中药复方君子汤诱导急性白血病细胞凋亡的实验研究及临床疗效观察
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摘要
背景:白血病是一类造血干细胞克隆性恶性疾病,是最常见的恶性肿瘤之一,近年来其发病率逐渐上升,造血过程中细胞分化及凋亡受阻是白血病的重要发病机制。多种类型的白血病细胞可在体内或体外经各种化合物的作用发生终末分化和凋亡,中药毒副作用小,随着人类对肿瘤认识的不断提高,中药诱导肿瘤细胞凋亡的研究也成为热点。
目的:观察中药复方君子汤(FFJZ)诱导白血病K562细胞凋亡的作用特点及其对急性白血病患者的疗效,以了解FFJZ对化疗的增效减毒作用,从而为FFJZ用于AL的治疗提供实验及临床依据,并初步探讨出中西医结合治疗急性白血病的方法。
方法:1、体外实验:(1)细胞形态学观察:将不同浓度FFJZ与K562细胞共同培养,于24小时、48小时、72小时,在倒置显微镜下观察细胞生长状况;(2)采用台盼蓝拒染法观察细胞活性;(3)采用MTT法观察K562细胞株增殖情况;(4)流式细胞仪(FCM)测定细胞内Annexin V-FITC/PI荧光强度,以检测细胞凋亡数。2、临床研究:对40例急性白血病患者,按随机原则分组治疗,其中治疗组20例,应用FFJZ配合化疗治疗;对照组20例,应用化疗治疗,观察两组的疗效。
结果:1、体外实验:(1)K562细胞经FFJZ作用48小时后,浓度在1mg/ml及以下组出现细胞形态呈梭状、棒状,伴有胞浆丝状突起形成伪足,2mg/mlFFJZ组细胞与对照组无明显差别,4mg/ml及6mg/mlFFJZ组,细胞出现皱缩、破碎;8mg/mlFFJZ及10mg/mlFFJZ组细胞经培养24小时后,即出现细胞明显变小,且均匀分散,以后时间里均呈现此种状况。(2)经0.01mg/ml-1mg/ml FFJZ作用后的K562细胞,不同时间计数活细胞数均于对照组无明显差异(P＞0.05),2mg/ml FFJZ作用于K562细胞,各时期活细胞计数均较对照组略低(P＜0.05)。4mg/ml FFJZ组与K562细胞共同培养不同时间活细胞计数较对照组低(P＜0.01),与As_2O_3(3uM)组细胞活力无明显差别(P＞0.05)。细胞培养24小时后,6mg/ml FFJZ组及8mg/ml FFJZ组细胞增殖受抑,活细胞计数下降,10mg/ml FFJZ组细胞最为明显,几乎无活细胞。(3)2mg/ml FFJZ组与空白对照组无显著差别(P＞0.05),其余FFJZ组对K562细胞的抑制率随药物浓度的加大而增加(各浓度组与未加药对照组相比有显著性差异(P＜0.01)。6mg/ml FFJZ组及8mg/ml FFJZ组与其它组相比有统计学意义(P＜0.05),4mg/ml FFJZ组与As_2O_3(3uM)组无明显差异(P＞0.05)。FFJZ对K562细胞的半数抑制浓度(IC50)约为5.6mg/ml。(4)FFJZ质量浓度2mg/ml时,诱导凋亡作用不明显,与对照组比较无差异(P＞0.05),当其质量浓度达到4mg/ml时,早期凋亡明显增多,与未加药组相比差异有统计学意义(P＜0.01),至8mg/ml组时,细胞早期凋亡率下降,与空白组相比仍有显著差异,(P＜0.01),晚期凋亡/死亡细胞显著增多,与未加药组有显著差异(P＜0.01)。培养细胞早期凋亡率显示4mg/ml FFJZ组与阳性对照As_2O_3(3uM)组相比差异无统计学意义(P＞0.05)。2、临床研究:(1)临床观察的结果治疗组总有效率(75%)明显高于对照组(45%),差异有统计学意义(P＜0.05);(2)治疗组达完全缓解8例,对照组达完全缓解6例,随访1年,两组达完全缓解时间及持续时间无统计学意义(P＞0.05);(3)两组发生不良反应例数无统计学意义(P＞0.05)。
结论:FFJZ可以抑制K562白血病细胞的生长和增殖,在体外一定浓度范围内可诱导K562细胞凋亡,低浓度FFJZ可诱导K562细胞发生类似分化的细胞形态学改变。FFJZ联合化疗能增强抗白血病药物的疗效,值得进一步临床研究。
Background:Leukemia is caused by the maglignant clone of the hemopoietic stem cells.It is one of the most common cancer and its morbidity is increased in recent year.The stagging of the differentiation and the blocking of apoptosis of leukemic cells are the two main mechanism of the leukemia development.All manner of leukemic cells can be inducted to terminal differentiation and apoptosis by miscellaneous drugs in vivo and vitro.Traditional Chinese drug has little side-effect.With the persistent improvement of knowing tumor,the traditional Chinese drug with apoptosis promotion of tumor cells has been given more and more attention.
Objective:The study is to investigate the effect about apoptosis induction by Chinese medicine compound FFJZ and the efficinency of FFJZ synergy the chemotherapy,in order to know synergetic effect and attenuation of FFJZ and to provide experimental and clinic evidence to treatment of acute leukemia,and to search a method on treatment of acute leukemia by Chinese medicine and western medicine.
Methods:1.Experimental study:(1)The growth states of leukemia cells K562 cultrued in vitro were observed by the invent microscope after cultured for 24,48,72 hours.(2)The number of live K562 cells were counted by trypan blue exclusion..(3)The vegetation of the cells K562 was estimated by MTT method.(4)FCM(flow cytometre methods) was used to assess the intracellular fluorescence intensity of Annexin V-FITC/PI,and to assess account of apoptotic cells.2.Clinical study:The 40 cases of acute leukemia were randomly divided into two groups.There were 20 cases in treatment group,treated with FFJZ plus chemotherapy;There were 20 cases in control group,treated with chemotherapy.The clinicial therapeutic effectiveness was observed between two groups.
Result:1.Experimental study:(1)After 48 hours culture,the morphology of K562 cells that FFJZ's concentration is 1mg/ml and less than 1mg/ml became fusiform and claviform.The K562 cells that FFJZ's concentration is 2mg/ml has no conspicuous different.The K562 cells that FFJZ's concentration is 4mg/ml and 6mg/ml took on shapes of crenation.(2) The count of the live K562 cells in As_2O_3 group grew slowly after 24 hours'culture.It was also found in FFJZ 4mg/ml,6mg/ml and 8mg/ml group.There was scarcely live cells in FFJZ 10mg/ml group after 24 hours'culture.(3)By the MTT assay,we found that the K562 cell growth and proliferation of all the experimental group except the FFJZ 2mg/ml group were inhibited in different extents.The inhibitions were significant differences in contrast with the control group(P＜0.05),and the inhibition rate of 8mg/ml group was higher than that of other experimental groups(P＜0.05).There are no significant differences berween FFJZ 4mg/ml group and As_2O_3 group(P＞0.05).The ICS0 of K562 cells was about 5.6mg/ml.(4) The FCM results demonstrated that the expression of early apoptosis of K562 cells in FFJZ 4mg/ml,6mg/ml and 8mg/ml group increased and there were significant differences in contrast with the control group(P＜0.01).Early apoptosis rate decreased when FFJZ'concentration was 8mg/ml,and the cells of late apoptosis/necrosis increased remarkblely in contrast with control group(P＜0.01).
2.Clincal study:(1)The effective rates of treatment group and control group are 75% and 45%,showing statistical difference(P＜0.05);(2)There are 8 cases getting Complete Remission(CR) in treatment group,and there are 6 cases in control group.Days of keeping CR shows no statistical difference afar observing one year(P＞0.05);(3)There are no statistical difference between two group of side effect(P＞0.05).
Conclusion:FFJZ can induct K562 cells to apoptosis and can conspicuously synergy the chemotherapy.FFJZ may become a safe and effective synergist with low toxicity in acute leukemia chemotherapy.
目的:观察中药复方君子汤(FFJZ)诱导白血病K562细胞凋亡的作用特点及其对急性白血病患者的疗效,以了解FFJZ对化疗的增效减毒作用,从而为FFJZ用于AL的治疗提供实验及临床依据,并初步探讨出中西医结合治疗急性白血病的方法。
方法:1、体外实验:(1)细胞形态学观察:将不同浓度FFJZ与K562细胞共同培养,于24小时、48小时、72小时,在倒置显微镜下观察细胞生长状况;(2)采用台盼蓝拒染法观察细胞活性;(3)采用MTT法观察K562细胞株增殖情况;(4)流式细胞仪(FCM)测定细胞内Annexin V-FITC/PI荧光强度,以检测细胞凋亡数。2、临床研究:对40例急性白血病患者,按随机原则分组治疗,其中治疗组20例,应用FFJZ配合化疗治疗;对照组20例,应用化疗治疗,观察两组的疗效。
结果:1、体外实验:(1)K562细胞经FFJZ作用48小时后,浓度在1mg/ml及以下组出现细胞形态呈梭状、棒状,伴有胞浆丝状突起形成伪足,2mg/mlFFJZ组细胞与对照组无明显差别,4mg/ml及6mg/mlFFJZ组,细胞出现皱缩、破碎;8mg/mlFFJZ及10mg/mlFFJZ组细胞经培养24小时后,即出现细胞明显变小,且均匀分散,以后时间里均呈现此种状况。(2)经0.01mg/ml-1mg/ml FFJZ作用后的K562细胞,不同时间计数活细胞数均于对照组无明显差异(P＞0.05),2mg/ml FFJZ作用于K562细胞,各时期活细胞计数均较对照组略低(P＜0.05)。4mg/ml FFJZ组与K562细胞共同培养不同时间活细胞计数较对照组低(P＜0.01),与As_2O_3(3uM)组细胞活力无明显差别(P＞0.05)。细胞培养24小时后,6mg/ml FFJZ组及8mg/ml FFJZ组细胞增殖受抑,活细胞计数下降,10mg/ml FFJZ组细胞最为明显,几乎无活细胞。(3)2mg/ml FFJZ组与空白对照组无显著差别(P＞0.05),其余FFJZ组对K562细胞的抑制率随药物浓度的加大而增加(各浓度组与未加药对照组相比有显著性差异(P＜0.01)。6mg/ml FFJZ组及8mg/ml FFJZ组与其它组相比有统计学意义(P＜0.05),4mg/ml FFJZ组与As_2O_3(3uM)组无明显差异(P＞0.05)。FFJZ对K562细胞的半数抑制浓度(IC50)约为5.6mg/ml。(4)FFJZ质量浓度2mg/ml时,诱导凋亡作用不明显,与对照组比较无差异(P＞0.05),当其质量浓度达到4mg/ml时,早期凋亡明显增多,与未加药组相比差异有统计学意义(P＜0.01),至8mg/ml组时,细胞早期凋亡率下降,与空白组相比仍有显著差异,(P＜0.01),晚期凋亡/死亡细胞显著增多,与未加药组有显著差异(P＜0.01)。培养细胞早期凋亡率显示4mg/ml FFJZ组与阳性对照As_2O_3(3uM)组相比差异无统计学意义(P＞0.05)。2、临床研究:(1)临床观察的结果治疗组总有效率(75%)明显高于对照组(45%),差异有统计学意义(P＜0.05);(2)治疗组达完全缓解8例,对照组达完全缓解6例,随访1年,两组达完全缓解时间及持续时间无统计学意义(P＞0.05);(3)两组发生不良反应例数无统计学意义(P＞0.05)。
结论:FFJZ可以抑制K562白血病细胞的生长和增殖,在体外一定浓度范围内可诱导K562细胞凋亡,低浓度FFJZ可诱导K562细胞发生类似分化的细胞形态学改变。FFJZ联合化疗能增强抗白血病药物的疗效,值得进一步临床研究。
Background:Leukemia is caused by the maglignant clone of the hemopoietic stem cells.It is one of the most common cancer and its morbidity is increased in recent year.The stagging of the differentiation and the blocking of apoptosis of leukemic cells are the two main mechanism of the leukemia development.All manner of leukemic cells can be inducted to terminal differentiation and apoptosis by miscellaneous drugs in vivo and vitro.Traditional Chinese drug has little side-effect.With the persistent improvement of knowing tumor,the traditional Chinese drug with apoptosis promotion of tumor cells has been given more and more attention.
Objective:The study is to investigate the effect about apoptosis induction by Chinese medicine compound FFJZ and the efficinency of FFJZ synergy the chemotherapy,in order to know synergetic effect and attenuation of FFJZ and to provide experimental and clinic evidence to treatment of acute leukemia,and to search a method on treatment of acute leukemia by Chinese medicine and western medicine.
Methods:1.Experimental study:(1)The growth states of leukemia cells K562 cultrued in vitro were observed by the invent microscope after cultured for 24,48,72 hours.(2)The number of live K562 cells were counted by trypan blue exclusion..(3)The vegetation of the cells K562 was estimated by MTT method.(4)FCM(flow cytometre methods) was used to assess the intracellular fluorescence intensity of Annexin V-FITC/PI,and to assess account of apoptotic cells.2.Clinical study:The 40 cases of acute leukemia were randomly divided into two groups.There were 20 cases in treatment group,treated with FFJZ plus chemotherapy;There were 20 cases in control group,treated with chemotherapy.The clinicial therapeutic effectiveness was observed between two groups.
Result:1.Experimental study:(1)After 48 hours culture,the morphology of K562 cells that FFJZ's concentration is 1mg/ml and less than 1mg/ml became fusiform and claviform.The K562 cells that FFJZ's concentration is 2mg/ml has no conspicuous different.The K562 cells that FFJZ's concentration is 4mg/ml and 6mg/ml took on shapes of crenation.(2) The count of the live K562 cells in As_2O_3 group grew slowly after 24 hours'culture.It was also found in FFJZ 4mg/ml,6mg/ml and 8mg/ml group.There was scarcely live cells in FFJZ 10mg/ml group after 24 hours'culture.(3)By the MTT assay,we found that the K562 cell growth and proliferation of all the experimental group except the FFJZ 2mg/ml group were inhibited in different extents.The inhibitions were significant differences in contrast with the control group(P＜0.05),and the inhibition rate of 8mg/ml group was higher than that of other experimental groups(P＜0.05).There are no significant differences berween FFJZ 4mg/ml group and As_2O_3 group(P＞0.05).The ICS0 of K562 cells was about 5.6mg/ml.(4) The FCM results demonstrated that the expression of early apoptosis of K562 cells in FFJZ 4mg/ml,6mg/ml and 8mg/ml group increased and there were significant differences in contrast with the control group(P＜0.01).Early apoptosis rate decreased when FFJZ'concentration was 8mg/ml,and the cells of late apoptosis/necrosis increased remarkblely in contrast with control group(P＜0.01).
2.Clincal study:(1)The effective rates of treatment group and control group are 75% and 45%,showing statistical difference(P＜0.05);(2)There are 8 cases getting Complete Remission(CR) in treatment group,and there are 6 cases in control group.Days of keeping CR shows no statistical difference afar observing one year(P＞0.05);(3)There are no statistical difference between two group of side effect(P＞0.05).
Conclusion:FFJZ can induct K562 cells to apoptosis and can conspicuously synergy the chemotherapy.FFJZ may become a safe and effective synergist with low toxicity in acute leukemia chemotherapy.
引文
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