摘要研究背景我国白血病近十年发病以2%的速度递增，是一种起源于造血前体细胞的恶性肿瘤，目前主要的治疗方法为造血干细胞移植和化疗。化疗是目前治疗白血病的主要手段，然而化疗的副作用及多药耐药的产生，给临床治疗白血病预期的完全缓解和巩固治疗带来了诸多困难。中药作为我国传统治疗白血病的手段，具有个体化、辩证施治的明显优势，实践证明中医中药在治疗白血病中显示出重要作用。近年来随着中药及其有效成分抗白血病的临床及实验研究日益广泛，部分研究发现温阳中药中的有效成分可以改变白血病细胞的生物学活性，诱导白血病细胞成熟分化和调亡。半夏是传统中草药材，内含多种有效活性成分，半夏提取物和生物总碱抑制白血病细胞增殖有相关的研究，但其具体作用机制缺乏研究。目的探索半夏提取物有效成份对阿霉素耐药的慢性髓系细胞白血病细胞K562增殖的抑制作用和诱导细胞调亡的作用，探寻其作用的分子水平机制：探讨半夏提取物在耐药的白血病治疗中可能的应用价值。研究方法用氯仿低温超声方法提取半夏有效成分，分别干预经阿霉素处理的K562-ADR细胞株设置0.1mgml、0.3mgml、0.5mgml、0.7mgml、1mgml、2mgml、3mgml7种浓度，尔后通过IC50计算得半数抑制浓度，再优化浓度范围。观察半夏提取物干预慢性髓系白血病耐药细胞K562-ADR形态学改变：CCK-8法检测半夏提取物对K562-ADR细胞增殖的抑制作用：实时荧光定量PCR检测半夏提取物对K562-ADR细胞检测BaX、BC-2、MDM2、BCR-ABL、PML基因表达量。研究结果1.CCK-8法检测结果显示：半夏提取物处理对K562、k562-ADR白血病细胞的增殖均有较强的抑制作用。2.由Graphpad Prism分别计算得半夏提取物对K562组IC50为1.83mg/ml,K562-ADR组1C50为2.36mgml。3.实时荧光定量PCR显示：①1、3、5mg/ml半夏提取物可能不调控Bx/Bcl-2途径使K562-ADR细胞调亡，5mg/ml在48h、72h干预时Bc-2有最大值(P<0.01)Bax表达量下调，提示该浓度下Bax/Bcl2途径可能不参与K562-ADR细胞调亡：②1、3、5mg/ml半夏提取物干预48h后影响K562、K562-ADR细胞MDM2、BCR-ABL、PML转录表达水平，结果显示1、3mg/ml半夏提取物可能激活K562-ADR细胞MDM2、BCR-ABL、PML表达。③0.5、0.7mg/ml半夏提取物干预48、72h后影响K562-ADR细胞转录Bax、Bcl2表达水平，BCL-2表达水平下调(P<0.05或P<0.01)。结论1.半夏提取物能抑制阿霉素耐药的慢性髓系白血病细胞增殖，在低浓度范围下通过Bx、BCL-2途径促进细胞调亡，且在4872小时的抑制生长曲线、调亡蛋白表达最为稳定。2.高浓度范围的半夏提取物对阿霉素耐药的慢性髓系白血病细胞，不通过Bx、BCL-2,的作用机制促进其调亡，通过对MDM2及BCR-ABL表达水平检测，推测可能通过P53非依赖性调亡通路促使细胞调亡。3.耐药的慢性髓系白血病细胞可能通过PML介导调控，但半夏提取物不能干预PML的表达水平。关键词：阿霉素耐药白血病：半夏提取物：Bax/BCl-2通路：MD2:BCR-ABL:细AbstractResearch backgroundThe incidence of leukemia in China has increased at a rate of 2%in the past decade.It is amalignant tumor originating from hematopoietic precursor cells.The main treatment methods atpresent are hematopoietic stem cell transplantation and chemotherapy.Chemotherapy is themain method to treat leukemia at present.However,the side effects of chemotherapy and theemergence of multidrug resistance have brought many difficulties to the complete remission andconsolidation of clinical treatment of leukemia.Traditional Chinese medicine,as a traditionaltreatment of leukemia in China,has obvious advantages of individualization and dialecticaltreatment.Practice has proved that traditional Chinese medicine plays an important role in thetreatment of leukemia.In recent years,with the clinical and experimental research of traditionalChinese medicine and its active ingredients against leukemia becoming more and moreextensive,some studies have found that the active ingredients in Wenyang traditional Chinesemedicine can change the biological activity of leukemia cells,induce the mature differentiationand apoptosis of leukemia cells.Pinellia temata is a traditional Chinese herbal medicine,whichcontains a variety of active ingredients.Pinellia temata extract and total alkaloids have relatedresearch on inhibiting the proliferation of leukemia cells,but its specific mechanism is lack ofresearch.ObjectiveTo explore the inhibitory effect of Pinellia extract on the proliferation and apoptosis ofadriamycin resistant chronic myeloid leukemia cells,explore the mechanism of its molecularlevel effect,and explore the possible application value of Pinellia extract in leukemia treatment.Research methodThe effective components of Pinellia temate were extracted by chloroform low-temperatureultrasound.The concentrations of 0.1 mg /ml,0.3 mg/ml,0.5 mg ml,0.7 mg ml,1 mg /ml,2 mg ml and 3 mg /ml in k562-adr cell line treated with adriamycin were intervenedrespectively.Then half of the inhibitory concentration was calculated by IC50,and theconcentration range was optimized.To observe the effect of Pinellia ternate extract on themorphology of k562-adr cells;CCK-8 method to detect the inhibitory effect of Pinellia temateextract on the proliferation of k562-adr cells;real-time fluorescence quantitative PCR to detectthe expression of Bax,Bcl-2,MDM2,ber-abl and PML genes in k562-adr cells.Research findings1.CCK-8 method showed that Pinellia ternate extract had a strong inhibitory effect on theproliferation of K562 and k562-adr leukemia cells.2.The IC50 of Pinellia ternata extract to K562 group was 1.83mg/ml,and that of K562ADR group was 2.36mg/ml.3.The drug-resistant chronic myeloid leukemia cells may be regulated by PML,but Pinelliaternata extract can not interfere with the expression of PML.3.Real time fluorescence quantitative PCR showed that:1 1,3,5mg /ml Pinellia temataextract may not regulate Bax Bcl-2 pathway to apoptosis of k562-adr cells,5mg /ml at 48h,72h intervention Bcl-2 had a maximum (P<0.01)Bax expression down-regulated,suggestingthat Bax Bcl-2 pathway might not participate in the apoptosis of k562-adr cells at thisconcentration;2 Pinellia ternata L.3 The expression of Bax and Bcl-2 in k562-adr cellswas influenced by 0.5 and 0.7mg/ml Pinellia ternata extract 48 and 72 hours later,and bcl-2expression was down regulated (P<0.05 or P<0.01).Conclusion1.Pinellia temate extract can inhibit the proliferation of doxorubicin resistant chronicmyeloid leukemia cells,promote cell apoptosis through Bax,bcl-2 pathway in the lowconcentration range,and the inhibition growth curve and the expression of apoptotic protein arethe most stable in 48-72 hours.2.The high concentration of Pinellia temate extract can promote the apoptosis of adriamycinresistant chronic myeloid leukemia cells without the mechanism of Bax and bcl-2.By detectingthe expression level of MDM2 and ber-abl,it is speculated that it may promote the apoptosis ofcells through p53 independent apoptosis pathway.Keywords:adriamycin resistant leukemia;Pinellia ternate extract;Bax Bcl-2 pathway;MDM2;bcr-abl;apoptosis