羟基脲诱导的K562细胞分化中PBK/TOPK的表达变化及其意义

目的研究PBK／TOPK在HU（羟基脲）诱导白血病细胞K562的分化时的表达及意义。方法用不同浓度的羟基脲对K562细胞进行诱导分化实验,通过联苯胺、吉姆萨-瑞氏染色和流式细胞仪证实其分化方向;用Western blot法检测PBK／TOPK在K562细胞分化前后表达的变化。结果400μmol／L的羟基脲对K562作用4d后具有良好的诱导分化效果,并诱导其向红系分化,且PBK／TOPK在K562细胞向成熟分化后表达不变,而Pho-PBK／TOPK和Pho-p38有少量增加。结论HU能抑制白血病细胞K562的增殖,并诱导其向红系分化,在此过程中,有激活的PBK／TOPK参与,推测很可能通过磷酸化下游分子p38而起作用。     

六亚甲基二乙酰胺对K562细胞作用的实验研究

目的：研究六亚甲基二乙酰胺（HMBA）体外对K562细胞的抑制增殖、诱导凋亡和促进分化的作用。方法：MTT法检测不同浓度HMBA对于K562细胞的增殖抑制率;流式细胞仪（FCM）检测HMBA对于K562细胞周期分布的影响;Annexin V／PI染色方法检测HM—BA诱导K562细胞凋亡的作用;通过瑞氏染色、联苯胺染色和FCM检测分化抗原研究K562细胞分化情况。结果：HMBA可以抑制K562细胞增殖,1、2、3和4mmol／L对K562细胞增殖抑制率分别为21．97％、36．05％、41．56％和47．59％;HMBA可以阻滞K562细胞周期,主要表现为G0／G1期的阻滞;HMBA诱导K562细胞凋亡的作用不明显,4mmol／L的HMBA对K562细胞诱导凋亡率〈5％;HMBA处理后,瑞氏染色显示K562细胞有一定程度的成熟表现,联苯胺染色基本为阴性;K562细胞膜表面CD11b、CD14、CD68和胞质内溶茵酶抗原的表达在HMBA处理前后均为阴性。结论：HM—BA可以抑制K562细胞的增殖,提示具有一定的治疗作用;HMBA对K562的作用机制和阻滞细胞周期有关,诱导凋亡不是主要作用机制;HMBA有促进K562细胞分化的迹象,但分化方向和机制有待进一步研究。     

水通道蛋白1基因过表达对K562细胞红系分化和增殖的影响

目的:探讨水通道蛋白1(aquaporin-1,AQP1)过表达对人慢性髓细胞白血病(chronic myeloidleukemia,CML) K562细胞红系分化和增殖的影响.方法:以人脑cDNA文库为模板,通过PCR扩增出AQP1基因的编码序列,构建pBABE-puro-AQP1真核表达载体;感染K562细胞,筛选建立稳定过表达AQP1基因的K562细胞株(命名为K562-AQP1);实时荧光定量PCR法、细胞免疫荧光染色法及蛋白质印迹法分别检测AQP1转录和蛋白表达水平.通过MTT法检测细胞生长增殖、实时荧光定量PCR法检测γ珠蛋白表达和分光光度法检测血红蛋白含量,研究AQP1过表达对K562细胞红系分化和增殖的影响.结果:与空载体对照组相比,pBABE-puro-AQP1转染入K562细胞后AQP1 mRNA和蛋白表达水平皆有显著升高(P＜0.01),K562-AQP1细胞中红系分化指标γ珠蛋白和血红蛋白表达水平明显增加,同时细胞生长速度明显降低(P＜0.05).结论:AQP1过表达可以显著促进K562细胞向红系分化,同时抑制细胞增殖.推测AQP1可能成为临床诱导分化治疗CML的基因靶点之一.     

miR-let-7c-5p靶向c-myc在白血病细胞定向单核/巨噬细胞分化中的作用机制研究

目的 探讨miR-let-7c-5p/c-myc信号轴在白血病细胞定向单核/巨噬细胞分化中的调控作用。方法 采用PMA+LPS+IFN-γ诱导THP-1白血病细胞向单核/巨噬细胞定向分化,PBS作为对照组。诱导48 h后CCK8法测定细胞增殖水平;流式细胞仪测定细胞CD11b与CD14分化抗原表达水平;RT-qPCR检测白血病细胞分化前后miR-let-7c-5p和c-myc的表达变化;蛋白质印迹法检测c-myc蛋白表达变化;双荧光素酶结合实验检测miR-let-7c-5p与c-myc的3′UTR靶向结合和活性调控关系;转染miR-let-7c-5p mimic观察c-myc表达变化后细胞的增殖分化水平变化;过表达c-myc拯救实验观察miR-let-7c-5p对PMA+LPS+IFN-γ诱导的THP-1细胞增殖分化的影响。THP-1细胞转染miR-let-7c-5p inhibitor,观察c-myc表达变化对THP-1定向分化为M1样巨噬细胞的影响。结果 与PBS对照组相比,PMA+LPS+IFN-γ诱导48 h后,THP-1细胞的增殖能力明显降低(0.64±0.01 vs 0.3...     

STI571与三氧化二砷对多药耐药bcr-abl阳性白血病细胞的协同抑制作用

目的　研究酪氨酸激酶抑制剂STI5 71与三氧化二砷 (As2 O3 )联合应用对多药耐药bcr abl阳性白血病细胞的协同效应。方法　采用MTT法比较STI5 71单独或与不同浓度的As2 O3 联合应用对bcr abl和mdr1共同阳性的白血病细胞系K5 6 2 n/VCR的抑制作用。结果　 1μmol/L的STI5 71对K5 6 2 n/VCR细胞无明显细胞毒作用 ,与 10 -5,10 -6,10 -7,10 -8mol/L的As2 O3 联合应用 ,细胞毒作用明显增强。As2 O3 单独对K5 6 2 n/VCR细胞的IC50 为 1.879μmol/L ,加STI5 71后 ,IC50 为 0 .15 5 μmol/L ,协同抑制作用为 12 .12倍。结论　STI5 71与As2 O3 联合应用对于bcr abl与mdr1共同阳性的白血病细胞有更强的抑制作用     

白细胞介素-24抑制K562细胞生长的机制

 目的　探讨白细胞介素-24（又称黑色素瘤分化相关基因-7,IL-24／mda-7）对白血病细胞系K562的周期阻滞作用机制。方法　利用基因芯片技术初步分析转染IL-24／mda-7与转染空载体的K562细胞之间基因表达差异,并以实时定量PCR验证;以Western blotting方法检测pRb的磷酸化水平。结果　转染IL-24／mda-7可使K562细胞的细胞周期相关基因p21WAF-1、BCCIP上调,cdk6、Smurf2下调,定量PCR证实了上述表达变化;IL-24／mda-7转染还可明显降低K562细胞pRb磷酸化水平。结论　IL-24／mda-7 可能通过调节细胞周期相关蛋白,即上调p21WAF-1、BCCIP,下调cdk6、Smurf2,使K562阻滞于G0／G1期,从而抑制细胞生长。     

孕酮对白血病细胞的抑制增殖和诱导分化作用

 目的探讨孕酮对K562和NB4白血病细胞系的抑制增殖和诱导分化作用。方法采用细胞计数,XTT实验观察孕酮对白血病细胞增殖能力的影响,采用Wright-Giemsa染色,联苯胺染色,NBT还原实验和流式细胞术观察孕酮对白血病细胞的诱导分化作用。结果不同浓度孕酮连续作用白血病细胞5天,液体培养显示细胞数明显减少,第5天处理组的XTT值显著低于对照组,并呈剂量依赖关系;液体培养和XTT均显示孕酮对NB4细胞的增殖并无显著影响。K562细胞联苯胺染色 OD值显著升高,与对照组比较差异有统计学意义（P<0.01）,NB4细胞NBT还原能力提高（P<0.01）,并呈剂量依赖性。形态学观察孕酮可诱导K562细胞和NB4细胞趋向成熟分化,流式细胞术检测孕酮处理后K562和NB4细胞膜CD71和CD11b分化抗原表达阳性率分别为85.72％和61.28％。结论孕酮可显著抑制K562白血病细胞增殖,诱导K562和NB4白血病细胞向成熟分化。     

苦参碱对白血病细胞癌基因和细胞周期调控蛋白表达的影响

目的：探讨在苦参碱诱导下,白血病细胞株K562增殖和分化时相关癌基因及细胞周期调控蛋白表达表达的改变及意义。方法：应用分子生物学Norhern Blot和DotBlot杂交技术检测人红白细胞病细胞株K562在苦参碱作用后的c-myc,N-ras及p53mRN表达;同时用Western BLotting-ECL分析苦参碱作用前、后24、48、72小时K562细胞Cyclin D1,CyclinE,Ddk2,Cdk5的不同表达水平。结果：在增殖的K562细胞（培养48小时）中,伴随着DNA合成增加,CyclinE和Cdk2保持高表达;而在苦参碱作用24小时分化启动的细胞中,随着DNA合成减少,N-ras,p53mRNA表达增强;c-myc mRNA表达明显受抑;同时CyclinD,Cdk5表达增强,结论：若参碱抑制K562细胞增殖并诱导分化可能与相关癌基因表达和CyclinD1/Cdk5,CyclinE/Cdk2不同表达有关。     

LncRNA GATA3-AS1通过调控miR-362-3p/FABP5轴抑制宫颈癌细胞增殖、迁移及侵袭

目的:探究长链非编码RNA GATA3反义RNA 1(lncRNA GATA3-AS1)调控微小RNA-362-3p(miR-362-3p)表达对宫颈癌细胞恶性生物学行为的影响。方法:qRT-PCR检测宫颈癌细胞中lncRNA GATA3-AS1、miR-362-3p、FABP5表达;双荧光素酶报告基因实验验证lncRNA GATA3-AS1和miR-362-3p的靶向关系、miR-362-3p和FABP5的靶向关系;将细胞分为pcDNA-NC组、pcDNA-GATA3-AS1组、si-NC组、si-GATA3-AS1组、si-GATA3-AS1+inhibitor-NC组、si-GATA3-AS1+miR-362-3p inhibitor组、miR-NC组、miR-362-3p mimics组、miR-362-3p mimics+pcDNA-NC组、miR-362-3p mimics+pcDNA FABP5组;Western blot检测蛋白表达;EdU法检测细胞增殖;Transwell检测细胞迁移侵袭。结果:在宫颈癌细胞系中,GATA3-AS1、FABP5均为高表达,miR-362...     

丝裂原活化蛋白激酶信号转导系统对Vp-16诱导分化作用的影响

目的:探讨丝裂原活化蛋白激酶(mitogen-acti-vated protein kinases,MAPKs)信号转导系统对依托泊苷(Vp-16)诱导K562细胞分化作用的影响。方法:采用四甲基偶氮唑盐(MTT)法测定细胞增殖活性;流式细胞仪解析细胞周期;硝基四氮唑蓝(NBT)还原实验检测细胞向单核/巨噬系统分化。结果:0·1~0·8μg/mL的Vp-16抑制K562细胞增殖,引起细胞G2/M期阻滞,诱导细胞向单核/巨噬系统分化;细胞外信号调节激酶(extracellular signal-regulated kinases,ERK)抑制剂PD98059降低Vp-16的诱导分化作用,P<0·05;p38丝裂原活化蛋白激酶(p38mitogen-activated protein kina-ses,p38MAPK)抑制剂SB203580增强Vp-16的作用,P<0·05;而C-JUN氨基末端激酶(c-jun N-terminal ki-nases,JNK)抑制剂SP600125对Vp-16的诱导分化作用无明显影响,P>0·05。结论:在Vp-16诱导K562细胞向单核/巨噬系统分化过程中,ERK正向,p38MAPK负向调节Vp-16的诱导分化作用。     

Effect on proliferation and erythroid differentiation of K562 Cells by IER3IP1-knockdown

Objective  To investigate the effect on erythroid differentiation and proliferation of K562 cells by IER3IP1-knockdown with RNA interference targeting at IER3IP1 gene. Methods  The shRNA eukaryotic expression vectors targeting at IER3IP1 gene were designed and constructed. Inhibitory effect was detected by semiquantitative RT-PCR. The impacts on K562 cells by RNAi were studied by MTT assay, benzidine staining, light microscope and electron microscopy observation, cell cycles analysis, colony formation assay and RT-PCR. The expressions of erythroid differentiation correlated genes Gfi-1B, GPA and γ-globin were studied after being exposed to 0.2 μmol/L imatinib for two days. Results  The shRNA eukaryotic expression vectors were successfully constructed. The expression of IER3IP1 gene was significantly inhibited with an inhibition efficiency of 76% (P<0.01). Compared with the control groups, bcr/abl mRNA level was increased in K562/shRNA-IER3IP1 group (P<0.01). The proliferation ability was enhanced (P<0.01) and the proportion of cells at G0/G1 phase decreased but S phase increased (P<0.05) in K562/shRNA-IER3IP1 group. Under electron microscopy, the amount of euchromatin increased but heterochromatin decreased. There were structural abnomalities in endocytoplasmic reticulum and clusters of vesicular. The percentage of benzidine staining positive cells and mRNA expression levels of Gfi-1B, GPA and γ-globin were all decreased after being exposed to 0.2 μmol/L STI571 for two days in K562/shRNA-IER3IP1 group (P<0.01). Conclusion  IER3IP1-knockdown can hinder the erythroid differentiation and elevate the proliferation level of K562 cells. IER3IP1 may play a role in erythroid differentiation and proliferation of K562 cells. This work was supported by the National Natural Science Foundation of China (No.30171150)     

外源性野生型p53基因在K562－n细胞表达的意义

目的研究外源性野生型ｐ５３基因在红白血病细胞表达的意义。方法采用重组逆转录病毒载体介导的方法将人野生型ｐ５３基因ｃＤＮＡ转移入Ｋ５６２－ｎ细胞，用形态学、流式细胞仪和联苯胺染色试验检测转基因后的Ｋ５６２－ｎ细胞。结果野生型ｐ５３基因可以诱导Ｋ５６２－ｎ细胞编程性细胞死亡和分化的形态特征，且伴有细胞周期Ｇ１期的生长阻滞。结论野生型ｐ５３基因能抑制人红白血病细胞生长的某些机制。     

Aminopeptidase inhibitors inhibit proliferation and induce apoptosis of K562 and STI571-resistant K562 cell lines through the MAPK and GSK-3beta pathways

A tyrosine kinase inhibitor, STI571, has been demonstrated to be effective for the treatment of chronic myelogenous leukemia (CML). STI571 inhibits tyrosine kinase activity of ABL and induces apoptosis of CML cells. However, drug resistance develops commonly in patients with blast phase CML, and has become a significant therapeutic problem. We examined the effects of aminopeptidase inhibitors on CML cell line (K562) and a STI571-resistant subline of K562. Ubenimex and the more potent aminopeptidase inhibitor, actinonin, inhibited proliferation of both K562 cells and STI571-resistant K562 cells and also induced their apoptosis in dose- and time-dependent manners. Ubenimex and actinonin induced the activation of caspase-3, and the induction of apoptosis was inhibited by pan-caspase inhibitor, indicating this apoptosis is caspase-dependent. We found that serine phosphorylation of both MAPK and glycogen synthase kinase-3β were suppressed by aminopeptidase inhibitors in parent K562 and STI571-resistant K562 cells. The expression level of cyclin D1 protein was also reduced by ubenimex and actinonin in both cell lines. These results indicated STI571-resistance does not confer the cross-resistance to aminopeptidase inhibitors in K562 cells and revealed the new findings of aminopeptidase inhibitor-induced intracellular signaling pathways.     

As2O3与STI571单用及联用对K562细胞bcr-abl磷酸化的影响

目的 研究As2O3单用及联用ST1571对K562细胞bcr-abl蛋白酪氨酸磷酸化的影响。探讨其抗白血病的分子机制,为As2O3和ST1571联合应用治疗CML提供理论依据。方法 采用细胞增生实验检测细胞生长;采用Annexin-V／PI双染实验、DNA的PI染色及DNA电泳等方法检测细胞凋亡：运用免疫沉淀和Western blot检测K562细胞bcr-abl蛋白酪氨酸磷酸化水平。结果 As2O3和ST1571明显抑制K562细胞生长。并检测出凋亡细胞群。DNA电泳出现“梯”状条带;bcr-Abl蛋白酪氨酸磷酸化水平出现时间剂量依赖性下调。结论 As2O3和ST1571能诱导K562细胞凋亡和抑制其增生．两药联用具有协同作用．机制似与下调K562细胞bcr-abl蛋白酪氨酸磷酸化有关。     

Differential expression of ras protooncogenes during in vitro differentiation of human erythroleukemia cells.

We have compared the expression of the ras protooncogene family (H-, K-, and N-ras) in leukemia cell differentiation utilizing as a model K562 and HEL erythroleukemia cells treated either with 1-beta-arabinofuranosylcytosine or 12-O-tetradecanoylphorbol-13-acetate (TPA). 1-beta-D-Arabinofuranosylcytosine induced terminal erythroid differentiation of K562 cells, while TPA induced myeloid differentiation of K562 and HEL cells, resulting in myelomonocytic-like cells expressing macrophagic and megakaryocytic markers. H-ras mRNA levels showed a dramatic decrease in K562 cells subjected to erythroid and myelomonocytic differentiation. The same result was found at the protein level for p21H-ras. Expression of K-ras and N-ras in K562 cells also decreased with differentiation, although significant mRNA levels remained despite cessation of cell proliferation. The decrease in K-ras expression was greater for TPA-treated cells than for 1-beta-arabinofuranosylcytosine-treated cells. TPA-induced myelomonocytic differentiation in HEL cells also resulted in a dramatic down-regulation of H-ras mRNA levels. Thus, by using a leukemia cell line able to differentiate along two different lineages, our results reveal a lineage-specific modulation of ras gene family expression.     

K562多药耐药细胞系中肿瘤干细胞样细胞对伊马替尼耐药机制的初步研究

Objective： To characterize a novel chronic myeloid leukemia （CML） cell line and to further elucidate the mechanisms of resistance to STI571. Methods： A novel K562 cell line （K562NP16） was achieved after exposure of the K562 cells to VP16. A small subpopulation （K562NP16 SP） that was capable of excluding Hoechst 33342 in the K562NP16 cell line was isolated by fiow cytometry sorting. The rest of the K562NP16 cells were classified as non-SP K562NP16. The mechanisms involved in K562NP16 SP cells which became resistant to STI571 were studied. Results： The levels of Bcr-Abl and Abl proteins were similar in the K562 cell line and in non-SP K562NP16 and K562NP16 SP cells. The multidrug-resistant gene 1 （MDR1） expression of the 170 kDa P-glycoprotein （P-gp） was detected in K562NP16 non-SP and K562NP16 SP cells but not in K562 cells. The expression levels of P-gp in the two K562NP16 cell lines were similar. Compared with non-SP K562/ VP16, the K562NP16 SP cells were more resistant to STI571. This resistance could hardly be reversed by many multidrug resistance inhibitors. In addition, in vivo study showed that the K562NP16 SP cells induced tumorigenesis in mice, while the K562NP16 non-SP cells failed to do so. Conclusion： A novel K562 cell line, K562NP16, was generated. A small side population K562NP16 SP cells, had high resistance to STI571 treatment and more tumorigenic than the K562 cells. It may represent the cancer stem cells of the K562NP16 cell line.     

三氧化二砷联合甲磺酸伊马替尼、咖啡因对K562白血病细胞的协同作用

 目的 探讨三氧化二砷（Arsenic trioxide,As2O3）与酪氨酸激酶抑制剂甲磺酸伊马替尼（实验药物代号STI571）以及细胞周期调节剂咖啡因联合诱导K562细胞凋亡的作用及机制,为寻找克服K562细胞对As2O3抵抗的有效手段提供实验依据。方法 以As2O3与STI571、咖啡因联合作用于K562细胞,采用MTT方法检测细胞增生活性,PI染色流式细胞仪检测细胞周期,PI和Annexin V双染色流式检测细胞凋亡,Western blot法检测细胞周期相关调节蛋白的表达。结果 5.0 μmol／L浓度的咖啡因对K562细胞增生无抑制作用,亦不能增强As2O3的抑制作用,单独以STI571 1.0 μmol／L处理,即可有效抑制K562细胞的生长,与As2O3联用能明显增加其抑制增生的效应;咖啡因与As2O3联合作用,不增加As2O3诱导的K562细胞凋亡率[（14.7±3.6）%vs（15.3±3.3）%,P＞0.05]; STI571具有轻度诱导K562细胞凋亡的作用[（18.3±4.5）%],与As2O3合用可显著增加诱导凋亡率[（14.7±3.6）%vs（42.8±4.2）%,P＜0.01],并明显降低As2O3诱导的G2／M期细胞比例。与单用As2O3比较,As2O3 + STI571明显抑制cdc2、cdc2-p及survivin蛋白表达,而As2O3与咖啡因合用不能诱导survivin蛋白表达下调,对cdc2、cdc2-p蛋白的表达无明显影响。结论 周期调节药物咖啡因对As2O3诱导K562细胞凋亡无增敏作用;酪氨酸激酶抑制剂STI571能协同As2O3诱导K562细胞凋亡,下调抗凋亡蛋白survivin的表达可能是其机制之一,值得深入研究其临床应用效果。     

Anti-proliferative effect of the abl tyrosine kinase inhibitor STI571 on the P-glycoprotein positive K562/ADM cell line

STI571, an abl tyrosine kinase inhibitor, is less effective in chronic myelogenous leukemia (CML) patients in the accelerated phase and in blastic crisis. We addressed whether STI571 is effective for the CML blastic crisis cell line K562 and the P-glycoprotein (P-gp) positive, multidrug resistance cell line K562/ADM. The present results demonstrate that P-gp positive K562/ADM cells were more resistant than K562 cells to the anti-proliferative and apoptotic effect of STI571, but the co-addition of a P-gp modulator augmented the sensitivity of K562/ADM cells to STI571. For patients in CML blastic crisis, simultaneous use of a P-gp modulator may increase the efficacy of STI571.