【作者】 胡婕；

【导师】 马保根；

【作者基本信息】 郑州大学 ， 内科学， 2011， 硕士

【摘要】 白血病是造血系统常见的恶性肿瘤,是一组异质性起源的恶性克隆性疾病,年发病率3-4/10万。目前治疗白血病的方法主要是化疗,随着化疗方案的改进和新化疗药的出现、支持治疗的加强及干细胞移植技术的应用,白血病的治疗效果有了明显的改善。然而,多药耐药的产生则成为目前部分白血病患者不能获得缓解,或缓解后复发的主要原因。因此,寻找新的抗肿瘤药物、探索新的治疗方案具有重要的意义。近年来,中药及其有效的成分逆转白血病多药耐药引起了人们的注意。雷公藤泛指卫矛科雷公藤属植物,是我国传统的中药材。雷公藤红素是雷公藤单体之一,是一种三萜类天然化合物,又名南蛇藤素,分子式为C29H38O4。,分子量为450。文献报道雷公藤红素有免疫抑制、抑制血管内皮细胞体外增值、抑制HMC-1细胞的黏附及其黏附分子的表达；雷公藤红素还能体外诱导非小细胞肺癌细胞株H1299、人急性髓系白血病细胞株HL-60、恶性胶质细胞瘤株、视网膜母细胞瘤等肿瘤的凋亡。本研究以人慢性粒细胞白血病急变K562细胞株和耐阿霉素K562/A02细胞株为靶细胞,采用CCK-8方法和流式细胞术,探讨雷公藤红素对K562/A02细胞的逆转作用,并检测了雷公藤红素对K562/A02细胞内ADM浓度和P-gp的表达的影响。方法：1、K562和K562/A02细胞的培养K562细胞培养于含10%新生牛血清、100U/ml青霉素和1000ug/ml链霉素的RPMI-1640培养基中,K562/A02细胞培养于含阿霉素(ADM 1μmol/L)的上述培养基中,将2种细胞置于37℃、5%CO2培养箱中常规培养,2-3天传代1次,其中K562/A02细胞在实验前两周改用无阿霉素的培养基培养。取对数生长期细胞用于实验。2、CCK-8法检测ADM、雷公藤红素的细胞毒性及耐药细胞的耐药倍数取对数生长期细胞,采用台盼兰染色活细胞计数方法。取30 u 1细胞悬液,用台盼兰染色后镜检100个细胞并计算活细胞百分率,其中拒染细胞为活细胞,染成蓝色的细胞为死细胞。取存活率大于95%的细胞为实验细胞。将K562和K562/A02细胞(2～3×109cells/孔)于96孔板中,由高到低浓度分别加入ADM(160、80、40、20、10、5、2.5、1.25μmol/L),雷公藤红素(320、160、80、40、20、10、5、2.5μmol/L),每孔设3个复孔,终体积150 u l。在37℃、5%CO2培养箱中培养72小时后,每孔加入CCK-8试剂10μ1,继续培养2小时,以空白孔调零,在全自动酶标仪450nm处测吸光度值,参比波长650nm。所测数据采用SPSS16.0统计软件计算生长抑制率在50%的药物浓度即半数抑制率(IC50)。抑制率(%)=(1-实验组0D值/对照组OD值)×100%；耐药倍数=(耐药细胞的IC50/敏感细胞的IC50)。实验重复3次,取平均值。3、CCK-8法检测雷公藤红素耐药逆转性取对数生长期、存活率大于95%的K562/A02细胞(2～3×104cells/孔)于96孔板中。实验分组：①K562/A02、②K562/A02+雷公藤红素、③K562/A02+VLP。所加ADM等药物浓度同上,每孔设3个平行孔,终体积150μl。并用维拉帕米(VLP)组5μg/L作为阳性对照。在37℃,5%CO2培养箱中孵育72小时后加入10μl CCK-8,继续培养2小时后,以空白孔调零,检测波长同上。计算半数抑制率(IC50)。实验重复3次。逆转倍数=(耐药细胞逆转前的IC50/耐药细胞逆转后的IC50)。4、流式细胞术检测K562和K562/A02细胞内的ADM浓度取对数生长期、存活率大于95%的细胞于24孔板中,实验分6组①K562/A02、②K562/A02+ADM、③K562/A02+ADM+雷公藤红素(IC50)、④K562/A02+ADM+雷公藤红素(IC50)、⑤K562、⑥K562+ADM。第③、④组分别先加入IC10和IC50的雷公藤红素,24h后在第②③④⑥组中再加入ADM10μg/ml,药物作用3小时,收集细胞1×106个细胞,冷PBS(4℃,0.01mol/L,Ph7.4)洗涤2次,再重悬于冷PBS液中,4℃保存至上样进行流式细胞仪检测(激发波长Ex=488nm,发射波长Em=575nm)。5、流式细胞术检测P-gp表达取对数生长期、存活率大于95%的细胞于24孔板中,实验分4组,分别为①K562/A02、②K562/A02+ADM(IC50)、③K562/A02+雷公藤红素(IC50)、④K562/A02+ADM(IC50)+雷公藤红素(IC50),置培养箱孵育48h后,收集细胞,约3×105个细胞,用冷的PBS液洗2遍,按操作说明加入P-gp抗体,室温下避光孵育30分钟后上流式细胞仪检测,并给出相应的P-gp表达率。结果：1、雷公藤红素对K562、K562/A02细胞的增值抑制作用结果雷公藤红素对K562和K562/A02细胞的IC50分别为411.59±26.551μmol/L和295.58±23.288μmol/L,两者差异明显(P<0.05)。ADM对K562、K562/A02的IC50分别为0.749±0.741μmol/L、59.755±6.883μmol/L,耐药倍数为79.78倍,说明K562/A02对ADM有明显的耐药性(P<0.05)。细胞毒剂量的雷公藤红素可降低ADM对K562/A02细胞的IC50,提高对ADM的敏感性,应用雷公藤红素后,IC50为0.507±0.070μmol/L,较单用ADM明显减低(P<0.05),耐药性逆转倍数(RF)为117.860倍；而加入5μg/L维拉帕米后的IC50为17.196±6.303μmol/L,也明显低于单用ADM组(P<0.05),RF为3.745倍。2、流式细胞仪检测细胞内阿霉素浓度和细胞膜P-gp的表达结果经流式细胞仪检测发现加入雷公藤红素后,细胞内的ADM的浓度明显增加,加入IC,。浓度的雷公藤红素和IC50浓度的雷公藤红素后细胞内的阿霉素浓度分别为73.727±8.626、102.86±4.518；增加倍数分别为1.102和1.537倍。且两组细胞内的阿霉素浓度有明显差异(P<0.05)。单用阿霉素时,K562/A02细胞的P-gp的表达波峰无明显影响；单用雷公藤红素后,K652/A02细胞的P-gp的表达波峰明显降低；当雷公藤红素和阿霉素合用后,K562/A02细胞的P-gp的表达也比单用阿霉素有明显的降低。结论：1、阿霉素和雷公藤红素对K562/A02、K562的半数抑制率浓度(IC50)均有显著差异。K562/A02细胞对ADM的耐药性是K562细胞的79.78倍。2、细胞毒剂量的雷公藤红素作用后,ADM对K562/A02细胞的IC50显著下降。3、细胞毒剂量和非细胞毒剂量的雷公藤红素处理后的K562/A02细胞和K562细胞内的ADM浓度显著增加。4、雷公藤红素能明显下调K562/A02细胞的P-gp表达。更多还原

【Abstract】 Leukemia is one of the most common malignant tumor in beings,which is a heterogeneous group of clonal origins of malignant disease. The incidence of leukemia is reported to be 3-4/100,000 per year. The main treatment method for leukemia is dependent on chemotherapy. With the development of chemotherapy plans and the apperence of new medicines, supportive care and stem cell transplantation to enhance the application of the treatment of leukemia has been significantly improved. However, the emergence of mulyidrug resistance is not become part of the leukemia patients in remission, or the main reason for relapse after remission. Therefore, the searching for new anticancer drugs, and exploring new treatment options is of great significant.In recent years, some of traditional Chinese medicine and its active ingredient reversal of multidrug resistance of leukemia caused people’s attentions. Celastrol or Tripterygium, one of traditional Chinese medicines, is belonging to Wilfordil hook and celastrol is one of the monomers of tripterygium. Chemically, Celastrol belongs to triterpene constituents with molecular weight 450 and its molecular formula is C29H3gO4.It reported that tripterine inhibite immune; inhibition of vascular endothelial cells in vitro value;inhibition of HMC-1 cells adhesion and adhesion molecule expression; Celastrol also induced apoptosis of tumor such as non-small cell lung cancer line H1299,human acute myeloid leukemia cell line HL-60,malignant glioma lines and retinoblastoma.In this study, we use human chronic myelogenous leukemia blast crisis cell lines K562 and K562/A02 cell line resistance to adrimycin as the target cell to determine the reverse effect of Tripterine by CCK-8 and flow cytometry,and detected the concentration of intracellular ADM and P-gp expression by Tripterine.Materials and Method1、Culture of K562 and K562/A02K562 cells were cultured with 10% newborn calf serum RPMI-1640 medium, which include in 100U/mL,100μg/mL penicillin and streptomycin per liter.K562/A02 cells were cultured with the same medium, but this medium add adriamycin(ADM1μmol/L).The two kinds of cells were cultured at 37℃,5%CO2 incubator in noemal culture. Every 2 to 3 days passage 1 times. K562/A02 cells changed with adriamycin-free medium two weeks before experiment. Logarithmic growth phase cells were used for experiments.2、The sensitivity of cells to ADM or Celastrol and the reversal of drug resistance were determined with CCK-8method.Logarithmic growth phase cells were used for experiments. Living cell counting methed based on Trypan bluedye staining staining was used for tumor cell killing test. Take 20μL cell suspension with the examinated under microscope to count 100 cells for calculating living cell persatages. Trypan bluedye stained cells deal and non-stained cells means living. Take activity greater than 95% of cells for experimental cell. K562 or K562/A02 cells of 2～3×109cells per well were inoculated into 96- well. High to low concentrations of ADM were added(such as 160、80、40、20、10、5、2.5、1.25μmol/L).The same method with Celastrol (such as 320、160、80、40、20、10、5、2.5μmol/L).Each bore hole had three repetitive wells. The volume of every wells was 150μl. The cells cultured in 37℃,5%CO2 incubator after 72 hours, each well by adding 10μl CCK-8 reagent. Continued to train 2 hours. Zero with an empty hole, put the 96-well in automatic microplate reader at absorbance measured at 450nm,and the reference wavelength of 650nm. The measured data using statistical software to calculate SPSS16.0.The SPSS 16.0 software could calculate the growth inhibition rate of 50% of the drug concentration that is half inhibition(IC50).The inhibition was calculated as follows:Inhibition=(1-Experimental group’s OD)/Control group’s OD);Resistance index=IC50 of K562/A02/IC50 of K562.The experiment was repeated three times and averaged the datas.3、The drug resistance of Celastrol were determined with CCK-8.Logarithmic growth phase K562/A02 cells, which activity greater than 95%. K562/A02 cells of 2～3×109cells per well were inoculated into 96- well. The experiment groups divided into the following groups:①K562/A02;②K562/A02+ Celastrol;③K562/A02+VLP.The concentration of ADM and other drugs as discussed earlier. Each bore hole had three repetitive wells. The volume of every wells was 150μL. The VLP(5μg/L) as positive control. The cells cultured in 37℃,5%CO2 incubator after 72 hours, each well by adding 10μl CCK-8 reagent. Continued to train 2 hours. Zero with an empty hole, and the detection wavelength as discussed earlier. The experiment was repeated three times. The reversal fold was calculated as follows: Reversal fold= The IC50 of before reversal of K562/A02/The IC50 of after reversal of K562/A02.4、The intracellular of ADM concentration of K562/A02 and K562 by Flow cytometry.Logarithmic growth phase K562/A02 and K562 cells inoculated into 24- well, which activity greater than 95%. The experiment groups divided into four groups:①K562/A02;②K562/A02+ADM(IC50);②K562/A02+ Celastrol (IC50);④K562/A02+ADM(IC50)+Celastrol (IC50). Following 48 hours in incubator, collected cells,washed with ice-cold PBS (4℃,0.01mol/L, Ph7.4) twice. Resuspended in ice-cold PBS.The samples were stored at 4℃first detected by flow cytometry.(Ex=488nm,Em=575nm)5、P-gp expression detected by flow cytometry Logarithmic growth phase K562/A02 cells inoculated into 24- well, which activity greater than 95%. The experiment groups divided into the following groups:①K562/A02;②K562/A02+ADM(IC50);③K562/A02+ Celastrol (IC50)④K562/A02+ADM(IC50)+ Celastrol (IC50). Following 48 hours in incubator, collected cells,washed with ice-cold PBS (4℃,0.01mol/L,Ph7.4) twice. Added the P-gp antibody following with the instruction. Used flow cytometry to detect the P-gp expression after incubated at room temperature away from light 30 min.Result1、The results of K562 and K562/A02 cells inhibition by CelastrolThe IC50 of Celastrol to K562/A02 and K562 cells were 295.58±23.288μmol/L、411.59±26.5511μmol/L.The results of two cells has a significant difference (P<0.05). This result explain that Celastrol has non-drug resistance in K562/A02 cells. The IC50 of ADM to K562/A02 and K562 cells were 59.755±6.883μmol/L、0.749±0.741μmol/L. The resistance of K562/A02 cells to ADM was 79.78 times of K562 cells. It explained that ADM has evident drug resistance in K562/A02. The cytotoxic does of Celastrol could reduce the IC50 of ADM in K562/A02. This cytotoxic does could increase the ADM’s sensitivity. After incubating with cytotoxic does of Celastrol, the IC50 of ADM was 0.507±0.070μmol/L,which decreased significantly than only used ADM (P<0.05), and the resistance was reversed by 117.860 times. And the IC50 of VLP(5μg/L)was 17.196±6.303μmol/L,which decreased significantly than only used ADM too(P<0.05).The resistance was reversed by 3.745 times.2、The results from concentration of intracellular ADM and P-gp expression were tested with flow cytometry (FCM)Tested with Celastrol, the concentration of intracellular ADM included obviously. After incubation of cytotoxic and non-cytotoxic does of Celastrol, the intracellular concentration of ADM were73.727±8.626、102.86±4.518.It increased 1.537 and 1.102 times in K562/A02 and K562 cells(P<0.05).When used ADM alone, the crest of P-gp expression has not obvious change. When used Celastrol alone, the crest of P-gp expression has obviously lower. And when Celastrol with ADM, the crest of P-gp expression lower obviously than ADM.Conclusion1. The IC50 of Tripterine to K562/A02 and K562 cells had a significant difference. The resistance of K562/A02 cells to ADM was 79.78 times of K562 cells.2. After incubating with cytotoxic does of Celastrol, the IC50 of ADM was obviously lower.3. After incubation of cytotoxic and non-cytotoxic does of Celastrol, the intracellular concentration of ADM increased obviously.4. Celastrol could down-regulate P-gp expression obviously.更多还原