补骨脂素逆转白血病HL60/HT耐药细胞研究

中药补骨脂主要成分补骨脂素对化疗药物耐药的急性白血病(AL)患者有明确增敏作用[1],本文采用HL60细胞株及相应耐药HT的细胞株HL60/HT,观察补骨脂素与化疗药物的协同作用.探讨补骨脂素对耐药细胞的逆转作用,初步分析其作用机理,为今后研究提供依据.     

补骨脂素对HL60/HT耐药细胞的逆转作用及对细胞内钙离子浓度的影响

目的研究补骨脂素对HL60/HT耐药细胞的逆转作用及对耐药细胞内Ca2 浓度的影响,探讨其可能的作用机制。方法采用MTT法测定补骨脂素对细胞增殖的抑制作用,高效液相色谱法检测细胞内三尖杉酯碱(HT)的量,激光共聚焦显微镜测定细胞内不同时段和不同时间点的Ca2 浓度。结果补骨脂素在1~20μmol/L能不同程度降低HT对HL60/HT细胞的IC50,并不同程度提高细胞内HT的量;1~20μmol/L补骨脂素作用不同时段(24、48、96h),HL60/HT细胞内Ca2 浓度与作用时间成负相关,10和20μmol/L补骨脂素作用HL60/HT不同时间(5、10、15、20、25min),细胞内Ca2 浓度与作用成负相关性。结论补骨脂素能逆转HL60/HT细胞的多药耐药,其作用机制与影响耐药细胞内Ca2 浓度、增加细胞内HT的量有关。     

中药活性成分逆转K562/ADM耐药性聚类模糊研究

 目的在具有耐药逆转作用的数种中药成分中进行聚类分组和优选排序。方法选用补骨脂素、苦参碱、人参皂苷Rb1、槲皮素、姜黄素、贝母碱、川芎嗪、大黄酸8种中药成分,MTT比色法测定药物对K562/ADM细胞的增殖抑制作用,流式细胞仪测定细胞内化疗药物ADM荧光强度,测定细胞P糖蛋白表达;就测定的相关数据,通过多元统计中的聚类分析方法进行药物分组,以及采用模糊理论中的集值统计方法进行优选排序。结果8种药物逆转倍数在4.4～15.4之间,K562/ADM细胞中ADM浓度在(8.53±3.51)～(26.17±9.21),K562/ADM细胞P-gp表达率在(26.91±12.19)～(66.91±12.29);8种药物之间的相似性聚类分析成4组:补骨脂素、人参皂苷Rb1、槲皮素3种药物为第1组,苦参碱、贝母碱、姜黄素3种药物为第2组,大黄酸为第3组,川芎嗪为第4组。集值统计优选药效次序为:第4组、第3组、第1组、第2组。结论根据药物间相关性可用聚类分析分组,集值统计方法可以结合药效结果的区间表示进行定量计算,进而得出组间排序。     

解毒化瘀复方含药血清对白血病HL60/Adr细胞耐药的逆转作用

目的 探讨解毒化瘀复方含药血清对白血病HL60/Adr细胞的耐药逆转作用.方法应用中药血清药理学方法制备解毒化瘀复方的兔血清,以MTT法检测经含药血清、干扰素处理后HL60/Adr细胞对化疗药物的敏感性;免疫组化方法检测P-gp、Bcl-2、P53耐药基因表达情况.结果MTT结果显示不同浓度解毒化瘀复方含药血清对HL60/Adr细胞均有耐药逆转作用,其逆转倍数随剂量增加而增大;免疫组化结果显示解毒化瘀复方含药血清能够降低HL60/Adr细胞耐药基因P-gp和抗凋亡基因Bcl-2的表达,但对P53基因无影响.结论解毒化瘀复方含药血清能够降低白血病HL60/Adr细胞P-170和bcl-2的表达,逆转HL60/Adr细胞的耐药.     

解毒化瘀药含药血清对白血病HL60/ADR细胞Survivin基因表达影响的研究

目的:探讨解毒化瘀药含药血清对白血病HL60/ADR细胞Survivin基因表达的影响。方法:应用半定量逆转录聚合酶链反应(RT-PCR)法检测解毒化瘀药含药血清处理后HL60/ADR细胞Survivin基因的表达,并与干扰素作对照。结果:解毒化瘀药含药血清能够降低HL60/ADR细胞Survivin基因的表达。结论:解毒化瘀药含药血清通过降低HL60/ADR细胞Survivin基因的表达,逆转白血病细胞耐药。     

解毒化瘀药含药血清对白血病HL60/ADR耐药 细胞NF-κB信号靶向干预研究

目的:探讨解毒化瘀药含药血清对白血病HL60/ADR细胞核周因子κB(NF-κB)信号基因表达的影响。方法:将HL60/ADR耐药细胞分为牛血清组、兔血清组、含药血清高、中、低剂量处理组和干扰素对照组,用Western blot法检测各组细胞NF-κB/p65,NF-κB/p50,IκBα的表达情况。结果:流式细胞仪检测结果显示非细胞毒作用的解毒化瘀药血清能够增加HL60/ADR细胞内柔红霉素的浓度。HL60/ADR耐药细胞NF-κB信号基因呈高表达,HL60敏感细胞未见表达,加用不同浓度解毒化瘀含药血清处理后,p65,p50和IKBa的表达均有不同程度下降,以高、中剂量组对p65的下调作用更明显。结论:解毒化瘀药对HL60/ADR细胞的耐药逆转作用是通过阻断NF-κB信号通路实现的。     

二十碳五烯酸体外逆转HL-60/ADM耐药性的研究

目的探讨不饱和脂肪酸二十碳五烯酸(EPA)逆转HL-60/ADM多药耐药性的效果。方法采用MTT法对EPA和阿霉素合用时肿瘤细胞的IC50值进行检测。结果浓度为10 mg/L的EPA能提高阿霉素对HL-60/ADM的细胞毒作用,提高倍数达6.8倍,浓度为20 mg/L的EPA提高倍数则达7.2倍。结论EPA在体外能部分逆转白血病耐药细胞对阿霉素的耐药性。     

解毒化淤药对白血病K562/A02、HL60/Adr细胞P-gp、Bcl-2、P53基因表达影响的研究

目的探讨解毒化淤中药复方含药血清对白血病K562/A02、HL60/Adr细胞P-gp、Bcl-2、P53基因表达的影响。方法应用中药血清药理学方法制备解毒化淤药的兔血清,以MTT法检测经含药血清处理后K562/A02、HL60/Adr细胞对化疗药物的敏感性;免疫组化方法检测P-gp、Bcl-2、P53耐药基因表达情况。结果 MTT结果显示不同浓度解毒化淤含药血清对K562/A02、HL60/Adr细胞均有耐药逆转作用,其逆转倍数随剂量增加而增大;免疫组化结果显示解毒化淤药含药血清能够降低K562/A02、HL60/Adr细胞耐药基因P-gp和抗凋亡基因Bcl-2的表达,但对P53基因无影响。结论解毒化淤方含药血清逆转白血病K562/A02、HL60/Adr细胞耐药的机制是降低P-gp和bcl-2的表达而逆转耐药的,对P53基因表达无影响。     

补骨脂素对HL60/HT耐药细胞逆转及对细胞内Ca2+浓度影响研究

 目的研究补骨脂素对HL60/HT耐药细胞逆转作用及对耐药细胞内Ca²⁺浓度影响,探讨其可能作用机制。方法采用MTT法测定补骨脂素对细胞增殖抑制作用,高效液相色谱法检测细胞内HT的浓度,激光共聚焦显微镜测定细胞内不同时段和不同时间点的Ca²⁺浓度。结果补骨脂素在1～20μmol·L^-1能不同程度降低HT对HL60/HT细胞的IC50,并不同程度提高细胞内HT浓度;1～20μmol·L^-1补骨脂素在不同作用时段(24,48,96 h)对HL60/HT细胞内Ca2+浓度与作用时间成负相关,10～20μmol·L^-1补骨脂素在作用HL60/HT不同时间点(5,10,15,20,25 min)细胞内Ca2+浓度与作用时间成负相关性。结论补骨脂素能逆转HL60/HT细胞的MDR,其作用机制与影响耐药细胞内Ca²⁺浓度,故而增加细胞内HT浓度有关。     

中药活性成分逆转肿瘤多药耐药的研究进展

中药因其低毒和综合作用的优势已在肿瘤多药耐药逆转方法的研究中逐步受到重视,目前已从多种中药中发现了具有逆转肿瘤多药耐药作用的活性成分。列举了几种研究较多的中药活性成分,从实验研究、耐药机制和毒性几个方面进行综述,最后探讨中药逆转肿瘤多药耐药所面临的问题。     

芦笋有效成分熊果酸诱导HL-60细胞凋亡的实验研究

目的：探讨芦笋有效成分熊果酸对ＨＬ－６０细胞增殖、凋亡的影响。方法：Ｅ和ＭＴＴ法、ＤＮＡ凝胶电泳、形态学方法等测定熊果酸对ＨＨＬ－６０细胞增殖、凋亡的影响。结果：熊果酸抑制ＨＬ－６０细胞增殖，对ＨＬ－６０细胞的半数生长的抑制剂量（ＩＣ５０）为８．２６μｍｏｌ／Ｌ，并能导其发生凋亡结论：熊果酸通过抑制ＨＬ－６０细胞增殖和诱导其凋亡发挥抗肿瘤作用。     

Antitumour effects of selected plant polyphenols,gallic acid and ellagic acid,on sensitive and multidrug‐resistant leukaemia HL60 cells

The aim of this study was to examine the antitumour effects of plant phenolic acids, gallic acid (GA) and ellagic acid (EA), on human promyelocytic leukaemia sensitive HL60 cell line and its resistant sublines exhibiting two MDR phenotypes: HL60/VINC (overexpressing P‐glycoprotein) and HL60/MX2 (characterized by the presence of mutated α isoform of topoisomerase II). Both studied compounds exerted comparable cytotoxic activities towards sensitive HL60 cells and their MDR counterparts. It was also found that GA and EA modulated the cellular level of reactive oxygen species in a dose‐dependent and time‐dependent manner. Furthermore, it was demonstrated that GA (IC₉₀) and EA (IC₅₀ and IC₉₀) significantly increased the percentage of sub‐G1 subpopulation of all studied leukaemia cells causing oligonucleosomal DNA fragmentation. Both compounds used at IC₉₀ triggered mainly the apoptotic death of these cells. However, GA had no effect on the activity of caspase‐3 as well as caspase‐8 in sensitive HL60 cells and their MDR counterparts. In contrast, EA provoked a significant activation of these caspases in all studied leukaemia cells. It was also found that lysosomes were not involved in triggering programmed death of sensitive HL60 and MDR cells by GA and EA.     

白及有效部位诱导HL60细胞凋亡及其作用机制的研究

目的：研究白及有效部位诱导HL60细胞凋亡及其作用机制。方法：采用乙醇回流提取白及,系统溶剂萃取法萃取后得到白及氯仿层,采用folin-酚比色法测定氯仿层的总酚含量。将白及氯仿层配成50、100、150μg/mL后,作用于HL60细胞,应用Hoechst33342染色和透射电子显微镜观察细胞的形态学变化,应用流式细胞术分析白及氯仿层对细胞凋亡的影响,并用RT-PCR法检测Bcl-2和BaxmRNA的表达。结果：白及氯仿层总酚含量为25.35%,药物对HL60细胞作用24h后,Hoechst33342染色后荧光显微镜下可见明显的胞核固缩、深染及碎裂等多种形态改变,透射电子显微镜下可见典型的凋亡细胞,流式细胞仪分析结果显示,白及氯仿层可显著诱导HL60细胞凋亡,凋亡率为24.9%,36.0%,51.3%,RT-PCR结果显示,Bcl-2mRNA的表达随着药物浓度增加而下降,BaxmRNA表达增加。结论：白及能有效诱导HL60细胞凋亡,抑制HL60细胞增殖,其机制可能与Bax基因表达增加及Bcl-2基因表达降低有关。     

In vitro antileukaemic activity of extracts from chokeberry (Aronia melanocarpa [Michx] Elliott) and mulberry (Morus alba L.) leaves against sensitive and multidrug resistant HL60 cells

The aim of the present study was to determine in vitro antileukaemic activities of extracts obtained from chokeberry (Aronia melanocarpa [Michx] Elliot) and mulberry (Morus alba L.) leaves against promyelocytic HL60 cell line and its multidrug resistant sublines exhibiting two different MDR phenotypes: HL60/VINC (overexpressing P-glycoprotein) and HL60/DOX (overexpressing MRP1 protein). It was found that the extracts from chokeberry and mulberry leaves were active against the sensitive leukaemic cell line HL60 and retained the in vitro activity against multidrug resistant sublines (HL60/VINC and HL60/DOX). The values of resistance factor (RF) found for these extracts were very low lying in the range 1.2-1.6.     

Apoptotic effect of Oldenlandia diffusa on the leukaemic cell line HL60 and human lymphocytes

Oldenlandia diffusa is traditionally prescribed in the treatment of a number of cancers and studies suggest that it exerts a cytotoxic action specific to cancer cells. To further investigate this suggested action, the effect(s) of Oldenlandia diffusa on leukaemic cells (HL60) and stimulated and unstimulated human blood lymphocytes (PBLs) was investigated. For the HL60s, cell growth, apoptotic induction, alterations in cell cycle characteristics and genotoxicity were investigated. For the PBLs, apoptotic induction and alterations in cell cycle characteristics were investigated. Preliminary chemical analysis to identify the cytotoxic constituents of Oldenlandia diffusa was also carried out. Results showed that Oldenlandia diffusa significantly inhibited the growth of the HL60s and induced apoptosis in a cell cycle-independent fashion, possibly through the induction of genotoxic damage. Oldenlandia diffusa did not induce apoptosis in the PBLs however progression through the cell cycle was not evident (in stimulated PBLs) suggesting some degree of cytotoxicity. Preliminary chemical analysis indicated that a number of compounds appear to be responsible for the cytotoxic action of Oldenlandia diffusa. These results indicate that HL60s are more sensitive to Oldenlandia diffusa than stimulated PBLs and thus support a cytotoxic action for Oldenlandia diffusa that has some degree of specificity.     

含益髓灵兔血清诱导HL-60细胞凋亡与对Bcl-2抑制研究

目的：探讨中药复方益髓灵治疗白血病作用机理：方法：在已知益髓灵具有诱导HL-60细胞凋亡的基础上,通过血清药理学方法,选择含不同浓度益髓灵初提物的兔药血清,与人急性早幼粒白血病HL-60细胞共同孵育不同时间后,观察兔药血清对HL-60细胞凋亡的影响,并对其发生凋亡机制进行初步探讨。结果：益髓灵兔药血清有较强的诱导HL-60细胞凋亡作用,以10％兔药血清作用48h效果最好,并发现其诱导凋亡机制与抑制Bcl-2基因表达相关。结论：益髓灵进入动物体内也有诱导白血病细胞凋亡作用,其诱导白血病细胞凋亡是治疗白血病的关键机制之一。