【作者】 程薇；

【导师】 马保根；

【作者基本信息】 郑州大学 ， 内科学， 2009， 硕士

【摘要】 背景与目的急性白血病是最常见的血液系统恶性肿瘤,是一组异源性起源的恶性克隆性疾病,年发病率3～4/10万。其中急性髓系白血病约占70%,是成人白血病的主要类型。化疗是目前治疗白血病的主要手段。随着化疗方案的不断改进和新的化疗药物的出现,白血病的预后已有非常明显的改善,但仍然存在白血病耐药及停药后白血病复发等问题,严重影响了病人的生活质量及长期生存。因此,寻找有效的抗肿瘤新药、探索相关治疗新方案具有重要意义。近年来,某些中药及其有效组分因具有诱导肿瘤细胞凋亡的作用备受关注。雷公藤（TWHF）泛指卫矛科雷公藤属植物,是我国传统中药材之一,具有抗炎、免疫抑制、抑菌和抗肿瘤等多种药理作用。雷公藤红素（celastrol）是从雷公藤的根部分离到的三萜类色素,分子式为C₂₉H₃₈O₄,分子量为450。有文献报道,红素不仅具有免疫抑制和抗炎作用,还对多种肿瘤细胞具有显著的体外抑制作用。目前有关雷公藤红素对白血病的研究尚处于基础研究阶段,可见到雷公藤红素用于部分白血病细胞株的报道,而该药用于急性髓系白血病原代细胞的研究国内外罕见报道,有待进一步探索。本研究将雷公藤红素作用于急性髓系白血病原代细胞,观察其在体外对急性髓系白血病原代细胞增殖及凋亡的影响,以探讨应用雷公藤红素治疗白血病的可能性,为临床应用雷公藤红素提供理论依据。方法1.人急性髓系白血病细胞分离与培养采用密度梯度法分离初诊急性髓系白血病患者骨髓单个核细胞,以1×10⁶/ml接种在培养瓶中,37℃、5%CO₂条件下RPMI-1640中悬浮培养,2～3天换液一次。取处于对数生长期,细胞活性大于95%的细胞进行实验。2.台盼蓝染色活细胞计数法采用台盼蓝染色进行活细胞计数。取40μl雷公藤红素处理过的急性髓系白血病原代细胞悬液用台盼蓝染色后,然后镜检100个细胞,其中拒染细胞为活细胞,染成蓝色细胞为死细胞,计算出活细胞百分率。3.甲基噻唑基四唑比色法（MTT法）取处于指数生长期的未处理急性髓系白血病原代细胞按1×10⁶/mL接种于96孔培养板,180μl/孔。实验孔红素终浓度分别为1μmol/L、2μmol/L、5μmol/L和10μmol/L,同时设置不加药物的对照孔和不加药物与细胞的调零孔,均设4个复孔。将培养板放至37℃、5%CO2温箱分别培养3h,6h,12h,24h后,每孔加入MTT 20μl,37℃继续培养4h,离心,弃上清,每孔加入150μl二甲基亚砜（DMSO）,低速震荡,使甲瓒晶体完全溶解,置酶标仪在490nm波长处测定每孔的光密度值（OD）。计算细胞增殖抑制率,并以药物浓度为横坐标,抑制率为纵坐标绘制细胞生长抑制曲线,找出雷公藤红素作用于急性髓系白血病原代细胞的最适时间和最适浓度。4.磷脂酸丝氨酸外翻技术（Annexin V-FITC/PI法）采用Annexin V-FITC/PI流式细胞术法。取终浓度为1μmol/L、2μmol/L、5μmol/L、10μmol/L的雷公藤红素作用于急性髓系白血病原代细胞6h及2μmol/L雷公藤红素作用3h、6h、12h、24h后的急性髓系白血病细胞悬液100μl,分别加入10μl Annexin V-FITC,室温避光10min后加入碘化丙啶染色液5μl,避光反应5min,加入400μl 1×Annexin V-FITC结合液,混匀,立即上流式细胞仪定量检测（一般不超过1h）,试验重复3次。同时以不加Annexin V-FITC及PI的一管作为阴性对照。5.细胞凋亡峰定量分析（PI单染法）采用PI单染法定量分析细胞凋亡峰。取终浓度为1μmol/L、2μmol/L、5μmol/L、10μmol/L的雷公藤红素作用于急性髓系白血病原代细胞6h的细胞悬液,用冰预冷的70%乙醇4℃固定24h,取固定后的细胞用PBS洗涤,加入RNAase 37℃消化30min,再加入PI染液,混匀后4℃避光染色15min。流式细胞仪进行细胞周期分析,低于G1期DNA含量细胞为凋亡细胞,实验重复3次。6.数据统计实验数据采用SPSS 13.0统计软件分析,所有统计结果以均数±标准（（?）±s）表示,多样本均数的比较采用单因素方差分析,α=0.05为检验水准。结果1.不同药物浓度及作用时间与细胞增殖抑制率变化的关系采用MTT比色法检测结果显示:不同浓度雷公藤红素组两两比较,2μmol/L、5μmol/L和10μmol/L的雷公藤红素对急性髓系白血病原代细胞增殖的抑制作用相比,差异无统计学意义（P＞0.05）,与1μmol/L组及对照组相比较,均具有显著性差异（P＜0.01）,最适作用浓度为2μmol/L。不同时间雷公藤红素组两两比较发现,6h、12h和24h时雷公藤红素对急性髓系白血病原代细胞的增殖抑制率相比较,差异无统计学意义（P＞0.05）,与3h时相比较,均具有显著性差异（P＜0.01）,最适作用时间为6h。总的来说,雷公藤红素可抑制急性髓系白血病原代细胞的生长,其作用的最适浓度为2μmol/L,最适时间为6h。2.不同药物浓度及作用时间与细胞凋亡率变化的关系流式细胞术检测结果显示1μmol/L、2μmol/L、5μmol/L、10μmol/L雷公藤红素作用6h,急性髓系白血病原代细胞均可出现凋亡现象,凋亡率分别为（34.03±17.07）%、（49.53±19.84）%、（50.56±15.80）%、（28.28±21.21）%,明显高于不加药物的对照组（4.95±1.85）%（P＜0.01）;2μmol/L、5μmol/L浓度组与其他组比较,差异具有统计学意义（P＜0.05）,二者之间比较,凋亡率无明显差异（P＞0.05）。雷公藤红素10μmol/L浓度组的细胞凋亡率较5μmol/L组明显降低（P＜0.05）。红素浓度为2μmol/L时,作用3h、6h、12h、24h后细胞凋亡率分别为（19.67±0.16）%、（37.44±1.01）%、（40.10±1.42）%、（46.53±0.37）%,各组之间相比较有统计学意义（P＜0.05）。3.流式细胞仪对细胞凋亡峰的定量分析用流式细胞仪PI单染法测定结果显示:1μmol/L、2μmol/L、5μmol/L、10μmol/L雷公藤红素作用于急性髓系白血病原代细胞6h后,均可出现G1亚峰（Apoptosis峰）,凋亡峰定量分析结果分别（19.34±5.28）%、（39.84±9.99）%、（46.40±2.67）%、（32.32±8.42）%,均明显高于不加药物的对照组（P＜0.01）;2μmol/L组和5μmol/L、10μmol/L组比较差异均无统计学意义（P＞0.05）,与1μmol/L组相比较,差异有统计学意义（P＜0.05）;雷公藤红素10μmol/L浓度组细胞凋亡率较5μmol/L组明显降低（P＜0.05）。结论1.雷公藤红素在（1～10μmol/L）浓度范围内可抑制急性髓系白血病原代细胞的增殖,最适作用浓度为2μmol/L,最适作用时间为6h。2.雷公藤红素在（1～10μmol/L）浓度范围内,可诱导急性髓系白血病原代细胞凋亡,提示诱导细胞凋亡可能是雷公藤红素抗白血病作用的重要机制。更多还原

【Abstract】 Background and objectiveAcute leukemia is one of the most common malignant tumors in human beings. The incidence of leukemia is reported to be 3～4/100,000 per year.Approximately 70%is AML,and it occurs mostly to adults.The main treatment method for AML is dependent on chemotherapy.With the development of chemotherapy plans and the appearance of new medicines,the prognosis of patients suffering from AML has being greatly improved.However,the multi-drug resistance and recurrence of leukemia remain to be serious and popular problem.So it is great importance to look for new anti-tumor drugs and establish the associated curative strategy.In the recent years,some traditional Chinese medicines have been confirmed to possess anti-tumor effects and this causes great attentions.Tripterygium,one of traditional Chinese medicines,is belonging to Wilfordil hook,and it possesses many pharmacological actions,such as anti-inflammation、immunological suppression、bacteriostasis and anti-tumor.Celastrol is one of the monomers extracted from the root of tripterygium.Chemically,Celastrol belongs to triterpene constituents with molecular weight 450 and its molecular formula is C₂₉H₃₈O₄.It is reported that Celastrol either inhibit immune or inflammatory responses vitro,it is found that celastrol show a function of growth inhibiting in many kinds of tumor cells.In overseas and domestic,the research of celastrol in treatment of leukemia is still in the stage of fundamental researches.And there are some reports about celastrol acting on some leukemia cell lines,but the researches about cells from AML patients in vitro are rare at home and abroad,So it remains to be explored further.In this study,we used cells from acute myelogenous leukemia patients as the target cells to determine the effect of Celastrol on growth depression and apoptosis in vitro,we attempted to explore the possibility that celastrol in treatment leukemia preliminary,and to provide the theory basis for celastrol in clinical application.Materials and methods1.Separate and culture of AML primary cellsBone marrow mononuclear cells were separated from bone marrow of untreated AML patients by Ficoll.The AML cells of 1×10⁶/ml were inoculated into suspension culture flask and then cultured at 37℃for 2～3 generation under the atmosphere of 5%CO₂.To experiment used the cells that cytoactive above 95% in exponential phase.2.Trypan bluedye staining methodLiving cell counting method based on Trypan bluedye staining was used. Brietly,40μl of each the cell suspensions was stained,and then detected under microscope to count 100 cells for calculating living cell percentages.Trypan bluedye stained cells were dead and non-stained cells means living.3.MTT assayThe AML primary cells grew at exponential phase of growth,whose viability were more than 95%were used in the MTT assay.The AML cells（1×10⁶/mL） were incubated in a 96-well plate and 180μl per well.There were 4 experiment groups and the final concentration were 1μmol/L,2μmol/L,5μmol/L,10μmol/L, respectively.Meanwhile the control groups without celastrol and the blank groups were set.experiments were repeated for four times.Add 20μl MTT was added to each wells after the cells were cultured for 3h,6h,12h and 24 h at the atmosphere of 37℃、5%CO2.centrifugated the cell suspensions,Discarded the supernatant.And then added 150μl DMSO to each well.Shaked lightly to solution the formazan.Optical density（OD） was detected by an ELISA at 490nm wavelength. Then the growth inhibitory rates were counted,and the optimal concentration and time was set from the curve which the abscissa was the concentrations and the ordinate was proliferation inhibited rates.4.Annexin V-FITC/PI法Flow cytometry using Annexin V-FITC/PI was performed to detect tumor cells apoptosis.Briefly,10μl Annexin V-FITC was added into 100μl AML primary cell suspensions which treated with 1μmol/L、2μmol/L、5μmol/L、10μmol/L Celastrol or treated with 2μmol/L celastrol for 3h、6h、12h、24h,and then incubated at 37℃for 10 min.The cells were washed with PBS and then resuspended.5μl of PI solution was added to stop the reaction,and flow cytometry was applied to detect the apoptotic cells as soon as possible,experiments were repeated for three times. Meanwhile the negative controls that without Annexin V-FITC and PI were set.5.PI单染法Flow cytometry using PI staining was performed to analyze quantitatively the Gl sub-peak（apoptotic peak） of AML primary cells.Treated the AML primary cells with 1μmol/L、2μmol/L、5μmol/L、10μmol/L celastrol for 6h,And then fixed the treated cells with 70%ethanl for about 24h.Washed by PBS for twice.Added RNAase at 37℃for 30min,and then added PI,mixed and stained for 15min at 4℃. Flow cytometry was performed to analyze quantitatively the apoptotic peak of AML primary cells.6.Statistical analysisAll the data were showed in the form of mean±standard deviation,The multi-samples mean values were compared with variance analysis by SPSS13.0 software.Takingα=0.05 as the significant standard of test.Results1.The proliferation inhibited rates of AML primary cells treated by Celastrol with different concentrations and different durations.After the AML primary cells were treated with different concentrations of celastrol,the results of MTT were analyzed,cells growth were significantly inhibited compared with blank control group（P＜0.01）.There was no statistical difference among 2μmol/L、5μmol/L and 10μmol/L celastrol（P＞0.05） except at the concentration of 1μmol/L（P＜0.01）.The optimal concentration was 2μmol/L of celastrol.After AML primary cells were treated with celastrol for 3h,6h,12h, 24h,the result showed that the difference among 6h,12h,24h groups had no statistical significance（P＞0.05）.But compared with 3h group,the difference had statistical significance（P＜0.01）,The optimal time was 6h.Conclusively,celastrol was capable of inhibiting the AML primary cells proliferation with optimal concentration at 2μmol/L and optimal time at 6h.2.correlation among the concentration and duration of Celastrol and the changes of apoptotic ratesThe detection results by flow cytometry indicated that the apoptosis was seen after treated with 1μmol/L、2μmol/L、5μmol/L、10μmol/L Celastrol for 6h, apoptotic rates were significantly higher with apoptotic rates at（34.03±17.07）%、（49.53±19.84）%、（50.56±15.80）%、（28.28±21.21）%,respectively.Compared with the control group at（4.95±1.85）%,the difference had statistical significance （P＜0.01）.there was no statistically significant difference between 2μmol/L and 5μmol/L groups（P＞0.05）.while the two groups were significantly higher than others（P＜0.05）.The apoptotic rate decreased significantly at the concentration of 10μmol/L than the at the concentration of 5μmol/L（P＜0.05）.After the cells were treated with 2μmol/L Celastrol for 3h、6h、12h、24h,the apoptotic rates were （19.67±0.16）%、（37.44±1.01）%、（40.10±1.42）%、（46.53±0.37）%,respectively, and there was statistical difference among apoptotic rates in different groups.3.The quantitative analysis of apoptotic peak by flow cytometryFlow cytometry was used to analyze quantitatively the G1 sub-peak（apoptotic peak） of treated-cells.The apoptotic peak was seen after treated with 1μmol/L、2μmol/L、5μmol/L、10μmol/L Celastrol for 6h,respectively.The apoptotic rates were（19.34±5.28）%、（39.84±9.99）%、（46.40±2.67）%、（32.32±8.42）%, respectively,and the difference was significant compared with the control（P＜0.01）. The apoptotic rates had no significant difference between the 2μmol/L celastrol group and 5μmol/L、10μmol/L groups（P＞0.05）,but the difference had statistical significance compared with 1μmol/L celastrol group（P＜0.05）.And the apoptotic rate was lower obviously in 10μmol/L group than in 5μmol/L group（P＜0.05）.Conclusions1.The proliferation of AML primary cells can be inhibited at the concentration of （1～10μmol/L） Celastrol,And the optimal concentration is 2μmol/L celastrol and the optimal time is 6h.2.When the concentrations of Celastrol is within 1～10μmol/L,apoptosis of the AML primary cells can be induced by celastrol.Prompting that inducing apoptosis may be revolved in the anti-leukemia mechanism of Celastrol.更多还原