紫杉醇对K562细胞增殖抑制及诱导凋亡的作用

目的　探讨紫杉醇体外抑制 K5 6 2细胞株及诱导凋亡的作用。方法　体外培养 K 5 6 2白血病细胞株 ,以不同浓度紫杉醇处理 12～ 72小时后 ,用 MTT法测定细胞生长抑制作用 ,用细胞形态学观察和 DNA琼脂糖凝胶电泳检测细胞凋亡 ,用流式细胞仪 (FCM)检测细胞 DNA含量及细胞周期。结果　 K5 6 2细胞经紫杉醇处理后细胞生长受抑 ,对 K5 6 2细胞的半数抑制浓度 IC50 为 0 .84μg/ ml。经药物作用后可见细胞染色质浓聚和凋亡小体形成 ,FCM显示细胞凋亡率明显增加 ,但电泳检测未见 DNA裂解。结论　紫杉醇能抑制 K5 6 2细胞的生长 ,诱导细胞凋亡可能为其抗肿瘤作用机制之一     

氨基葡萄糖硫酸盐诱导K562细胞凋亡的实验研究

目的　探讨氨基葡萄糖硫酸盐(GS)对慢性髓系白血病K 5 62细胞凋亡诱导作用和分子机理。方法　采用台盼蓝拒染法测定细胞生长曲线来观察GS对K5 62细胞的生长抑制作用;取对数生长期K 5 62细胞,在0 .5 ,1.0 ,5 .0mmol/LGS不同浓度作用48h后,利用细胞计数、电镜、吖啶橙/溴化乙啶(AO/EB)染色、DNA梯状电泳、流式细胞仪等方法来观察GS对K 5 62细胞的抑制效应和促凋亡作用。并用Westernblot检测活化的caspase 3和bcl- 2基因的表达。结果　0 .5 ,1.0 ,5 .0mmol/LGS对K 5 62细胞的生长有抑制作用,并呈时间和剂量依赖性(P <0 .0 1)。采用光镜、电镜和AO /EB染色法发现5mmol/LGS作用K 5 62细胞后,出现典型的凋亡形态学改变。AnnexinV染色后流式细胞仪能检测到早期凋亡细胞,细胞周期显示:G1期细胞比例增高,S期比例减低,并有凋亡峰。有明显的梯状DNA。同时GS诱导K 5 62细胞凋亡后出现活化的Caspase 3 ,而Bcl- 2蛋白表达减弱,甚至消失。结论　GS能诱导白血病K- 5 62细胞凋亡,该过程伴有Caspase- 3的活化和Bcl- 2蛋白表达降低。     

苦参碱对K562及其多药耐药细胞K562／vin,K562／dox的诱导调？…

目的：明确中药苦参碱对化疗敏感和耐药细胞的诱导调亡作用。方法：采用ＭＴＴ法测定苦参碱对各细胞的ＩＣ５０值。Ｋ５６２、Ｋ５６２／ｋｉｎ、Ｋ５６２／ｄｏｘ细胞与浓度苦参碱共同孵育于１６４０培养液中,一定时间后对细胞行Ｗｒｉｇｈｔ’ｓ－Ｇｉｅｍｓａ染色,做形态学观察,同时行ＤＮＡ凝胶电泳、流式细胞仪ＤＮＡ含量分析以检测调亡。结果：细胞形态学观察可见０．２５￣１．００ｍｇ／ｍｌ浓度苦参碱作用后的细胞体积缩     

紫草素诱导人白血病细胞K562的凋亡

目的: 研究紫草素对人白血病细胞株K562增殖抑制和诱导凋亡作用.方法: 四氮甲唑蓝(MTT)观察细胞的生长状况,应用彗星分析法、TUNEL技术和流式细胞仪检测细胞凋亡.结果: 紫草素在(1-9)×10-5mol/L浓度范围内能明显抑制K562细胞的增殖,并具有时间和浓度依赖性,9 X 10-5mol/L紫草素作用72h的K562细胞株A值最低;彗星分析法显示经紫草素作用的K562细胞出现长的彗尾条带;原位细胞凋亡可见细胞变小,核固缩为一个或多个染色质团块;流式细胞仪检测细胞凋亡主要发生在G1/S期.结论: 紫草素在体外一定浓度范围内能抑制K562细胞增殖,诱导凋亡.     

TNFα诱导KS62／VCR和K562细胞凋亡及逆转其耐药作用的实验研究

目的　探讨肿瘤坏死因子 α(TNFα)体外诱导 K5 6 2 / VCR及 K5 6 2细胞凋亡 ,及其逆转 K5 6 2 / VCR细胞多药耐药 (MDR)的作用机制。方法　以光镜、电镜、流式细胞仪观察细胞凋亡 ;流式细胞仪检测 P- gp和 bcl- 2表达。MTT检测药物敏感性。结果　 (1) TNFα浓度 >10 0 U/ m l,作用时间 >4 8小时 ,2株细胞均可见典型凋亡现象 ,以 K5 6 2 / VCR细胞明显。 (2 )以 TNFα10 0 0 U/ ml处理后 ,K5 6 2 / VCR和 K5 6 2细胞凋亡率分别为 2 9.4 %和10 .7%。 (3) K5 6 2 / VCR经 TNFα10 0 0 U/ ml处理 72小时 P- gp表达率从 93.6 %降至 82 .4 % ,bcl- 2表达率从32 .9%降至 7.0 %。 (4 ) K5 6 2 / VCR和 K5 6 2细胞分别经 TNFα10 0 U/ ml和 10 0 0 U/ ml处理后 ,VCR、Ara- C和VP- 16的药物敏感性增加 (P<0 .0 5 )。结论　 (1) TNFα可诱导 K5 6 2 / VCR和 K5 6 2细胞凋亡 ,以前者更加敏感 ,且存在时间和剂量依赖性。 (2 )高浓度 TNFα可下调 bcl- 2表达 ,但不显著改变 P- gp表达。 (3) TNFα能增加 K5 6 2 /VCR和 K5 6 2细胞对 VCR、Ara- C和 VP- 16的药物敏感性 ,以 K5 6 2 / VCR细胞更明显。 (4 ) TNFα逆转 K5 6 2 / VCR多药耐药是通过诱导细胞凋亡 ,降低 bcl- 2表达 ,而非下调 P- gp表达途径实现     

蟾蜍毒素对P-gp阳性白血病K562细胞的杀伤作用

目的 :探讨蟾蜍毒素混合物对白血病K5 62敏感株和耐柔红霉素株细胞的杀伤作用。方法 :采用四甲基偶氮唑盐 (MTT )法检测细胞的生长率 ,流式细胞仪测定细胞的DNA含量 ,细胞涂片经Wright Giemsa染色观察细胞凋亡。结果 :蟾蜍毒素混合物 >0 0 2 μg/mL以上时 ,抑制K5 62 /S和K 5 62 /D细胞的增殖 ;在0 0 4μg/mL以上浓度时 ,K5 62 /D细胞的生长率明显降低 ,与K5 62 /S相比差异有统计学意义。当 0 0 4μg/mL的蟾蜍毒素混合物处理K5 62 /S和K5 62 /D细胞 2 4h ,仅K5 62 /D细胞发生明显的G2 /M期阻滞 ;处理 48h ,K5 62 /S和K5 62 /D均发生明显的G2 /M期阻滞。 0 4μg/mL的蟾蜍毒素混合物使K5 62 /D细胞在 2 4h就发生明显凋亡 ,而K 5 62 /S细胞未见凋亡。结论 :蟾蜍毒素混合物对K 5 62 /S和K 5 62 /D细胞均具有杀伤作用 ,与K5 62 /S相比 ,对K5 62 /D的杀伤作用明显增强 ,并且不受P gp影响。     

MTX和IFN-α-2b诱导K562细胞凋亡及p73基因表达的研究

目的 :研究甲氨蝶呤 (MTX)和干扰素 α 2b(IFN α 2b)诱导人慢性粒细胞白血病急变细胞株 (K5 62 )凋亡的作用及凋亡相关基因p73mRNA在凋亡前后表达的变化。方法 :采用形态学观察法、DNA琼脂糖凝胶电泳和流式细胞仪检测K5 62细胞凋亡 ,采用半定量逆转录PCR方法 (RT PCR)检测p73mRNA的表达。结果 :4μg/mLMTX和60 0IU/mLIFN α 2b分别作用于K5 62细胞 6、12和 2 4h ,通过Wright’s Giemsa染色可见部分细胞染色质明显固缩。DNA琼脂糖凝胶电泳可见清晰的梯形条带。流式细胞仪检测 ,MTX作用细胞6、12和 2 4h ,细胞凋亡率 (AR)分别为 5 15 %、14 70 %和 3 5 49% ;而IFN α 2b作用 6、12和 2 4h ,AR分别为 2 3 3 %、11 90 %和 3 5 15 %。对照组AR分别为 0 93 %和 1 0 9%。半定量RT PCR测定表明 ,IFN α 2b作用 2 4h组p73mRNA表达较对照组下调 ( 0 43± 0 0 5vs 0 66± 0 11,P <0 0 5 ) ,其余各组无明显变化。结论 :MTX、IFN α 2b可诱导K5 62细胞凋亡 ;在MTX、IFN α 2b诱导K5 62细胞凋亡的早期 ,p73mRNA表达无明显变化 ;IFN α 2b作用 2 4h ,p73mRNA表达下调。     

TNFα诱导K562/VCR和K562细胞凋亡及逆转其耐药作用的实验研究

目的探讨肿瘤坏死因子α(TNFα)体外诱导K562/VCR及K562细胞凋亡,及其逆转K562/VCR细胞多药耐药(MDR)的作用机制.方法以光镜、电镜、流式细胞仪观察细胞凋亡;流式细胞仪检测P-gp和bcl-2表达.MTT检测药物敏感性.结果 (1)TNFα浓度>100 U/ml,作用时间>48小时,2株细胞均可见典型凋亡现象,以K562/VCR细胞明显.(2)以TNFα 1 000 U/ml处理后,K562/VCR和K562细胞凋亡率分别为29.4%和10.7%.(3)K562/VCR经TNFα 1 000 U/ml处理72小时P-gp表达率从93.6%降至82.4%,bcl-2表达率从32.9%降至7.0%.(4)K562/VCR和K562细胞分别经TNFα 100 U/ml和1 000 U/ml处理后,VCR、Ara-C和VP-16的药物敏感性增加(P<0.05).结论 (1)TNFα可诱导K562/VCR和K562细胞凋亡,以前者更加敏感,且存在时间和剂量依赖性.(2)高浓度TNFα可下调bcl-2表达,但不显著改变P-gp表达.(3)TNFα能增加K562/VCR和K562细胞对VCR、Ara-C和VP-16的药物敏感性,以K562/VCR细胞更明显.(4)TNFα逆转K562/VCR多药耐药是通过诱导细胞凋亡,降低bcl-2表达,而非下调P-gp表达途径实现.     

木黄酮作用于K562细胞机制的初步研究

目的：探讨酪氨酸激酶抑制剂木黄酮（Genistein)作用于慢性粒细胞性白血病（CML）的机制。方法：通过细胞增殖、活力检测、形态学观察、半固体集落培养及DNA凝胶电泳和流式细胞术,观察木黄酮对K562细胞生长的影响。结果：（1）经≥5mg/L木黄酮处理2d的K562细胞,增殖抑制率达到50％以上,且与作用剂量和时间呈正相关;形态渐趋向凋亡但体积涨大;(2)K562细胞集落抑制达50％时的木黄酮浓度为10mg/L,也与时间和剂量呈正相关;（3）K562细胞经10mg/L木黄酮处理4d,DNA凝胶电泳可见清晰的梯状条带;（4）K562细胞分别经2．5mg/L、5mg/L和10mg/L木黄酮处理1d和2d后,流式细胞术检测细胞凋亡率分别为0．62％、1．64％、2．71％和0．68％、4．09％、8．4％;2d后G2期细胞分别占总细胞数的17％、34．5％和79．5％,而G1期和S期细胞逐渐明显减少。结论：木黄酮对K562细胞具有显的抑制增殖作用,其机制与诱导凋亡有关。     

氧化苦参碱对人卵巢癌细胞SKOV-3体外活性的影响

目的研究氧化苦参碱对人卵巢癌细胞SKOV-3体外活性的影响.方法MTT显色法检测不同浓度组药物对SKOV-3细胞的生长抑制作用;透射电镜观察细胞超微结构改变;流式细胞仪分析细胞周期变化.结果氧化苦参碱1mg/ml对细胞生长无明显影响,2mg/ml、5mg/ml、10mg/ml抑制细胞生长,呈量效关系;2mg/ml药物作用72小时后,流式细胞仪可检测到细胞凋亡现象并发现显著的S期阻滞;透射电镜可见胞核固缩,凋亡小体形成等凋亡形态学变化.结论氧化苦参碱在体外能够抑制人卵巢癌细胞SKOV-3生长,将其阻滞在S期,并能诱导细胞发生凋亡.     

K562及其耐药细胞系多药耐药机制的蛋白质组学研究

 目的 探讨K562及其耐药细胞系蛋白差异表达情况。方法 应用二维荧光差异电泳结合质谱技术,经过相应软件处理找出K562和K562耐药细胞之间蛋白质表达质和量的变化。结果 二维电泳发现11个差异蛋白点,质谱鉴定出9个表达差异的蛋白质,其中4种蛋白质能量代谢有关;3种蛋白质与细胞信号转导有关;2种蛋白与细胞增殖相关。9种差异表达蛋白质中,6种在K562/ADM中表达增加,3种表达降低。结论 研究表明应用2D-DIGE结合质谱技术为白血病多药耐药机制研究是一种新的可靠手段,对于揭示耐药细胞与敏感细胞蛋白组学变化和蛋白质之间的相互作用提供新的思路。     

Effects of inducers of erythroid differentiation of human leukemia K562 cells on vincristine-resistant K562/VCR cells

K562/VCR cells, which are resistant to the cytotoxicity of vincristine, were isolated from human erythroleukemia K562 cells. Various compounds that induce erythroid differentiation of K562 cells were tested on K562/VCR cells. Differentiation of K562/VCR cells was not induced by actinomycin D or adriamycin alone, but the resistance of these cells to the inducers was overcome by verapamil. In contrast, mitomycin C, butyric acid and hemin induced differentiation of K562/VCR cells as effectively as that of K562 cells. These results suggest that therapy by induction of differentiation of leukemic cells is effective for leukemic cells that have acquired resistance to therapeutic drugs.     

INDEPENDENT AND SYNERGIC INHIBITION OF DIPYRIDAMOLE AND RADIATION ON K562-AND K562/ADM CELL LINES IN VITRO

It is first demonstrated that dipyridamole (DP) and radiation were capable of significantly inhibiting, independently and synerglcally, clonogenlc growth in the two kinds of K562 cell lines, adriamycin (ADM) -sensitive and ADM- resistant. DP or radiation alone Increased clonogenlc Inhibition rate (CIR) in the two kinds of cell lines in a dose- dependent fashion. DP potentiated radiosensitivity and radiation increased inhibition of DP in the two kinds of cell lines. K562/ ADM cell lines were higher sensitive to DP. radiation and combination of them than K562 cell lines (P<0. 01). There was stronger synergic inhibition of clonogenlc growth in the two kinds of cell lines when pretreated with DP than when posttreated with DP (P<0. 01).     

Characterization of an Etoposide-resistant Human K562 Cell Line, K/eto

An etoposide-resistant K562 cell line (K/eto) was obtained by stepwise exposure, in culture, to increasing concentrations of etoposide, without the use of mutagens. This cell line was resistant to etoposide, and slightly resistant to adriamycin, but sensitive to anti-cancer drugs such as camptothecin, vincristine, actinomycin D and so on. P-GIycoprotein, the mdr 1 gene product, was not detected in this cell line, as assessed by immunocytochemistry, itnmunoprecipitation and flow cytometry. Overexpression of mdr 1 mRNA was also not found. Interestingly, expression of 85 kD protein recognized by MRK 20 monoclonal antibody was noted. The level of DNA topoisomerase II protein, detected by antibody staining, decreased concomitantly with a general decrease in DNA topoisomerase II unknotting activity, while DNA topoisomerase I activity was not affected. Cellular accumulation of [³H]etoposide was reduced by 75% in the resistant line compared with parental K562. Karyotype analysis showed that the number of chromosomes in K/eto was 55 and neither a homogeneous staining region nor double-minute chromosomes were detected. These results indicate that this resistance is not due to an altered interaction between the drug and cellular transport machinery, i.e. MDR1, associated with the "classic" multiple drug resistance phenotype, but rather is due to the existence of other mechanism(s) of resistance, decreased transport of the drug and decreased target enzyme, DNA topoisomerase II.     

PTEN/PI3K/Akt信号通路对K562细胞凋亡调控的研究

 目的 探讨PTEN/PI3K/Akt信号传导通路对人慢性粒细胞白血病细胞系K562的增殖、凋亡调控的研究及可能的分子作用机制。 方法 将携带有野生型PTEN及绿色荧光蛋白的腺病毒（Ad-PTEN-GFP）及空载体（Ad-GFP）腺病毒,转染人慢性粒细胞白血病细胞系K562。通过MTT检测细胞生长曲线,流式细胞术检测细胞凋亡率和细胞增殖指数,同时用细胞光镜、电镜形态等方法检测细胞凋亡,荧光定量PCR（FQ-PCR）检测PTEN及凋亡相关基因Bcl-2、Bcl-_xL、Bax mRNA水平变化,Western blot检测PTEN及Akt、p-Akt蛋白水平变化。 结果 与Ad-GFP组相比,Ad-PTEN-GFP 转染K562细胞后,细胞增殖受抑,增殖指数降低,凋亡率增加,p-Akt表达降低,抗凋亡相关基因Bcl-2、Bcl-_xL mRNA表达降低,促凋亡基因Bax mRNA表达增加。 结论 过表达PTEN可能通过抑制PI3K/Akt通路抑制K562细胞系增殖,促进细胞凋亡。     

Characterization of an etoposide-resistant human K562 cell line, K/eto.

An etoposide-resistant K562 cell line (K/eto) was obtained by stepwise exposure, in culture, to increasing concentrations of etoposide, without the use of mutagens. This cell line was resistant to etoposide, and slightly resistant to adriamycin, but sensitive to anti-cancer drugs such as camptothecin, vincristine, actinomycin D and so on. P-Glycoprotein, the mdr1 gene product, was not detected in this cell line, as assessed by immunocytochemistry, immunoprecipitation and flow cytometry. Overexpression of mdr1 mRNA was also not found. Interestingly, expression of 85 kD protein recognized by MRK 20 monoclonal antibody was noted. The level of DNA topoisomerase II protein, detected by antibody staining, decreased concomitantly with a general decrease in DNA topoisomerase II unknotting activity, while DNA topoisomerase I activity was not affected. Cellular accumulation of [3H]etoposide was reduced by 75% in the resistant line compared with parental K562. Karyotype analysis showed that the number of chromosomes in K/eto was 55 and neither a homogeneous staining region nor double-minute chromosomes were detected. These results indicate that this resistance is not due to an altered interaction between the drug and cellular transport machinery, i.e. MDR1, associated with the "classic" multiple drug resistance phenotype, but rather is due to the existence of other mechanism(s) of resistance, decreased transport of the drug and decreased target enzyme, DNA topoisomerase II.     

Independent and synergic inhibition of verapamil and electric beam radiation on clonogenic growth in K562 and K562/ADM cell linesin vitro

It was first reported here that verapamil (VP) and electric beam radiation (EBR) were capable of inhibiting, independently or synergically, clonogenic growth in two kinds of K562 cell lines, adriamycin (ADM)-sensitive and ADM-resistant (K562/S and K562/ADM). Results showed that clonogenic rate (CGR) decreased by 3%–99.9% in the presence of dependent dose-ADM (3.8 μg/ml) in K562/ADM cell lines, while treated with 0.5μM–6μM of VP. VP was capable of potentiating radiosensitivity in K562/S and K562/ADM cell lines, whether before or after exposure of them to electric beam radiation, and significantly reduced CGR in these kinds of cell lines (P<0.01).