清毒扶正方药对ANLL-CR患者BMNC来源DC的诱导作用
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摘要
第一部分文献研究
急性白血病(AL)是血液科常见肿瘤之一,具有病情较重,发展较快,缓解后易于复发,长期生存率不高等特点,随着中、西医理论的完善、白血病的治疗方法不断开发和发展,使急性白血病的完全缓解率(CR)和长期生存率有了相当程度的提高,甚至完全治愈。但大部分患者即使完全缓解仍有复发,其根本原因就在于微量残留的白血病细胞的存在,对这种白血病的微小残留状态(MRD)防治是目前白血病研究的热点之一。
国内外比较一致的意见认为,微小残留白血病的存在除了与机体免疫功能有关外,与白血病细胞本身的免疫逃逸机制有更密切关系,所以杀灭微小残留白血病细胞必须依靠机体自身的免疫监视功能的正常发挥。而在微小残留白血病阶段,肿瘤负荷较小,并且经过长期化疗后残存下来的白血病细胞对药物的敏感性已经很低,此时应用免疫治疗辅助机体杀灭残存的肿瘤细胞是最好的时机。
中医基于对急性白血病正气虚弱,邪毒内侵的病因病机认识,目前对微小残留病的病理机制的认识基本一致,机体处于“邪去正衰”、“正气虚弱”而“余邪未尽”的病理生理状态。基于正气虚弱而邪毒未净是微量残留白血病的基本病理,多认为扶正祛邪治法是其基本的治疗。
第二部分实验研究
清毒扶正方药对ANLL-CR患者BMNC来源DC的诱导作用
1.研究目的
本实验用清毒饮、养正片含药血清配合细胞因子对急性髓细胞性白血病缓解期患者骨髓单个核细胞(BMNC)进行体外培养,观察树突细胞(DC)的诱导转化情况。以诱导转化的DC与纯化的正常人T细胞、患者自体T细胞共同培养,以激发T细胞,观察激发后的T细胞对人白血病细胞株K562细胞的杀伤作用。
清毒饮、养正片是本院研制的治疗急性白血病的有效中药制剂,本实验从中医药结合免疫治疗白血病的新途径出发,从提高缓解后患者的树突状细胞抗原提呈能力的新切入点,探讨中医药抗白血病的新作用机制,寻求中医治疗缓解后白血病并防止其复发的有效方法。
2.研究方法：
2.1标本来源
2.1.1骨髓单个核细胞来源于广州中医药大学第一临床医学院血液科住院急性髓系白血病患者缓解后(AML-CR)骨髓两例。患者均符合急性白血病诊断标准经标准方案化疗后完全缓解,符合急性白血病完全缓解标准。
2.1.2 T细胞来自正常成人外周血及白血病患者完全缓解后外周血各一例。
2.1.3白血病细胞株K562细胞。
2.2制备中药含药血清
取新西兰兔分组,分别以清毒饮、养正片、清毒饮合养正片按低、中、高剂量灌胃动物,制备三个组别的不同浓度的含药血清。
2.3分离骨髓去T细胞单个核细胞
应用羊红细胞玫瑰花结程序从骨髓中分离出去T细胞的骨髓单个核细胞(TD-MNC)。
2.4培养来源于AML-CR患者TD-MNC的DC
将TD-MNC细胞与不同中药含药血清与细胞因子联合培养,分为：清毒饮组、GM-CSF+IL-4+IFN-α+TNF-α组、清毒饮+GM-CSF+IL-4+IFN-α+TNF-α组、养正片组、养正片+GM-CSF+IL-4+IFN-α+TNF-α组、清毒饮合养正片+GM-CSF+IL-4+IFN-α+TNF-α组。各含药血清组均分低、中、高剂量组。
2.5 DC鉴定：包括形态观察、免疫学检测、及其分泌的细胞因子IL-2、IL-6的测定。
2.6外周血T细胞的分离纯化(磁珠分选法)
正常人、AML-CR患者各取一份外周血50ml,经Ficoll密度梯度离心获得外周血单个核细胞(PBMNC)后,采用磁珠阴性选择法制得纯化的外周血T细胞。流式细胞仪检测纯度。
2.7同种异体T细胞及自体T细胞的激发
分别收集纯化的同种异体T细胞、AML-CR患者自体T细胞,与AML-CR患者TD-MNC来源的DC以10：1比例加入24孔培养板培养5d,培养基中加入10OU/ml的IL-2。
2.8激发T细胞对K562细胞的杀伤作用(MTT法)
将各组T细胞与K562细胞分别以10：1、20：1、40:1效：靶比例培养,计算细胞杀伤率。
3.研究结果
本实验结果发现清毒饮+养正片组的低剂量组联合细胞因子可促进DC高表达CD1a、HLA-DR、CD80、CD86,并能促使DC高分泌细胞因子IL-2、IL-6,与对照组(细胞因子组)相比表达均明显升高,有统计学意义,说明清毒扶正中药可促进树突细胞的增殖和表达,提高树突细胞的抗原提呈能力。并且,各中药组内效靶比越高,杀伤率越高,这也说明T细胞浓度越大,对肿瘤细胞的杀伤率也越高。各中药组与对照组相比的结果说明,在某种浓度中药联合细胞因子可以比单纯用细胞因子更好地促进DC细胞的诱导转化,从而更强地激发T细胞,产生更大的杀伤力。
第三部分临床观察
附：清毒扶正方药对AL-CR患者骨髓细胞CD抗原表达及增殖的影响
1.研究目的
以清毒扶正为立法依据的清毒饮+养正片对照观察完全缓解后急性白血病患者治疗前后骨髓细胞CD34、CD34/CD2、CD34/CD19、CD13/CD33表达率,了解清毒扶正治法对微小残留白血病的治疗效果；通过对照观察患者治疗前后骨髓细胞增殖周期的变化,进一步探求该法治疗微小残留白血病的可能机制。
2.研究方法
本研究采用随机对照观察清毒扶正方药清毒饮+养正片对完全缓解的急性白血病患者骨髓细胞CD抗原表达及增殖周期的影响。
2.1病例来源来源于广州中医药大学第一附属医院血液科住院及门诊患者。
2.2治疗措施
2.2.1对照组：按常规化疗方案巩固维持治疗(ANLL用DA或MA或HA方案,ALL用VDP或VDLP方案)进行化疗,如出现感染、出血等情况,则按常规抗感染及止血治疗。
2.2.2观察组：西医治疗原则同对照组。巩固维持化疗的同时口服清毒饮、养正片方煎剂,日一剂,分二次服,持续4周。
2.2.3分别采集两组患者治疗前、治疗四周后的骨髓组织,进行实验观察。
2.3骨髓细胞CD抗原表达检测流式细胞仪测定患者治疗前后骨髓细胞CD34、CD34/CD2、CD34/CD19、CD13/CD33表达率。
2.4骨髓细胞增殖周期检测流式细胞仪检测患者治疗前后骨髓细胞增殖周期各时相表达率。
3.实验结果
本实验结合流式细胞仪对患者骨髓细胞增殖周期的测定,表明清毒扶正方药有抑制CD34表达的作用,说明清毒饮+养正片可能抑制了白血病细胞的增殖,从而使能代表髓系白血病细胞的CD13/CD14/CD33、代表淋巴系白血病细胞的CD3/CD19表达率下降。因而,扶正清毒方法可能对稳定病情、改善生活质量,甚至对提高急性髓性白血病患者生存期有宜。
第四部分研究结论
目前尚未见关于中药单体或复方在白血病肿瘤抗原提呈方面的作用研究。本论文用血清药理学方法率先研究方药复方制剂清毒饮、养正片协同细胞因子对完全缓解后急性白血病患者骨髓单个核细胞来源树突细胞诱导作用及其生物学特性的相关研究,并观察清毒扶正复方制剂在临床上对完全缓解后急性白血病患者骨髓细胞免疫表型及增殖周期的影响,探求清毒扶正方法对急性髓细胞白血病缓解后的可能治疗机制,为进一步开拓急性白血病缓解后的中医治疗方法做出努力。
PartⅠLiterature
Acute leukemia (AL) is one of the common hematology tumors, which has some characteristics, as like with serious condition and the fast development, easy to relapse after remission, low long-term survival rate. As the improvement in Chinese medicine and Western medicine theory, continuous development of the treatment to leukemia is keeping up so that the rate of acute leukemia in complete remission (CR) and long-term survival is with a considerable degree of improvement or even cure. However, most patients still relapse after complete remission, for which the fundamental reason is the existence of minimal residual leukemia cells. To control such a state of minimal residual leukemia (MRD) is one of the hotspots of leukemia research.
The international consensus is going like this; the matter of immune function besides, the presence of minimal residual leukemia has more to do with leukemia cell immune escape mechanisms, so that to kill the minimal residual leukemic cells must rely on how the body's own immune surveillance functions are performing. In this stage, after surviving a long-term chemotherapy, drug sensitivity of leukemic cells has become very low and this is the best time for the body to kill residual tumor cells assisting by immune therapy aids.
Based on the knowledge about pathogenesis, such like righteousness weak, cult poison invasion of, Chinese Medicine for minimal residual disease at present has the basically same understanding of the pathogenesis. The body is on "evil to being bad," "righteousness weak" and "not entirely over evil "pathophysiological state.
PartⅡExperimental study Qingdu Fuzheng recipe on ANLL-CR patients BMNC induction of DC source
1. Research purposes
In this experiment, Qing du Drink and Yang zheng Troche in acute myeloid leukemia patients in remission bone marrow mononuclear cells (BMNC) were cultured in vitro to observe the dendritic cells (DC) induced transformation of the situation. To induce transformation of normal DC and purified T cells were cultured with autologous T cells after stimulation was observed after stimulating T cells against human leukemia cell line K562 cells in vitro.
Qing du Drink and Yang zheng Troche are the effective hospital treatment of acute leukemia which is developed by our hospital. This study is researching from the new ways of treatment of leukemia combined by Chinese medicine, and from a new entry point that is to increase remission in patients with antigen presenting ability of dendritic cells. The purpose is to look for the effective ways for leukemia remission but to prevent its recurrence.
2. Method
2.1 The source of specimen
2.1.1 Bone marrow mononuclear cells derived from the Guangzhou University of Traditional
Chinese Medicine Department of Hematology, First Clinical Medical College of patients hospitalized with acute myeloid leukemia after complete remission (AML-CR) of bone marrow in two cases. Patients meet diagnostic criteria for acute leukemia after complete remission after standard chemotherapy, consistent with acute leukemia in complete remission criteria.
2.1.2 T cells from normal adult peripheral blood and peripheral blood of leukemia patients with complete remission after one each.
2.1.3 Leukemia cell line K562 cells.
2.2 Preparation of herb medicine
Group of New Zealand rabbits, respectively Qingdu drink, keep positive, keep positive Qingdu drink and by low, medium and high doses of fed animals, preparation of the three groups with different concentrations of serum.
2.3 Separation of bone marrow mononuclear cells to T cells
Application procedures for sheep erythrocyte rosette separated from the bone marrow T cells in the bone marrow mononuclear cells (TD-MNC).
2.4 Training from AML-CR patients with TD-MNC in DC
The TD-MNC cells with different Chinese medicines containing serum and cytokines in joint training, divided into:drinking group Qingdu, GM-CSF IL-4+IFN-α+TNF-αgroup and disinfection drink+GM-CSF+IL-4+IFN-α+TNF-αgroup, support positive group, the raising of positives+GM-CSF+ IL-4+IFN-α+TNF-αgroup and disinfection drink and keep positive+GM-CSF +IL-4+IFN-α+TNF-αgroup. Equalization of the group with serum is low and medium and high dose group.
2.5 DC identification include
morphology, immunological detection, and the secretion of cytokines IL-2, IL-6 determination.
2.6 Purification of peripheral blood T cells (magnetic activated cell sorting method)
Normal, AML-CR patients are depicting a blood 50ml, by Ficoll density gradient centrifugation of peripheral blood mononuclear cells (PBMNC), the use of bead negative selection method were purified peripheral blood T cells.
2.7 allogeneic T cells and stimulate autologous T cells
Were collected purified allogeneic T cells, AML-CR T cells from patients with AML-CR patients with TD-MNC derived DC to 10:1 ratio by adding 24-hole incubated 5d, medium supplemented with 100U/ml of IL-2.
2.8 stimulate T cells to K562 Cells (MTT)
T cells in each group with K562 cells, respectively 10:1,20:1,40:1 effect: target ratio of culture based on cell kill rate.
3. The results
The experimental results show that the group of Qing du Drink and Yang zheng Troche combined with cytokines can promote DC expression of CDla, HLA-DR, CD80, CD86, and to promote DC high secretion of cytokines IL-2, IL-6 is significantly higher than the cell group, which is statistically significant. Also, the Chinese medicine group is higher than the cell group on efficiency butt and the killing rate. The Chinese medicine group also explains the greater the concentration of T cells, tumor cell-killing rate is increased. The Chinese group compared with the control group shows that the concentration of medicine in a joint cell factor can be better than simply promote the use of DC cytokines.
PartⅢClinical Observation
Attachment:Qing du fu zheng recipe on AL-CR in patients with bone marrow cells of CD antigen expression and proliferation
1. Research purposes
We observe the expression rate of CD34, CD34/CD2, CD34/CD19, CD13/CD33 of acute leukemia patients' bone marrow cells after complete remission to understand the effect of the treatment for tiny residual leukemia; To compare the difference of bone marrow cell proliferation cycle between before and after treatment, we try to deeply study the possible mechanism of this treatment to tiny residual leukemia.
2. Research Methods
This study adopts random comparison to observe Qing du Drink and Yang zheng Troche's impact on bone marrow cells CD antigen's expression and proliferation cycle of acute leukemia patients after the complete remission.
2.1 Case from Traditional Chinese Medicine from the University Hospital and Department of Hematology, First Affiliated Hospital outpatients.
2.2 treatments
2.2.1 control:according to conventional chemotherapy to consolidate maintenance treatment (ANLL with the DA or MA or HA program, ALL or VDLP program with VDP) in chemotherapy, such as infection, bleeding, etc. according to conventional anti-infection and hemostasis.
2.2.2 Observer Group:Traditional Chinese principle of treatment with the control group. Consolidation chemotherapy, while maintaining oral Qing du Drink and Yang zheng Troche, Japan 1, sub-second service, for 4 weeks.
2.2.3 The two groups were collected before treatment and four weeks after the bone marrow, experimental observation.
2.3 Detection of bone marrow cells the expression of CD antigens by flow cytometry before and after treatment in patients with bone marrow cells CD34, CD34/CD2, CD34/CD19, CD13/CD33 expression.
2.4 Detection of bone marrow cell cycle by flow cytometry before and after treatment of bone marrow cell proliferation cycle in the expression.
3. Experimental results
The study combined with the measurement of patient's bone marrow cell cycle by flow the cytometry machine indicates that Qing du Drink and Yang zheng Troche have the capability of restraining the CD34's expression, indicates that Qing du Drink and Yang zheng Troche might restrain the proliferation of leukemia cells so that it could decrease the expression rate of CD13/CD14/CD33, which could represent myeloid leukemia cells and CD3/ CD19, which could represent lymphobiastic leukemia cell.
PartⅣConclusions
The research on Chinese monomer or compound in leukemia tumor antigen presenting has not been seen yet. This paper firstly used the serum of pharmacology to study compound prescription drug drinking clean, keep positive synergistic cytokines to Inducement of bone marrow mononuclear sourcing dendritic cells of acute leukemia and its biological characteristics after complete remission and observe the effects of clear Fuzheng drug compound in clinical to bone marrow cells immunophenotype and generation cycle of acute leukemia patients after complete remission, try to discover the mechanism of this treatment to acute myelocytic leukemia after complete remission, in order to make a further improvement of Chinese medicine treatment to this disease.
急性白血病(AL)是血液科常见肿瘤之一,具有病情较重,发展较快,缓解后易于复发,长期生存率不高等特点,随着中、西医理论的完善、白血病的治疗方法不断开发和发展,使急性白血病的完全缓解率(CR)和长期生存率有了相当程度的提高,甚至完全治愈。但大部分患者即使完全缓解仍有复发,其根本原因就在于微量残留的白血病细胞的存在,对这种白血病的微小残留状态(MRD)防治是目前白血病研究的热点之一。
国内外比较一致的意见认为,微小残留白血病的存在除了与机体免疫功能有关外,与白血病细胞本身的免疫逃逸机制有更密切关系,所以杀灭微小残留白血病细胞必须依靠机体自身的免疫监视功能的正常发挥。而在微小残留白血病阶段,肿瘤负荷较小,并且经过长期化疗后残存下来的白血病细胞对药物的敏感性已经很低,此时应用免疫治疗辅助机体杀灭残存的肿瘤细胞是最好的时机。
中医基于对急性白血病正气虚弱,邪毒内侵的病因病机认识,目前对微小残留病的病理机制的认识基本一致,机体处于“邪去正衰”、“正气虚弱”而“余邪未尽”的病理生理状态。基于正气虚弱而邪毒未净是微量残留白血病的基本病理,多认为扶正祛邪治法是其基本的治疗。
第二部分实验研究
清毒扶正方药对ANLL-CR患者BMNC来源DC的诱导作用
1.研究目的
本实验用清毒饮、养正片含药血清配合细胞因子对急性髓细胞性白血病缓解期患者骨髓单个核细胞(BMNC)进行体外培养,观察树突细胞(DC)的诱导转化情况。以诱导转化的DC与纯化的正常人T细胞、患者自体T细胞共同培养,以激发T细胞,观察激发后的T细胞对人白血病细胞株K562细胞的杀伤作用。
清毒饮、养正片是本院研制的治疗急性白血病的有效中药制剂,本实验从中医药结合免疫治疗白血病的新途径出发,从提高缓解后患者的树突状细胞抗原提呈能力的新切入点,探讨中医药抗白血病的新作用机制,寻求中医治疗缓解后白血病并防止其复发的有效方法。
2.研究方法：
2.1标本来源
2.1.1骨髓单个核细胞来源于广州中医药大学第一临床医学院血液科住院急性髓系白血病患者缓解后(AML-CR)骨髓两例。患者均符合急性白血病诊断标准经标准方案化疗后完全缓解,符合急性白血病完全缓解标准。
2.1.2 T细胞来自正常成人外周血及白血病患者完全缓解后外周血各一例。
2.1.3白血病细胞株K562细胞。
2.2制备中药含药血清
取新西兰兔分组,分别以清毒饮、养正片、清毒饮合养正片按低、中、高剂量灌胃动物,制备三个组别的不同浓度的含药血清。
2.3分离骨髓去T细胞单个核细胞
应用羊红细胞玫瑰花结程序从骨髓中分离出去T细胞的骨髓单个核细胞(TD-MNC)。
2.4培养来源于AML-CR患者TD-MNC的DC
将TD-MNC细胞与不同中药含药血清与细胞因子联合培养,分为：清毒饮组、GM-CSF+IL-4+IFN-α+TNF-α组、清毒饮+GM-CSF+IL-4+IFN-α+TNF-α组、养正片组、养正片+GM-CSF+IL-4+IFN-α+TNF-α组、清毒饮合养正片+GM-CSF+IL-4+IFN-α+TNF-α组。各含药血清组均分低、中、高剂量组。
2.5 DC鉴定：包括形态观察、免疫学检测、及其分泌的细胞因子IL-2、IL-6的测定。
2.6外周血T细胞的分离纯化(磁珠分选法)
正常人、AML-CR患者各取一份外周血50ml,经Ficoll密度梯度离心获得外周血单个核细胞(PBMNC)后,采用磁珠阴性选择法制得纯化的外周血T细胞。流式细胞仪检测纯度。
2.7同种异体T细胞及自体T细胞的激发
分别收集纯化的同种异体T细胞、AML-CR患者自体T细胞,与AML-CR患者TD-MNC来源的DC以10：1比例加入24孔培养板培养5d,培养基中加入10OU/ml的IL-2。
2.8激发T细胞对K562细胞的杀伤作用(MTT法)
将各组T细胞与K562细胞分别以10：1、20：1、40:1效：靶比例培养,计算细胞杀伤率。
3.研究结果
本实验结果发现清毒饮+养正片组的低剂量组联合细胞因子可促进DC高表达CD1a、HLA-DR、CD80、CD86,并能促使DC高分泌细胞因子IL-2、IL-6,与对照组(细胞因子组)相比表达均明显升高,有统计学意义,说明清毒扶正中药可促进树突细胞的增殖和表达,提高树突细胞的抗原提呈能力。并且,各中药组内效靶比越高,杀伤率越高,这也说明T细胞浓度越大,对肿瘤细胞的杀伤率也越高。各中药组与对照组相比的结果说明,在某种浓度中药联合细胞因子可以比单纯用细胞因子更好地促进DC细胞的诱导转化,从而更强地激发T细胞,产生更大的杀伤力。
第三部分临床观察
附：清毒扶正方药对AL-CR患者骨髓细胞CD抗原表达及增殖的影响
1.研究目的
以清毒扶正为立法依据的清毒饮+养正片对照观察完全缓解后急性白血病患者治疗前后骨髓细胞CD34、CD34/CD2、CD34/CD19、CD13/CD33表达率,了解清毒扶正治法对微小残留白血病的治疗效果；通过对照观察患者治疗前后骨髓细胞增殖周期的变化,进一步探求该法治疗微小残留白血病的可能机制。
2.研究方法
本研究采用随机对照观察清毒扶正方药清毒饮+养正片对完全缓解的急性白血病患者骨髓细胞CD抗原表达及增殖周期的影响。
2.1病例来源来源于广州中医药大学第一附属医院血液科住院及门诊患者。
2.2治疗措施
2.2.1对照组：按常规化疗方案巩固维持治疗(ANLL用DA或MA或HA方案,ALL用VDP或VDLP方案)进行化疗,如出现感染、出血等情况,则按常规抗感染及止血治疗。
2.2.2观察组：西医治疗原则同对照组。巩固维持化疗的同时口服清毒饮、养正片方煎剂,日一剂,分二次服,持续4周。
2.2.3分别采集两组患者治疗前、治疗四周后的骨髓组织,进行实验观察。
2.3骨髓细胞CD抗原表达检测流式细胞仪测定患者治疗前后骨髓细胞CD34、CD34/CD2、CD34/CD19、CD13/CD33表达率。
2.4骨髓细胞增殖周期检测流式细胞仪检测患者治疗前后骨髓细胞增殖周期各时相表达率。
3.实验结果
本实验结合流式细胞仪对患者骨髓细胞增殖周期的测定,表明清毒扶正方药有抑制CD34表达的作用,说明清毒饮+养正片可能抑制了白血病细胞的增殖,从而使能代表髓系白血病细胞的CD13/CD14/CD33、代表淋巴系白血病细胞的CD3/CD19表达率下降。因而,扶正清毒方法可能对稳定病情、改善生活质量,甚至对提高急性髓性白血病患者生存期有宜。
第四部分研究结论
目前尚未见关于中药单体或复方在白血病肿瘤抗原提呈方面的作用研究。本论文用血清药理学方法率先研究方药复方制剂清毒饮、养正片协同细胞因子对完全缓解后急性白血病患者骨髓单个核细胞来源树突细胞诱导作用及其生物学特性的相关研究,并观察清毒扶正复方制剂在临床上对完全缓解后急性白血病患者骨髓细胞免疫表型及增殖周期的影响,探求清毒扶正方法对急性髓细胞白血病缓解后的可能治疗机制,为进一步开拓急性白血病缓解后的中医治疗方法做出努力。
PartⅠLiterature
Acute leukemia (AL) is one of the common hematology tumors, which has some characteristics, as like with serious condition and the fast development, easy to relapse after remission, low long-term survival rate. As the improvement in Chinese medicine and Western medicine theory, continuous development of the treatment to leukemia is keeping up so that the rate of acute leukemia in complete remission (CR) and long-term survival is with a considerable degree of improvement or even cure. However, most patients still relapse after complete remission, for which the fundamental reason is the existence of minimal residual leukemia cells. To control such a state of minimal residual leukemia (MRD) is one of the hotspots of leukemia research.
The international consensus is going like this; the matter of immune function besides, the presence of minimal residual leukemia has more to do with leukemia cell immune escape mechanisms, so that to kill the minimal residual leukemic cells must rely on how the body's own immune surveillance functions are performing. In this stage, after surviving a long-term chemotherapy, drug sensitivity of leukemic cells has become very low and this is the best time for the body to kill residual tumor cells assisting by immune therapy aids.
Based on the knowledge about pathogenesis, such like righteousness weak, cult poison invasion of, Chinese Medicine for minimal residual disease at present has the basically same understanding of the pathogenesis. The body is on "evil to being bad," "righteousness weak" and "not entirely over evil "pathophysiological state.
PartⅡExperimental study Qingdu Fuzheng recipe on ANLL-CR patients BMNC induction of DC source
1. Research purposes
In this experiment, Qing du Drink and Yang zheng Troche in acute myeloid leukemia patients in remission bone marrow mononuclear cells (BMNC) were cultured in vitro to observe the dendritic cells (DC) induced transformation of the situation. To induce transformation of normal DC and purified T cells were cultured with autologous T cells after stimulation was observed after stimulating T cells against human leukemia cell line K562 cells in vitro.
Qing du Drink and Yang zheng Troche are the effective hospital treatment of acute leukemia which is developed by our hospital. This study is researching from the new ways of treatment of leukemia combined by Chinese medicine, and from a new entry point that is to increase remission in patients with antigen presenting ability of dendritic cells. The purpose is to look for the effective ways for leukemia remission but to prevent its recurrence.
2. Method
2.1 The source of specimen
2.1.1 Bone marrow mononuclear cells derived from the Guangzhou University of Traditional
Chinese Medicine Department of Hematology, First Clinical Medical College of patients hospitalized with acute myeloid leukemia after complete remission (AML-CR) of bone marrow in two cases. Patients meet diagnostic criteria for acute leukemia after complete remission after standard chemotherapy, consistent with acute leukemia in complete remission criteria.
2.1.2 T cells from normal adult peripheral blood and peripheral blood of leukemia patients with complete remission after one each.
2.1.3 Leukemia cell line K562 cells.
2.2 Preparation of herb medicine
Group of New Zealand rabbits, respectively Qingdu drink, keep positive, keep positive Qingdu drink and by low, medium and high doses of fed animals, preparation of the three groups with different concentrations of serum.
2.3 Separation of bone marrow mononuclear cells to T cells
Application procedures for sheep erythrocyte rosette separated from the bone marrow T cells in the bone marrow mononuclear cells (TD-MNC).
2.4 Training from AML-CR patients with TD-MNC in DC
The TD-MNC cells with different Chinese medicines containing serum and cytokines in joint training, divided into:drinking group Qingdu, GM-CSF IL-4+IFN-α+TNF-αgroup and disinfection drink+GM-CSF+IL-4+IFN-α+TNF-αgroup, support positive group, the raising of positives+GM-CSF+ IL-4+IFN-α+TNF-αgroup and disinfection drink and keep positive+GM-CSF +IL-4+IFN-α+TNF-αgroup. Equalization of the group with serum is low and medium and high dose group.
2.5 DC identification include
morphology, immunological detection, and the secretion of cytokines IL-2, IL-6 determination.
2.6 Purification of peripheral blood T cells (magnetic activated cell sorting method)
Normal, AML-CR patients are depicting a blood 50ml, by Ficoll density gradient centrifugation of peripheral blood mononuclear cells (PBMNC), the use of bead negative selection method were purified peripheral blood T cells.
2.7 allogeneic T cells and stimulate autologous T cells
Were collected purified allogeneic T cells, AML-CR T cells from patients with AML-CR patients with TD-MNC derived DC to 10:1 ratio by adding 24-hole incubated 5d, medium supplemented with 100U/ml of IL-2.
2.8 stimulate T cells to K562 Cells (MTT)
T cells in each group with K562 cells, respectively 10:1,20:1,40:1 effect: target ratio of culture based on cell kill rate.
3. The results
The experimental results show that the group of Qing du Drink and Yang zheng Troche combined with cytokines can promote DC expression of CDla, HLA-DR, CD80, CD86, and to promote DC high secretion of cytokines IL-2, IL-6 is significantly higher than the cell group, which is statistically significant. Also, the Chinese medicine group is higher than the cell group on efficiency butt and the killing rate. The Chinese medicine group also explains the greater the concentration of T cells, tumor cell-killing rate is increased. The Chinese group compared with the control group shows that the concentration of medicine in a joint cell factor can be better than simply promote the use of DC cytokines.
PartⅢClinical Observation
Attachment:Qing du fu zheng recipe on AL-CR in patients with bone marrow cells of CD antigen expression and proliferation
1. Research purposes
We observe the expression rate of CD34, CD34/CD2, CD34/CD19, CD13/CD33 of acute leukemia patients' bone marrow cells after complete remission to understand the effect of the treatment for tiny residual leukemia; To compare the difference of bone marrow cell proliferation cycle between before and after treatment, we try to deeply study the possible mechanism of this treatment to tiny residual leukemia.
2. Research Methods
This study adopts random comparison to observe Qing du Drink and Yang zheng Troche's impact on bone marrow cells CD antigen's expression and proliferation cycle of acute leukemia patients after the complete remission.
2.1 Case from Traditional Chinese Medicine from the University Hospital and Department of Hematology, First Affiliated Hospital outpatients.
2.2 treatments
2.2.1 control:according to conventional chemotherapy to consolidate maintenance treatment (ANLL with the DA or MA or HA program, ALL or VDLP program with VDP) in chemotherapy, such as infection, bleeding, etc. according to conventional anti-infection and hemostasis.
2.2.2 Observer Group:Traditional Chinese principle of treatment with the control group. Consolidation chemotherapy, while maintaining oral Qing du Drink and Yang zheng Troche, Japan 1, sub-second service, for 4 weeks.
2.2.3 The two groups were collected before treatment and four weeks after the bone marrow, experimental observation.
2.3 Detection of bone marrow cells the expression of CD antigens by flow cytometry before and after treatment in patients with bone marrow cells CD34, CD34/CD2, CD34/CD19, CD13/CD33 expression.
2.4 Detection of bone marrow cell cycle by flow cytometry before and after treatment of bone marrow cell proliferation cycle in the expression.
3. Experimental results
The study combined with the measurement of patient's bone marrow cell cycle by flow the cytometry machine indicates that Qing du Drink and Yang zheng Troche have the capability of restraining the CD34's expression, indicates that Qing du Drink and Yang zheng Troche might restrain the proliferation of leukemia cells so that it could decrease the expression rate of CD13/CD14/CD33, which could represent myeloid leukemia cells and CD3/ CD19, which could represent lymphobiastic leukemia cell.
PartⅣConclusions
The research on Chinese monomer or compound in leukemia tumor antigen presenting has not been seen yet. This paper firstly used the serum of pharmacology to study compound prescription drug drinking clean, keep positive synergistic cytokines to Inducement of bone marrow mononuclear sourcing dendritic cells of acute leukemia and its biological characteristics after complete remission and observe the effects of clear Fuzheng drug compound in clinical to bone marrow cells immunophenotype and generation cycle of acute leukemia patients after complete remission, try to discover the mechanism of this treatment to acute myelocytic leukemia after complete remission, in order to make a further improvement of Chinese medicine treatment to this disease.
引文
[1]邓家栋,杨崇礼,杨天楹等.邓家栋临床血液学,上海：上海科学技术出版社,2001,7,第1版：942.
[2]刘贵建.急性白血病的MICM分型[J].中华检验医学杂志,2004；27(6)：398－400.
[3]Garand R, Bene MC, the GEIL. A new approach of acute lymphoblastic leukemia immunophenotypic classification:1984-1994 the GEIL experience[J]. Leukemia lymphoma,1994; 13 [Suppl1]:1 - 5.
[4]Bene MC, Castoldi G, Knapp W, et al. Proposals for the immunological classification of acute leukemia European Group for the Immunological Characterization of Leukemias(EGIL) [J]. Leukemia,1995; 9:1783-1786.
[5]张之南,郝玉书.血液病学,北京：人民卫生出版社,2003,第1版：994－1053.
[6]张之南,郝玉书,血液病学,北京：人民卫生出版社,2003,第1版：1063-1064.
[7]张国材,罗绍凯,李娟,等.异基因外周血干细胞与异基因骨髓移植治疗白血病疗效的比较[J].中山大学学报-医学科学版,2003；24(2)：172-174.
[8]陈灏珠主编.实用内科学,北京：人民卫生出版社,2005,第12版：2319.
[9]陈运贤,丁倩.白血病治疗新靶点—白血病干细胞[J].新医学,2008；39(9)：613-614.
[10]松村道等(于佩瑶译).白血病[J].日本医学介绍,2006；27(11)：500-503.
[11]Ravandi F, Estrov Z. Eradication of leukemia stem cells as a new goal of therapy in leukemia[J]. Clin Cancer Res,2006; 12(2):340-344.
[12]Misaghian N, Ligreti G, Steelman LS, et al. Targeting the leukemic stem cell:the Holy Grail of leukemia therapy. Leukemia,2009; 23(1):25-42.
[13]Ishikawa F, Yoshida S, Saito Y, et al. Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region [J]. Nat Biotechnol,2007; 25:1315-1321.
[14]邱林,马军.急性髓细胞白血病治疗的新进展—2009年ASCO年会点评[J].中国处方药,2009；89(8)：38-39.
[15]沈志祥,王振义.成人急性白血病的治疗[J].继续医学教育,2007；21(4)4-7.
[16]Didier B, Norbert V, Catherine F, et al. Current status of reduced intensity conditioning allogeneic stem cell transplan tation for acute myeloidleukemia[J]. Haematologica,2007; 92(4):533-541.
[17]崔彦芹,陈福雄.白血病免疫治疗的研究进展[J].广州医学院学报,2003；31(2)：101-104.
[18]Patricia Yotnda, Pierre Mintz, Kalliopi Grigoriadou, et al. Analysis of T-cell defects in the specific immune response against acute lymphoblastic leukemia cells [J]. Experimental Hematology,1999; 27:1375-1383.
[19]Ronald H, Schwartz. Costimulation of T lymphocytes:The role of CD28, CTLA -4, and B7/BBI in interleukin-2 production and immunotherapy[J]. Cell, 1992; 71:1065-1068.
[20]Angelo A Cardoso, Joachim L Sehultze, et al. Pre-B ALL lymphoblastic leukemia cells may induce T cell an-ergy to alloantigen[J]. Blood,1996; 88(1):41-48.
[21]Miwako Narita, Masuhiro Takahashi, Aichun Liu, et al. Leukemia blast-induced T-cell anergy demonsated by leukemia-derived dendritic cells in acute myelogenous leukemia[J]. Experimental Hematology,2001; 29:709-719.
[22]王斌,杨建民编译.成人急性髓性白血病治疗新策略[J].世界临床药物,2006；27(5)：289-291.
[23]William J. Kell,Alan K. Burnett, et al. A feasibility study of simultaneous administration of gemtuzumab ozogamicin with intensive chemotherapy in induction and consolidation in younger patients with acute myeloid leukemia.Blood,2003 (12); 102:4277-4283.
[24]Karp J, Lancet J, Kaufmann SH, et al. Clinical and biologic activity of the farnesyltransferase inhibitor R115777 in adults with refractory and relapse acute leukemias:a phase 1 clinical laboratory correlative trial. Blood,2001; 97 (6):3361-3369.
[25]Jeffrey E. Lancet, Ivana Gojo, Jason Gotlib, et al.A phase 2 study of the farnesyltransferase inhibitor tipifarnib in poor-risk and elderly patients with previously untreated acute myelogenous leukemia. Blood,2007; 109 (2):1387-1394.
[26]Walter Fiedler, Rolf Mesters, Heike Tinnefeld, et al. A phase 2 clinical study of SU5416 in patients with refractory acute myeloid leukemia. Blood, 2003; 102 (10):2763-2767.
[27]Ellen Weisberg, Christina Boulton, Louise M Kelly, et al. Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. Cancer Cell,2002; 1(5):433-443.
[28]Mark Levis, Jeffrey Allebach, Kam-Fai Tse, et al. A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. Blood, 2002; 99 (5):3885-3891.
[29]Stefan Frohling, Claudia Scholl, Ross L. Levine, et al. Identification of Driver and Passenger Mutations of FLT3 by High-Throughput DNA Sequence Analysis and Functional Assessment of Candidate Alleles. Cancer Cell,2007; 12(6):501-513.
[30]Keith W. Pratz, Takashi Sato, Kathleen M. Murphy, et al. FLT3-mutant allelic burden and clinical status are predictive of response to FLT3 inhibitors in AML. Blood,2010; 115 (2):1425-1432.
[31]Elihu Estey, Hartmut Dohner.Acute myeloid leukemia[J]. Lancet.2006; 368 (11):1894-1907.
[32]Hagop Kantarjian, Varsha Gandhi, Jorge Cortes, et al. Phase 2 clinical and pharmacologic study of clofarabine in patiens with refractory or replapsed acute leukemia. Blood,2003; 102 (10):2379-2386.
[33]杨天楹,张之南,郝玉书主编.临床血液学进展,中国协和医科大学[M].北京：北京医科大学联合出版社,1992,12：168.
[34]秘营昌,卞寿庚.急性髓系白血病完全缓解后治疗周期的初步探讨[J].中华血液学杂志,2001；22(10)：520-523.
[35]Guzman ML, Swiderski CF, Howard DS, et al. Preferential induction of apoptosis for primary human leukemic stem cells[J]. Proc Nail Acad Sci USA, 2002; 99:16220-16225.
[36]Jordan CT, Yamasaki G, Minamoto D. High-resolution cell cycle analysis of defined phenotypic subsets within primitive human hematopoietic cell populations [J]. Exp Hematol,1996; 24:1347-1355.
[37]Dean M, FojoT, Bates S. Tumour stem cells and drug resistance[J]. Nat Rev Cancer,2005; 5:275-284.
[38]古莹,邱曦,徐荣臻.白血病干细胞分子生物学特性及其治疗策略研究进展.基础医学与临床,2008；28(9)：904-907.
[39]叶海燕,陈焯文,肖平,等.流式细胞仪检测急性白血病微小残留病的意义[J].实用医学杂志,2009；25(13)：2075-2077.
[40]Cignetti A, Bryant E, Allione B, et al. CD34+ acute myeloid and lymphoid leukemic blasts can be induced to differentiation in-to dendritic cells[J]. Blood,2001; 94:2084-2055.
[41]童向民,金洁,薛永权,等.急性髓细胞白血病来源的树突状细胞的研究[J].中 华内科杂志,2004；43(1)：45-48.
[42]何学鹏,尤胜国,卞寿庚,等.急性髓系白血病患者树突状细胞诱导抗自身白血病T细胞免疫的实验研究[J].中华血液学杂志,2001；2(12)：629-632.
[43]Harrison BD, Adams JA, Mark B, et al. Stimulation of auto-logous proliferative and cytotoxic T -cell responses by "leukemia dendritic cells" derived from blast cells in acute myeloid leukemia [J]. Blood,2001; 97(9): 2764-2771.
[44]Montagna D, Maccario R, Locatelli F, et al. EX vivopriming for long-term maintenance of antileukemia human cytoxie T cell suggests a general procedure for adoptive immunotherapy[J]. Blood,2001; 98(12):3359 - 3366.
[45]吴明媛,黄勇,朱庭,等.CD34-+HPC来源的树突状细胞体外扩增和功能研究[J].苏州大学学报(医学版),2005；25(1)：4-5.
[36]张蓉,刘莉,张岩实,等.成人外周血来源的树突状细胞的体外诱导[J].实用医学杂志,2005;21(13)：1381-1382.
[47]贺白,邱国强,谢晓宝.冻融抗原负载树突状细胞诱导细胞毒性T淋巴细胞特异性杀伤急性白血病细胞的实验研究[J].生物医学工程学杂志,2004；21(6)：974-975.
[48]赵绍琴.中医中药治疗白血病的新认识[J].中医药研究,1992；2：11-12.
[49]谢天亮.白血病的中医论治[J].基层医学论坛,2008;12(11)：1018-1019.
[50]陆运鑫,罗昌国.试析白血病耐药的中医病机[J].辽宁中医药大学学报,2008；10(5)：10-11.
[51]胡乃平,王展.周霭祥治疗白血病经验[J].中医杂志,2000；41(6)：433-434.
[52]曹方,李彦,贾斌.唐由君治疗急性白血病的经验[J].国医论坛,2001；16(2)：11-12.
[53]李敬花,瞿东升.中医辨证治疗白血病的临床体会[J].中国中医药信息杂志,1999；6(2)：62-62.
[54]肖倩.中药配合化疗治疗急性白血病38例疗效观察[J].中医杂志,1998；39(5)：283-285.
[55]吴登峰,吴登蛟.HA方案加中药治疗急性非淋巴细胞白血病30例[J].黑龙江中医药,1998；(5)：19-20.
[56]李宇涛,甘慧娟,吴同玉等.急性白血病常用中医辨证方法探讨[J].现代中西医结合杂志,2009；18(10)：1107-1108.
[57]朱文锋.辨证统一体系的创立[J].中国中医基础医学杂志,2001；7(4)：4-8.
[58]麻柔,刘锌,薛向军.中医对微小残留向血病的认识和治疗[J].中医杂志,1994;35(2)：748-749.
[59]张姗姗,许勇钢,杨晓红.微小残留白血病患者细胞免疫功能与中医分型关系的研究[J].中国中西结合杂志,2004；24(1)：36-40.
[60]黄礼明.试论急性白血病缓解后之中医论治[J].江苏中医药,2003；24(5)：6-8.
[61]王乙忠,周霭祥,王志平,等.扶正抑癌冲剂配合化疗治疗微小残留白血病的临床与实验研究[J].中医杂志,1996；37(9)：535-538.
[62]陆灿红,李怡,巫蓉.残留白血病的中医治疗思路[J].四川中医,2000；18(1)：10-11.
[63]孙淑贞,麻柔.微小残留白血病中医药研究概况[J].北京中医药,2009；28(5)：387-390.
[64]黄礼明,胡莉文,丘和明.微小残留白血病的病因病机及治疗探讨[J].中医杂志,2005;46(6)：465-466.
[65]刘红,刘朝晖.滋阴补肾清热凉血法治疗微小残留白血病18例观察[J].实用中西医结合临床,2005；5(5)：24-25.
[66]华昭,黄礼明.治疗急性白血病缓解期贵在透邪与存阴并进[J].云南中医中药杂志,2009；30(3)：4-5.
[67]黄海茵,于尔辛.中药对NK、LAK细胞及IL-2活性的影响[J].中国中西医结合杂志.1993；13(4)：253-255.
[68]马武开.中医药防治白血病的思路及方法[J].中国中医药信息杂志.2004；11(3)：229-230.
[69]于志峰,戴锡珍,郝志强,等.扶正解毒方治疗微小残留白血病的疗效及其免疫机制的研究[J].中国中医药信息杂志,2000；7(11)：29-30.
[70]于志峰,戴锡孟,戴锡珍.益气养阴清热解毒法治疗微小残留白血病的凋亡机制研究[J].天津中医,2001；18(4)：29-30.
[71]于志峰,戴锡珍,戴锡孟,等.六神丸治疗微小残留白血病凋亡机制的实验研究[J].中医研究,2000；13(5)：18-20.
[72]王欣,褚建新,傅汝林,等.清热解毒方对可移植性小鼠微小残留白血病实验研究[J].实用中西医结合临床,2004；4(5)：1-3.
[73]王欣,褚建新,傅汝林,等.清热解毒方对可移植性小鼠淋巴细胞白血病及其微小残留病疗效的比较研究[J].中医药临床杂志,2005；17(2)：148-149.
[74]王东生.中药及其有效成分抗白血病的分子机制[J].国际中医中药杂志,2007；29(3)：160-163.
[75]潘世扬,黄佩王君,唐云章等.小儿急性淋巴细胞性白血病微小残留病免疫治疗的初步研究[J].南京医科大学学报,2001；21(6)；492-495.
[76]乌庆超,于书增,褚建新,等.骨髓根除性治疗恶性血液病临床研究[J].临床 血液学杂志,2000；12(4)：171.
[77]邓成珊,等.当代中西医结合血液病学,北京：中国医药科技出版社,1997：9.
[78]王斌,胡岳山,李杰芬.灵芝三萜对小鼠脾脏树突状细胞增殖的影响[J].中药材,2005；7(28)：577-578.
[79]李松林.清毒饮、养正片治疗急性白血病30例疗效观察[J].陕西中医函授,2000;1：13-14.
[80]麻柔,刘锋,杨经敏,等.扶正抗白冲剂对急性白血病患者长期存活影响的临床研究[J].中国中西医结合杂志,1998；18(5)：276-278.
[81]李振波,丘和明,张萍.L7212白血病小鼠脾细胞中肿瘤坏死因子α活性及mRNA表达和复方中药清毒饮对其影响[J].中国实验血液学杂志,1999；7(3):234-237.
[82]袁健,熊文,陈建波,等.染色体分析及FISH法对急性早幼粒细胞白血病诊断的临床意义[J].实用癌症杂志,2007；22(3)：266-268.
[83]李倩,杜兵,杨军军,等.应用序列探针检测慢性淋巴细胞白血病基因异常[J].中国卫生检验杂志,2007；17(4)：672-674.
[84]陈蕾蕾,孙雪梅,陈军浩,等.FISH在免疫表型特殊的急性早幼粒细胞白血病诊断中的应用[J].标记免疫分析与临床,2005；12(4)：193-195,T0001.
[85]刘启发,宋兰林,张钰,等.双色间期荧光原位杂交检测异基因造血干细胞移植后性染色体嵌合状态的临床意义[J].中华医学遗传学杂志,2005；22(2)：212-215.
[86]王慧萍,李国霞,乔振华,等.联合应用细胞遗传学、巢式RT-PCR和FISH技术检测慢性髓系白血病治疗过程中的肿瘤负荷[J].中国实验血液学杂志,2007；15(2)：237-241.
[87]李栋.常用微小残留白血病的检测方法[J].泰山卫生,2001；25(1)：17-20.
[88]丁肖华.白血病的检测方法[J].四川解剖学杂志,2006；14(3)：27-29.
[89]费新红,许小平.微小残留白血病的检测方法及临床应用进展[J].白血病·淋巴瘤,2003；12(5)：311-314.
[90]张秋堂,冯磊,李涛,等.三色荧光标记抗体在急性白血病免疫分型中的应用[J].临床血液学杂志,2004；17(5)：252-254.
[91]Bruce D. Cheson, John M. Bennett, Kenneth J., et al. Revised recommendations of the International Working Group for diagnosis, standardization of response criteria, treatment outcomes, and Reporting Standards for Therapeutic Trials in acute myeloid leukemia[J]. Clinical oncology,2003; 21 (12):4642 - 4649.
[92]Guido Marcucci, Krzysztof Mrozek, Amy S. Ruppert, et al. Abnormal cytogenetics at date of morphologic complete remission predicts short overall and disease-free survival, and higher relapse rate in adult acute myeloid leukemia:results from Cancer and Leukemia Group B Study 8461[J]. Clinical Oncology,2004; 22 (6):2410-2418.
[93]陈志炉,史亦谦,周郁鸿.中药诱导白血病细胞凋亡的研究近况[J].实用中西医结合临床,2007；7(3)：91-93.
[94]高娜,茆俊卿,张育.中药逆转白血病多药耐药的研究进展[J].江苏中医药,2007；39(3)：60-62.
[95]杜振兰,王瑞,陈虎.中医药治疗白血病的现状[J].第一军医大学分校学报,2005；28(1)：97-100.
[96]杨振江,曹克俭,郭岳峰,等.瘀毒清对微小残留白血病作用的实验研究[J].浙江中西医结合杂志,2001；11(10)：613-614,625.
[97]于志峰,戴锡孟,戴锡珍.益气养阴活血化瘀方法治疗微小残留白血病免疫机制的实验研究[J].天津中医学院学报,2001；20(1)：22-24.
[98]周岱翰.中药抗肿瘤研究的评析与前景[J].新中医,2008；40(11)：1-3.
[99]杨洪涌,潘习龙,丘和明,等.清毒饮和养正片诱导L712小鼠自血病细胞凋亡及其对Bcl-2、P53基因表达的影响[J].新中医；2002；34(6)：69-71.
[100]梁毅,陈志雄,丘和明.清毒饮、养正片影响白血病K562细胞增殖周期、细胞凋亡的实验研究[J].中国中医基础医学杂志,2004；10(1)：44-47.
[101]后盾,丘和明,聂梦仑.清毒饮、养正片联合化疗治疗急性髓细胞白血病32例及对血清乳酸脱氢酶的影响[J].中国中医药杂志,2005；12(5)：313-314.
[102]李振波,丘和明.不同浓度清毒饮和养正片对L7212白血病小鼠脾脏B细胞功能的影响[J].新中医,1999；31(8)：31-32.
[103]Geissler K pathophysiology and treatment of apastic anemia[J]. wiener kliniche wochen sehrift,2003; 115(13-14):444-450.
[104]Gabrii Ovich D. Mechanisms and functional significance of tumour induced dendritic-cell defects[J]. Nat Rev Immunol,2004; 4(12):941-951.
[105]Zou W. Immunosuppregsive networks in the tumour environment and their therapeutic relevance [J]. Nat Rev Cancer,2005,5(4):263-274.
[106]李启亮,单保恩,张超,等.不同刺激剂诱导肺癌患者引流淋巴结细胞分泌细胞因子的研究[J].细胞与分子免疫学杂志,2006；22(5)：609-612.
[107]Sabel Ms, Arora A, su G, et al. Adoptive immunotherapy Of breast cancer with lymph node cells primed by cryoablation of the primary tumor[J]. Cryobiology,2006; 53(3):360-366.
[108]翁开枝,谢晓宝,邱国强,等.临床注射用α-干扰素和粒-巨集落刺激因子诱导慢性髓性白血病细胞向树突状细胞分化[J].肿瘤,2007；27(1)：55-58.
[109]Jain P, Ahuja J, Khan ZK, et al. Modulation of dendritic cell maturation and function by the Tax protein of human T cell leukemia virus type 1[J]. J Leukoe Biol,2007；82(1):44-56.
[110]Zhou LJ, Tedder TF. CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells[J]. Proc Natl Acad Sci USA,1996； 93:2588.
[111]张群豪,钟蓓,陈可冀,等.用血清药理方法观察血府逐瘀浓缩丸对实验性动脉粥样硬化家兔主动脉平滑肌细胞增殖的影响[J].中国中西医结合杂志,1996；16(3)：156-159.
[112]谭余庆,霍海如.中药血清药理学研究方法及其应用[J].中国实验方剂学杂志,1997;3(6)：41-43.
[113]王力倩,李仪奎,符胜光,等.血清药理学方法研究探索[J].中国中医药科技,1997;13(3)：29-31.
[2]刘贵建.急性白血病的MICM分型[J].中华检验医学杂志,2004；27(6)：398－400.
[3]Garand R, Bene MC, the GEIL. A new approach of acute lymphoblastic leukemia immunophenotypic classification:1984-1994 the GEIL experience[J]. Leukemia lymphoma,1994; 13 [Suppl1]:1 - 5.
[4]Bene MC, Castoldi G, Knapp W, et al. Proposals for the immunological classification of acute leukemia European Group for the Immunological Characterization of Leukemias(EGIL) [J]. Leukemia,1995; 9:1783-1786.
[5]张之南,郝玉书.血液病学,北京：人民卫生出版社,2003,第1版：994－1053.
[6]张之南,郝玉书,血液病学,北京：人民卫生出版社,2003,第1版：1063-1064.
[7]张国材,罗绍凯,李娟,等.异基因外周血干细胞与异基因骨髓移植治疗白血病疗效的比较[J].中山大学学报-医学科学版,2003；24(2)：172-174.
[8]陈灏珠主编.实用内科学,北京：人民卫生出版社,2005,第12版：2319.
[9]陈运贤,丁倩.白血病治疗新靶点—白血病干细胞[J].新医学,2008；39(9)：613-614.
[10]松村道等(于佩瑶译).白血病[J].日本医学介绍,2006；27(11)：500-503.
[11]Ravandi F, Estrov Z. Eradication of leukemia stem cells as a new goal of therapy in leukemia[J]. Clin Cancer Res,2006; 12(2):340-344.
[12]Misaghian N, Ligreti G, Steelman LS, et al. Targeting the leukemic stem cell:the Holy Grail of leukemia therapy. Leukemia,2009; 23(1):25-42.
[13]Ishikawa F, Yoshida S, Saito Y, et al. Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region [J]. Nat Biotechnol,2007; 25:1315-1321.
[14]邱林,马军.急性髓细胞白血病治疗的新进展—2009年ASCO年会点评[J].中国处方药,2009；89(8)：38-39.
[15]沈志祥,王振义.成人急性白血病的治疗[J].继续医学教育,2007；21(4)4-7.
[16]Didier B, Norbert V, Catherine F, et al. Current status of reduced intensity conditioning allogeneic stem cell transplan tation for acute myeloidleukemia[J]. Haematologica,2007; 92(4):533-541.
[17]崔彦芹,陈福雄.白血病免疫治疗的研究进展[J].广州医学院学报,2003；31(2)：101-104.
[18]Patricia Yotnda, Pierre Mintz, Kalliopi Grigoriadou, et al. Analysis of T-cell defects in the specific immune response against acute lymphoblastic leukemia cells [J]. Experimental Hematology,1999; 27:1375-1383.
[19]Ronald H, Schwartz. Costimulation of T lymphocytes:The role of CD28, CTLA -4, and B7/BBI in interleukin-2 production and immunotherapy[J]. Cell, 1992; 71:1065-1068.
[20]Angelo A Cardoso, Joachim L Sehultze, et al. Pre-B ALL lymphoblastic leukemia cells may induce T cell an-ergy to alloantigen[J]. Blood,1996; 88(1):41-48.
[21]Miwako Narita, Masuhiro Takahashi, Aichun Liu, et al. Leukemia blast-induced T-cell anergy demonsated by leukemia-derived dendritic cells in acute myelogenous leukemia[J]. Experimental Hematology,2001; 29:709-719.
[22]王斌,杨建民编译.成人急性髓性白血病治疗新策略[J].世界临床药物,2006；27(5)：289-291.
[23]William J. Kell,Alan K. Burnett, et al. A feasibility study of simultaneous administration of gemtuzumab ozogamicin with intensive chemotherapy in induction and consolidation in younger patients with acute myeloid leukemia.Blood,2003 (12); 102:4277-4283.
[24]Karp J, Lancet J, Kaufmann SH, et al. Clinical and biologic activity of the farnesyltransferase inhibitor R115777 in adults with refractory and relapse acute leukemias:a phase 1 clinical laboratory correlative trial. Blood,2001; 97 (6):3361-3369.
[25]Jeffrey E. Lancet, Ivana Gojo, Jason Gotlib, et al.A phase 2 study of the farnesyltransferase inhibitor tipifarnib in poor-risk and elderly patients with previously untreated acute myelogenous leukemia. Blood,2007; 109 (2):1387-1394.
[26]Walter Fiedler, Rolf Mesters, Heike Tinnefeld, et al. A phase 2 clinical study of SU5416 in patients with refractory acute myeloid leukemia. Blood, 2003; 102 (10):2763-2767.
[27]Ellen Weisberg, Christina Boulton, Louise M Kelly, et al. Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. Cancer Cell,2002; 1(5):433-443.
[28]Mark Levis, Jeffrey Allebach, Kam-Fai Tse, et al. A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. Blood, 2002; 99 (5):3885-3891.
[29]Stefan Frohling, Claudia Scholl, Ross L. Levine, et al. Identification of Driver and Passenger Mutations of FLT3 by High-Throughput DNA Sequence Analysis and Functional Assessment of Candidate Alleles. Cancer Cell,2007; 12(6):501-513.
[30]Keith W. Pratz, Takashi Sato, Kathleen M. Murphy, et al. FLT3-mutant allelic burden and clinical status are predictive of response to FLT3 inhibitors in AML. Blood,2010; 115 (2):1425-1432.
[31]Elihu Estey, Hartmut Dohner.Acute myeloid leukemia[J]. Lancet.2006; 368 (11):1894-1907.
[32]Hagop Kantarjian, Varsha Gandhi, Jorge Cortes, et al. Phase 2 clinical and pharmacologic study of clofarabine in patiens with refractory or replapsed acute leukemia. Blood,2003; 102 (10):2379-2386.
[33]杨天楹,张之南,郝玉书主编.临床血液学进展,中国协和医科大学[M].北京：北京医科大学联合出版社,1992,12：168.
[34]秘营昌,卞寿庚.急性髓系白血病完全缓解后治疗周期的初步探讨[J].中华血液学杂志,2001；22(10)：520-523.
[35]Guzman ML, Swiderski CF, Howard DS, et al. Preferential induction of apoptosis for primary human leukemic stem cells[J]. Proc Nail Acad Sci USA, 2002; 99:16220-16225.
[36]Jordan CT, Yamasaki G, Minamoto D. High-resolution cell cycle analysis of defined phenotypic subsets within primitive human hematopoietic cell populations [J]. Exp Hematol,1996; 24:1347-1355.
[37]Dean M, FojoT, Bates S. Tumour stem cells and drug resistance[J]. Nat Rev Cancer,2005; 5:275-284.
[38]古莹,邱曦,徐荣臻.白血病干细胞分子生物学特性及其治疗策略研究进展.基础医学与临床,2008；28(9)：904-907.
[39]叶海燕,陈焯文,肖平,等.流式细胞仪检测急性白血病微小残留病的意义[J].实用医学杂志,2009；25(13)：2075-2077.
[40]Cignetti A, Bryant E, Allione B, et al. CD34+ acute myeloid and lymphoid leukemic blasts can be induced to differentiation in-to dendritic cells[J]. Blood,2001; 94:2084-2055.
[41]童向民,金洁,薛永权,等.急性髓细胞白血病来源的树突状细胞的研究[J].中 华内科杂志,2004；43(1)：45-48.
[42]何学鹏,尤胜国,卞寿庚,等.急性髓系白血病患者树突状细胞诱导抗自身白血病T细胞免疫的实验研究[J].中华血液学杂志,2001；2(12)：629-632.
[43]Harrison BD, Adams JA, Mark B, et al. Stimulation of auto-logous proliferative and cytotoxic T -cell responses by "leukemia dendritic cells" derived from blast cells in acute myeloid leukemia [J]. Blood,2001; 97(9): 2764-2771.
[44]Montagna D, Maccario R, Locatelli F, et al. EX vivopriming for long-term maintenance of antileukemia human cytoxie T cell suggests a general procedure for adoptive immunotherapy[J]. Blood,2001; 98(12):3359 - 3366.
[45]吴明媛,黄勇,朱庭,等.CD34-+HPC来源的树突状细胞体外扩增和功能研究[J].苏州大学学报(医学版),2005；25(1)：4-5.
[36]张蓉,刘莉,张岩实,等.成人外周血来源的树突状细胞的体外诱导[J].实用医学杂志,2005;21(13)：1381-1382.
[47]贺白,邱国强,谢晓宝.冻融抗原负载树突状细胞诱导细胞毒性T淋巴细胞特异性杀伤急性白血病细胞的实验研究[J].生物医学工程学杂志,2004；21(6)：974-975.
[48]赵绍琴.中医中药治疗白血病的新认识[J].中医药研究,1992；2：11-12.
[49]谢天亮.白血病的中医论治[J].基层医学论坛,2008;12(11)：1018-1019.
[50]陆运鑫,罗昌国.试析白血病耐药的中医病机[J].辽宁中医药大学学报,2008；10(5)：10-11.
[51]胡乃平,王展.周霭祥治疗白血病经验[J].中医杂志,2000；41(6)：433-434.
[52]曹方,李彦,贾斌.唐由君治疗急性白血病的经验[J].国医论坛,2001；16(2)：11-12.
[53]李敬花,瞿东升.中医辨证治疗白血病的临床体会[J].中国中医药信息杂志,1999；6(2)：62-62.
[54]肖倩.中药配合化疗治疗急性白血病38例疗效观察[J].中医杂志,1998；39(5)：283-285.
[55]吴登峰,吴登蛟.HA方案加中药治疗急性非淋巴细胞白血病30例[J].黑龙江中医药,1998；(5)：19-20.
[56]李宇涛,甘慧娟,吴同玉等.急性白血病常用中医辨证方法探讨[J].现代中西医结合杂志,2009；18(10)：1107-1108.
[57]朱文锋.辨证统一体系的创立[J].中国中医基础医学杂志,2001；7(4)：4-8.
[58]麻柔,刘锌,薛向军.中医对微小残留向血病的认识和治疗[J].中医杂志,1994;35(2)：748-749.
[59]张姗姗,许勇钢,杨晓红.微小残留白血病患者细胞免疫功能与中医分型关系的研究[J].中国中西结合杂志,2004；24(1)：36-40.
[60]黄礼明.试论急性白血病缓解后之中医论治[J].江苏中医药,2003；24(5)：6-8.
[61]王乙忠,周霭祥,王志平,等.扶正抑癌冲剂配合化疗治疗微小残留白血病的临床与实验研究[J].中医杂志,1996；37(9)：535-538.
[62]陆灿红,李怡,巫蓉.残留白血病的中医治疗思路[J].四川中医,2000；18(1)：10-11.
[63]孙淑贞,麻柔.微小残留白血病中医药研究概况[J].北京中医药,2009；28(5)：387-390.
[64]黄礼明,胡莉文,丘和明.微小残留白血病的病因病机及治疗探讨[J].中医杂志,2005;46(6)：465-466.
[65]刘红,刘朝晖.滋阴补肾清热凉血法治疗微小残留白血病18例观察[J].实用中西医结合临床,2005；5(5)：24-25.
[66]华昭,黄礼明.治疗急性白血病缓解期贵在透邪与存阴并进[J].云南中医中药杂志,2009；30(3)：4-5.
[67]黄海茵,于尔辛.中药对NK、LAK细胞及IL-2活性的影响[J].中国中西医结合杂志.1993；13(4)：253-255.
[68]马武开.中医药防治白血病的思路及方法[J].中国中医药信息杂志.2004；11(3)：229-230.
[69]于志峰,戴锡珍,郝志强,等.扶正解毒方治疗微小残留白血病的疗效及其免疫机制的研究[J].中国中医药信息杂志,2000；7(11)：29-30.
[70]于志峰,戴锡孟,戴锡珍.益气养阴清热解毒法治疗微小残留白血病的凋亡机制研究[J].天津中医,2001；18(4)：29-30.
[71]于志峰,戴锡珍,戴锡孟,等.六神丸治疗微小残留白血病凋亡机制的实验研究[J].中医研究,2000；13(5)：18-20.
[72]王欣,褚建新,傅汝林,等.清热解毒方对可移植性小鼠微小残留白血病实验研究[J].实用中西医结合临床,2004；4(5)：1-3.
[73]王欣,褚建新,傅汝林,等.清热解毒方对可移植性小鼠淋巴细胞白血病及其微小残留病疗效的比较研究[J].中医药临床杂志,2005；17(2)：148-149.
[74]王东生.中药及其有效成分抗白血病的分子机制[J].国际中医中药杂志,2007；29(3)：160-163.
[75]潘世扬,黄佩王君,唐云章等.小儿急性淋巴细胞性白血病微小残留病免疫治疗的初步研究[J].南京医科大学学报,2001；21(6)；492-495.
[76]乌庆超,于书增,褚建新,等.骨髓根除性治疗恶性血液病临床研究[J].临床 血液学杂志,2000；12(4)：171.
[77]邓成珊,等.当代中西医结合血液病学,北京：中国医药科技出版社,1997：9.
[78]王斌,胡岳山,李杰芬.灵芝三萜对小鼠脾脏树突状细胞增殖的影响[J].中药材,2005；7(28)：577-578.
[79]李松林.清毒饮、养正片治疗急性白血病30例疗效观察[J].陕西中医函授,2000;1：13-14.
[80]麻柔,刘锋,杨经敏,等.扶正抗白冲剂对急性白血病患者长期存活影响的临床研究[J].中国中西医结合杂志,1998；18(5)：276-278.
[81]李振波,丘和明,张萍.L7212白血病小鼠脾细胞中肿瘤坏死因子α活性及mRNA表达和复方中药清毒饮对其影响[J].中国实验血液学杂志,1999；7(3):234-237.
[82]袁健,熊文,陈建波,等.染色体分析及FISH法对急性早幼粒细胞白血病诊断的临床意义[J].实用癌症杂志,2007；22(3)：266-268.
[83]李倩,杜兵,杨军军,等.应用序列探针检测慢性淋巴细胞白血病基因异常[J].中国卫生检验杂志,2007；17(4)：672-674.
[84]陈蕾蕾,孙雪梅,陈军浩,等.FISH在免疫表型特殊的急性早幼粒细胞白血病诊断中的应用[J].标记免疫分析与临床,2005；12(4)：193-195,T0001.
[85]刘启发,宋兰林,张钰,等.双色间期荧光原位杂交检测异基因造血干细胞移植后性染色体嵌合状态的临床意义[J].中华医学遗传学杂志,2005；22(2)：212-215.
[86]王慧萍,李国霞,乔振华,等.联合应用细胞遗传学、巢式RT-PCR和FISH技术检测慢性髓系白血病治疗过程中的肿瘤负荷[J].中国实验血液学杂志,2007；15(2)：237-241.
[87]李栋.常用微小残留白血病的检测方法[J].泰山卫生,2001；25(1)：17-20.
[88]丁肖华.白血病的检测方法[J].四川解剖学杂志,2006；14(3)：27-29.
[89]费新红,许小平.微小残留白血病的检测方法及临床应用进展[J].白血病·淋巴瘤,2003；12(5)：311-314.
[90]张秋堂,冯磊,李涛,等.三色荧光标记抗体在急性白血病免疫分型中的应用[J].临床血液学杂志,2004；17(5)：252-254.
[91]Bruce D. Cheson, John M. Bennett, Kenneth J., et al. Revised recommendations of the International Working Group for diagnosis, standardization of response criteria, treatment outcomes, and Reporting Standards for Therapeutic Trials in acute myeloid leukemia[J]. Clinical oncology,2003; 21 (12):4642 - 4649.
[92]Guido Marcucci, Krzysztof Mrozek, Amy S. Ruppert, et al. Abnormal cytogenetics at date of morphologic complete remission predicts short overall and disease-free survival, and higher relapse rate in adult acute myeloid leukemia:results from Cancer and Leukemia Group B Study 8461[J]. Clinical Oncology,2004; 22 (6):2410-2418.
[93]陈志炉,史亦谦,周郁鸿.中药诱导白血病细胞凋亡的研究近况[J].实用中西医结合临床,2007；7(3)：91-93.
[94]高娜,茆俊卿,张育.中药逆转白血病多药耐药的研究进展[J].江苏中医药,2007；39(3)：60-62.
[95]杜振兰,王瑞,陈虎.中医药治疗白血病的现状[J].第一军医大学分校学报,2005；28(1)：97-100.
[96]杨振江,曹克俭,郭岳峰,等.瘀毒清对微小残留白血病作用的实验研究[J].浙江中西医结合杂志,2001；11(10)：613-614,625.
[97]于志峰,戴锡孟,戴锡珍.益气养阴活血化瘀方法治疗微小残留白血病免疫机制的实验研究[J].天津中医学院学报,2001；20(1)：22-24.
[98]周岱翰.中药抗肿瘤研究的评析与前景[J].新中医,2008；40(11)：1-3.
[99]杨洪涌,潘习龙,丘和明,等.清毒饮和养正片诱导L712小鼠自血病细胞凋亡及其对Bcl-2、P53基因表达的影响[J].新中医；2002；34(6)：69-71.
[100]梁毅,陈志雄,丘和明.清毒饮、养正片影响白血病K562细胞增殖周期、细胞凋亡的实验研究[J].中国中医基础医学杂志,2004；10(1)：44-47.
[101]后盾,丘和明,聂梦仑.清毒饮、养正片联合化疗治疗急性髓细胞白血病32例及对血清乳酸脱氢酶的影响[J].中国中医药杂志,2005；12(5)：313-314.
[102]李振波,丘和明.不同浓度清毒饮和养正片对L7212白血病小鼠脾脏B细胞功能的影响[J].新中医,1999；31(8)：31-32.
[103]Geissler K pathophysiology and treatment of apastic anemia[J]. wiener kliniche wochen sehrift,2003; 115(13-14):444-450.
[104]Gabrii Ovich D. Mechanisms and functional significance of tumour induced dendritic-cell defects[J]. Nat Rev Immunol,2004; 4(12):941-951.
[105]Zou W. Immunosuppregsive networks in the tumour environment and their therapeutic relevance [J]. Nat Rev Cancer,2005,5(4):263-274.
[106]李启亮,单保恩,张超,等.不同刺激剂诱导肺癌患者引流淋巴结细胞分泌细胞因子的研究[J].细胞与分子免疫学杂志,2006；22(5)：609-612.
[107]Sabel Ms, Arora A, su G, et al. Adoptive immunotherapy Of breast cancer with lymph node cells primed by cryoablation of the primary tumor[J]. Cryobiology,2006; 53(3):360-366.
[108]翁开枝,谢晓宝,邱国强,等.临床注射用α-干扰素和粒-巨集落刺激因子诱导慢性髓性白血病细胞向树突状细胞分化[J].肿瘤,2007；27(1)：55-58.
[109]Jain P, Ahuja J, Khan ZK, et al. Modulation of dendritic cell maturation and function by the Tax protein of human T cell leukemia virus type 1[J]. J Leukoe Biol,2007；82(1):44-56.
[110]Zhou LJ, Tedder TF. CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells[J]. Proc Natl Acad Sci USA,1996； 93:2588.
[111]张群豪,钟蓓,陈可冀,等.用血清药理方法观察血府逐瘀浓缩丸对实验性动脉粥样硬化家兔主动脉平滑肌细胞增殖的影响[J].中国中西医结合杂志,1996；16(3)：156-159.
[112]谭余庆,霍海如.中药血清药理学研究方法及其应用[J].中国实验方剂学杂志,1997;3(6)：41-43.
[113]王力倩,李仪奎,符胜光,等.血清药理学方法研究探索[J].中国中医药科技,1997;13(3)：29-31.