列空白对照组,将酶标板放置于37℃、5%CO2浓度,饱和湿度条件下的培养箱内培养后,分别加入不同浓度的实验药物(实验药物包括:①生理盐水组②蟾酥高剂量组③蟾酥中剂量组④蟾酥低剂量组⑤蟾酥高剂量+柔红霉素组⑥蟾酥中剂量+柔红霉素组⑦蟾酥小剂量+柔红霉素组)分别培养不同时间后,于自动酶标仪检测各标本吸光度(OD)值。根据公式计算各实验组的药物对白血病细胞的细胞毒作用。
(3)流式细胞术检测细胞凋亡率;
收集以上分组作用后的细胞,用Annexin V和PI染色双染法对细胞进行一系列处理后,送流式细胞仪检测其各组的凋亡率。
(4)用RT—PCR检测Survivin mRNA的表达。
提取以上加入药物作用后的白血病细胞的RNA,逆转录反应cDNA,然后进行RT—PCR反应,获得数据。
通过这些观察一般的项目和较新的、水平较高、较为公认的稳定的分子生物学指标,力求客观的反映中药蟾酥抗难治/复发性急性白血病的分子作用机制。
结果
(1)蟾酥可抑制K562/ADR耐药细胞的生长,药物浓度、作用时间与K562/ADR细胞生长抑制率之间均呈时效关系;
(2)蟾酥作用48h后显微镜下观察到细胞形态出现凋亡特征;
(3)随着药物浓度的增加和时间的推移,K562/ADR细胞中Survivin mRNA表达下降;
(4)蟾酥随着药物浓度的增加,K562/ADR细胞凋亡率也增加。
结论
蟾酥可抑制K562/ADR细胞生长,逆转其耐药,促进细胞凋亡,呈剂量依赖性,其机制可能与Survivin mRNA表达下降有关。
Objective:
Chronic myeloid leukemia is the Primary malignant disease of Hematopoietic system,35 years of age the incidence of the highest mortality rate of malignant. K562 cells in chronic myeloid leukemia cell lines in a CML cell line,human hematopoietic stem cells to erythroid differentiation by Friends virus infection,to stop the formation of differentiation.After years of research, traditional Chinese medicine has a strong clinical advantage on the Treatment of leukemia.A lot of Chinese medicine treatment of leukemia has obvious effects.To study the effect of chansu on growing,the reverse and the apoptosis of the K562/ADR cells,in order to provide useful based of cytology,molecular biology for multidrug resistant leukemia.
Methods:
The main experiments is in vitro cell culture in this study,the study includes:
(1) Chansu experimental serum pharmacological method
Serum pharmacology of Chinese medicine is a better way to study Chinese medicine,however,must distinguish between the different drugs and different levels of preparation program.At the same time in the study of pharmacological, The Chinese medicine can be combined with the Chemical serum,the active ingredients can be qualitative or quantitative analysis.This is the development of Chinese medicine is more practical significance.
(2) Analyse the growth inhibition of the cells by MTT assay;
From logarithmic phase cells,Adjust the cell number is 1×105/ml, Adding 100ul to 96' ELISA plate,Each set of parallel 8-hole,ELISA plate will be placed in 37℃,5%CO2,adding different concentrations of experimental drugs,training the different times,detection by the Automatic ELISA' OD. Calculated in accordance with the experimental group of drugs on the leukemia cell cytotoxicity.
(3) Detect the cell apoptosis by flow cytometry;
Collecting more than the role of the above of division,With Annex in V and PI to treat a series of cells,send their to FCM apoptosis rate in each group.
(4) The molecular assay of RT-PCR was used to examine the mRNA level of gene Survivin mRNA.
Extract the above division of cells' RNA.Reverse transcription reaction, Followed by RT-PCR reaction,to get the data.
Through these observations,in order to reflect the molecular mechanism of the reverse of resistance using chansu in multidrug resistant leukemia.
Results:
(1)Treatment of K562/ADR cells with chansu inhibited the cell growth in a dose-dependent manner.
(2)Apoptotic K562/ADR Cells were found after incubation with drugs.
(3)Positive findings of Survivin mRNA decreased after incubation with drugs.
(4)The percent of apoptotic K562/ADR cells increased after incubation with drugs.
Conclusion:
Chansu can inhibit the K562/ADR cell growth,reverse the resistance and induce the apoptosis in a dose-and time-depentent manner,while positive findings of Survivin mRNA decreased.
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