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。     

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。     

TPA对原代白血病细胞的诱导分化作用

目的：探讨TPA对原代白血病细胞的生长抑制和诱导分化作用。方法：取28例白血病患者原代白血病细胞,分别应用TPA、RA和TPA联合RA进行药物干预培养,用MTT比色法测定各组细胞活力．并观察细胞形态学和组织化学变化。结果：干预培养的原代白血病细胞均出现不同程度的生长抑制。TPA组和TPA RA组的28例白血病细胞均在12h-48h后出现聚集现象。23例ANLL细胞中TPA组19例出现细胞贴壁和胞质丝状突起．TPA RA组22例出现细胞贴壁和丝状突起。TPA组和TPA RA组的NAE活性增强。5例ALL均出现细胞聚集反应,NAE染色阴性。结论：TPA可以抑制体外原代急性白血病细胞的生长和增生,并有促使其向单核巨噬细胞分化作用,联合应用RA其抑制效果更明显。     

新型维甲酸衍生物ATPA对白血病细胞株K562增殖及分化的影响

目的:本研究探讨新型维甲酸衍生物4-氨基-2-三氟甲基苯基依曲替酸酯(4-amino-2-trifluoromethyl-phenyl acitretinate,ATPA)对白血病K562细胞体外增殖和分化的作用。方法:用不同浓度的ATPA作用白血病K562细胞后,在体外通过MTT法检测和绘制细胞生长曲线来分析细胞增殖;瑞特染色法观察细胞形态学改变;氯化硝基四氮唑蓝(nitroblue tetrazolium chloride,NBT)还原实验分析细胞的分化指标;采用FCM法检测细胞表面分化抗原及细胞周期的变化。结果:ATPA呈浓度依赖性抑制K562细胞增殖,明显的抑制作用从药物作用48h后开始出现,在72h后作用更明显。倒置显微镜下观察发现ATPA作用后K562细胞形态趋向成熟,NBT阳性细胞率增加;G0/G1期细胞表达量增加,S期细胞表达量减少,呈G0/G1期阻滞;细胞表面分化抗原CD71表达减少。结论:ATPA对白血病K562细胞具有抑制增殖和一定的诱导分化作用。     

骨髓成纤维样基质细胞系对急性髓细胞白血病细胞系增殖与分化影响的研究

目的：以急性髓细胞白血病（acute myeloid leukaemia,AML）细胞系为研究对象,观察正常人骨髓成纤维样细胞系（human bone marrow fibro—blastoid stromal cell line,HFCL）对急性单核细胞白血病细胞系U937、急性粒细胞白血病敏感细胞HL-60和多药耐药细胞HL-60／VCR增殖和分化的影响。方法：建立U937、HL-60和HL-60／VCR细胞与HFCL细胞的共培养模型,实验分对照组、直接接触组和transwell组。采用台盼蓝拒染法测定生长曲线;硝基四氮唑蓝（NBT）确定细胞分化;流式细胞仪检测细胞周期和CD11b、CD13、CD14和CD33细胞表面抗原进一步鉴定细胞分化;west-ernblot检测增殖细胞核抗原（PCNA）和P-糖蛋白（P-gP）的表达。结果：与HFCL细胞共培养96h后,U937、HL-60和HL-60／VCR细胞的生长受抑,且与HFCL细胞直接接触组的抑制作用大于用tr-answell组,P〈0．01。同时发现AML细胞系与HFCL细胞共培养后,G0／G1期细胞比例均增高,而S期细胞均减少,P〈0．01;尤其是直接接触组CD11b和CD14表达也均增高（P〈0．01）,CD13和CD33变化不大,NBT阳性细胞轻度增高,且差异有统计学意义。Western blot检测结果显示,3种AML细胞系PCNA表达下调;以直接接触组为甚。提示介导着白血病细胞和骨髓基质细胞间的相互作用的整合素p1（VLA-4）和p2（LFA-1）,在HFCL细胞对AML细胞生长作用影响中起着不可忽视的作用。结论：HFCL对3种具有代表性的AML细胞系U937、HL-60和HL-60／VCR有增殖抑制和诱导分化作用,除了能抑制AML细胞的生长,抑制PCNA的表达,出现G0／G1期阻滞外,还能诱导其部分向单核细胞分化。     

六磷酸肌醇对人结肠腺癌LoVo细胞增殖分化影响

目的观察六磷酸肌醇(IP6)对人结肠腺癌细胞株LoVo体外增殖、分化的影响.方法直接细胞记数法、MTT法、平皿集落形成实验、肠型ALP比活性测定等方法观察LoVo细胞增殖、分化特性.结果与对照组相比,IP6处理组在6h后细胞增殖与集落形成能力呈现抑制状态,并呈时间、剂量依赖关系.分化标志酶ALP比活性增高.结论IP6对LoVo细胞具有细胞毒作用,能明显抑制细胞增殖、诱导细胞分化.     

更昔洛韦对慢性粒细胞白血病细胞生长和分化影响及其机制研究

目的：探讨更昔洛韦（ＧＣＶ）对人白血病细胞的抗增殖和诱导分化作用。方法：ＧＣＶ加入Ｋ５６２细胞培养４ｄ,测定其活细胞数目、克隆形成率、联苯胺染色阳性率,观察光镜形态、组织化学染色,并地行流式细胞分析、端粒酶检测。结果：在ＧＣＶ的作用下,Ｋ５６２细胞地增殖受抑,生长分数降低,并且向具有合成血红蛋白能力的细胞分化。结论：ＧＣＶ对Ｋ５６２细胞具有抑制增殖和诱导分化作用,该结果为慢性粒细胞白血病（ＣＭＬ）的治疗探索新的途径。     

精氨酸酶对人APL原代细胞生长和分化的影响

了解精氨酸酶对人急性早幼粒白血病原代细胞生长和分化的影响。方法 :在RPMI 16 40培养液中补加 10 %胎牛血清和精氨酸酶 5 0 0U/L ,与对照组培养液同时置于 37℃孵箱中孵育 2 4h ,以活细胞密度 5× 10 8/L接种来自 18例急性早幼粒白血病病人的原代白血病细胞 ,在 37℃ ,5 %CO2 和饱和湿度下培养。结果 :培养 4d计数活细胞 ,发现其密度为起始密度的 (6 4 2 8± 3 12 ) % ,而对照组为 (10 8 5 6± 12 2 7) % (P <0 0 0 1) ;收集细胞行Wright Giemsa、DNA、POX、NAE及NaF抑制试验、墨汁吞噬试验和NBT还原试验等细胞化学染色 ,油镜下观察发现 ,白血病细胞向成熟分叶核粒细胞分化。结论 :精氨酸酶对APL原代细胞有抑制生长和引导分化作用     

精氨酸酶对人APL原工细胞生长和分化的影响

目的 :了解精氨酸酶对人急性早幼粒白血病原代细胞生长和分化的影响。方法 :在RPMI 16 40培养液中补加 10 %胎牛血清和精氨酸酶 5 0 0U/L ,与对照组培养液同时置于 37℃孵箱中孵育 2 4h ,以活细胞密度 5× 10 8/L接种来自 18例急性早幼粒白血病病人的原代白血病细胞 ,在 37℃ ,5 %CO2 和饱和湿度下培养。结果 :培养 4d计数活细胞 ,发现其密度为起始密度的 (6 4 2 8± 3 12 ) % ,而对照组为 (10 8 5 6± 12 2 7) % (P <0 0 0 1) ;收集细胞行Wright Giemsa、DNA、POX、NAE及NaF抑制试验、墨汁吞噬试验和NBT还原试验等细胞化学染色 ,油镜下观察发现 ,白血病细胞向成熟分叶核粒细胞分化。结论 :精氨酸酶对APL原代细胞有抑制生长和引导分化作用     

HL-60细胞高三尖杉酯碱诱导后CD44表达变化及机制的研究

目的:研究高三尖杉酯碱(homohar-ringtonine,HHT)诱导HL-60细胞分化后CD44表达变化及其与细胞周期调控的关系,探讨HHT的作用机制。方法:建立HHT诱导分化模型,瑞氏染色观察细胞形态,MTT法检测细胞增殖变化,NBT还原实验测定细胞分化状态,流式细胞术检测细胞表面CD11b表达及细胞周期变化,RT-PCR检测CD44、p27、p21和Cyclin E基因在mRNA水平的表达变化。结果:MTT实验显示,HHT对HL-60细胞产生增殖抑制作用,HL-60细胞G0/G1期细胞百分比明显上升,S期细胞百分比明显下降,P<0.05。半定量分析PCR扩增产物光密度相对表达量显示,HHT诱导后HL-60细胞中CD44基因表达逐渐减弱,p27和p21基因逐渐增强,Cyclin E基因逐渐减弱。CD44与p27表达呈明显负相关,r=-0.686,P<0.05。结论:HHT可能通过下调CD44基因,进而提高p27和p21表达,抑制Cyclin E活性而对HL60细胞产生诱导分化作用。     

3,7-二硝基二苯并溴五环盐诱导K562肿瘤细胞凋亡作用研究

目的：研究３,７－二硝基二苯半溴五环盐对人慢性粒细胞红白血病细胞株Ｋ５６２增殖的影响及其凋亡机理。方法：采用台盼攻拒染法,ＭＴＴ比色法及克隆形成率等方法,并用化学、荧光及电子显微镜观察照相,流式细胞术等方法分析ＤＮＡ断裂的数值。结果：ｃＢｒ对Ｋ５６２具有杀伤作用,可明显抑制Ｋ５６２细胞增殖,呈现时间和剂量效应：克隆形成率测得ＩＣ５０为０．４９７ｍｍｏｌ／Ｌ,流式细胞术测定凋亡率为７０．３４％;荧光显微镜和电镜观察到明显凋亡特征。结论：ｃＢｒ抑制肿瘤增殖是通过诱导肿瘤细胞凋亡实现的。     

Renin expression in hematological malignancies and its role in the regulation of hematopoiesis

It has been demonstrated that some myeloid blasts express renin, but normal bone marrow (BM) does not display this expression. The aim of the present work was to analyze the renin expression in different hematological malignancies and different myeloid cell lines. We investigated the expression of renin by RT-PCR in BM from patients with hematological malignancies (106 patients), in nine normal BM from healthy donors and in leukemic cell lines (K562, KU812, MEG-01, U-937 and HL60), as well in K562 cell line subjected to differentiation treatments. We have observed renin expression in cells from acute myeloid leukemia (AML), chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) cases. The highest frequency was observed in AML-non acute promyelocytic leukemia(APL) cases (47.2% of the cases). The disappearance of this expression was associated with the status of complete remission of AML. Renin is expressed in some myeloid human leukemia cell lines such as K562, KU812 and MEG-01. However, when K562 cells were treated with inducers of growth inhibition and/or differentiation, the expression did not disappear, indicating that renin expression is associated with a blastic phenotype rather than with cell proliferation. The obtained findings suggest that the renin expression could have a role on the disease development and could be used as an aberrant marker of leukemia.     

A novel aminosteroid is active for proliferation inhibition and differentiation induction of human acute myeloid leukemia HL-60 cells

A novel aminosteroid, 2beta-(4'-methyl-1'piperazinyl)-3alpha,17beta-dihydroxyl+ ++-5alpha-androstane (HY), was found to inhibit proliferation of HL-60 leukemia cells and induce these cells to differentiate toward macrophage-like cells from the following evidence. (1) It inhibited HL-60 cell proliferation by cell counts, colony counts and MTT assay; (2) It caused morphological changes toward macrophage-like cells after culture for 6 days; (3) It induced NBT reduction activity; (4) It induced alpha-naphthyl acetate esterase activity and (5) it induced CD11b and CD14 expression indicated by flow cytometry analysis. There is potential for this novel aminosteroid in the treatment of myeloid leukemia.     

硼替佐米对伊马替尼耐药细胞株K562/G01增殖抑制和诱导凋亡作用的研究

 目的　探讨蛋白酶体抑制剂硼替佐米（BOR）对慢性粒细胞白血病（CML）伊马替尼耐药细胞株K562／G01的增殖抑制和诱导凋亡作用。方法　采用MTT法观察细胞的生长抑制效应;流式细胞术（FCM）检测细胞周期与凋亡。结果　K562／G01细胞对伊马替尼不敏感,伊马替尼对K562／G01和K562细胞的IC50分别是（20.09±1.38）、（0.54±0.13）μmol／L;BOR对K562／G01细胞具有增殖抑制作用,并于48 h时作用效果达高峰,IC50（23.10±2.71）nmol／L,随着BOR浓度和作用时间的增加,FCM检测可见G2／M 期细胞周期阻滞及明显的凋亡峰。结论　BOR对CML伊马替尼耐药细胞株具有增殖抑制和诱导凋亡的作用,其机制可能与细胞周期G2／M 期的阻滞有关。     

Polyphenol tri-vanillic ester 13c inhibits P-JAK2V617F and Bcr–Abl oncokinase expression in correlation with STAT3/STAT5 inactivation and apoptosis induction in human leukemia cells

Constitutive activity of kinases has been reported in many types of cancers, so that inhibition of “onco-kinases” became a validated anti-cancer strategy. We found that the polyphenol 13c, a tri-vanillate derivative, inhibited kinase phosphorylation in leukemia cells. P-JAK2, P-Src and P-PI3Kp85 inhibition occurred independently of phosphatase involvement in JAK2V617F expressing HEL cells while 13c inhibited Bcr–Abl expression without inhibition of phosphorylation in chronic myelogenous leukemia cell lines (K562, MEG-01). In correlation with kinase inhibition, 13c abolished constitutive P-STAT3/P-STAT5 expression, down-regulated Mcl-1 and c-Myc gene expression and induced apoptosis. Altogether, polyphenol 13c displays potential antitumor activities by affecting onco-kinases and STAT activities.     

Colony growth characteristics in chronic myelomonocytic leukemia

Using cell culture studies specific in-vitro characteristics have been reported for Philadelphia chromosome positive myelogenous leukemia (Ph+ CML) and for juvenile chronic myelogenous leukemia (JCML) previously. We performed cell culture studies in four patients with chronic myelomonocytic leukemia (CMML) and demonstrated the following in-vitro features: excessively increased circulating CFU-C, while BFU-E and CFU-mix were either moderately increased or not detectable; CFU-C colony formation from CMML mononuclear cells (MNC) without addition of exogenous colony stimulating activity (CSA), even after depletion from adherent cells; failing inhibition of CMML MNC on normal BFU-E colony formation. These in-vitro characteristics point to CMML as a distinct entity. In two CMML-patients investigated CFU-C proliferation appeared to some extent inhibited by the addition of IFN-alpha, IFN-gamma and TNF-alpha to cell cultures.     

漆姑草醇提物对白血病细胞K562的诱导分化作用

目的： 研究漆姑草醇提物（HSJ）对人慢性髓系白血病细胞（K562）的诱导分化作用。方法： 实验设HSJ不同浓度（250、125、62.5 μg/mL）实验组,阴性对照组（等体积1640培养基）,作用48 h后,采用四甲基噻唑蓝（MTT）法检测K562细胞增殖抑制率;瑞氏-吉姆萨染色观察K562细胞形态;硝基四氮唑蓝（NBT）还原实验法测定K562细胞分化能力;流式细胞术检测K562细胞分化相关抗原CD11b、CD14、CD41a和CD42b阳性细胞的表达率;Western blot法检测K562细胞中珠蛋白转录因子1（GATA1）和造血转录因子1（PU.1）蛋白的表达。结果： 与阴性对照组比较,各浓度HSJ均能有效抑制K562细胞的增殖（P < 0.05）;经HSJ作用后,各实验组K562细胞核浆比例减小,出现分叶状细胞核,部分细胞核凹陷出现明显的细胞分化形态;与阴性对照组相比,3个不同浓度HSJ组的NBT还原率均提高（P < 0.05）;CD11b、CD14、CD41a和CD42b阳性细胞表达率均增加（P < 0.05）;GATA1和PU.1蛋白相对含量亦增加,差异均具有统计学意义（P < 0.05）。结论： 漆姑草醇提物能诱导K562细胞向成熟细胞方向分化。     

Induction of granulocytic differentiation in acute promyelocytic leukemia cells (HL-60) by water-soluble chitosan oligomer

Water-soluble chitosan oligomer (WSCO) has been reported to have anticancer activity, immuno-enhancing effect and antimicrobial activity. However, other biological activities are unknown. Herein, we have shown that WSCO is able to inhibit proliferation of human leukemia HL-60 cells and induce these cells to differentiate. Treatment with WSCO for 4 days resulted in a concentration-dependent reduction in HL-60 cell growth as measured by cell counting and MTT assay. This effect was accompanied by a marked increase in the proportion of G(0)/G(1) cells as measured by flow cytometry. WSCO also induced differentiation of the cells as measured by phorbol ester-dependent reduction of NBT, morphological changes as examined by Wright-Giemsa staining and expression of CD11b but not of CD14 as analysed by flow cytometry, indicating differentiation of HL-60 cells toward granulocyte-like cells. A combination of low dose of WSCO with all-trans retinoic acid, a differentiating agent toward granulocyte-like cells, exhibited a synergistic effect on the differentiation. In addition, treatment of HL-60 cells with WSCO for 6 or 8 days resulted in the induction of apoptosis as assayed qualitatively by agarose gel electrophoresis and quantitatively by Annexin V technique using flow cytometry. Collectively, there is a potential for WSCO in the treatment of myeloid leukemia.