Role of calcium in differentiation of murine erythroleukemia cells

Calcium plays a crucial role in the normal and abnormal cell metabolism. The role of calcium in the differentiation process of murine erythroleukemia cells(MELC) remains controversial. Here, based upon quantitative measurement of fluorescence in single cells, a method was developed to investigate the intracellular free calcium [Ca2+]i concentration and DNA contents simultaneously, by employing the fluorescent probe, fluo-3 acetoxymethyl ester and DNA dye Hoechst 33342. During MELC differentiation, [Ca2+]i concentration incresed. We also demonstrated that calcium ionophore, A23187, enhanced the HMBA-induced MELC differentiation, while verapamil, an inhibitor of calcuim uptake, slightly reduced differentiation. These results suggested that an increase in the [Ca2+]i level was an essential step in HMBA-induced MELC differentiation.

The distribution of calmodulin and Ca^(2+)-activated calmodulin in cell cycle of mouse erythroleukemia cells

Cell proliferation is accompanied with changing levels of intracellular calmodulin (CaM) and its activation. Prior data from synchronized cell population could not actually stand for various CaM levels in different phases of cell cycle. Here, based upon quantitative measurement of fluorescence in individual cells, a method was developed to investigate intracellular total CaM and Ca2+-activated CaM contents. Intensity of CaM immunoflurescence gave total CaM level, and Ca2+ -activated CaM was measured by fluorescence intensity of CaM antagonist trifluoperazine (TFP). In mouse erythroleuke-mia (MEL) cells, total CaM level increased from G1 through S to G2 M, reaching a maximum of 2-fold increase, then reduced to half amount after cell division. Meanwhile, Ca2+-activated CaM also in creased through the cell cycle (G1 , S, G2M). Increasing observed in G1 meant that the entry of cells from G1 into S phase may require CaM accumulation, and, equally or even more important, Ca2+-dependent activation of CaM. Ca2+- activated CaM decreased after cell divi-sion. The results suggested that CaM gene expression and Ca2+-modulated CaM activation act synergistically to accomplish the cell cycle progression.

THE PRELIMINARY STUDY ON HMBA-INDUCED DIFFERENTIATION OF THE DRUG-PRETREATED MURINE ERYTHROLEUKEMIA CELLS

Several drug-resistant variants have been developed by growing the parental MEL cells in presence of colchicine, adriamycin and vincristine respectively with stepwise increasing concentration. Both the colchicine-resistant Sc9(ColO) and vincristine-resis-tant Sc9(VCR5) cells displayed an accelerated HMBA-induced commitment to terminal cell differentiation, whereas the adriamycin-resistant SC9 (A 120) showed no acceleration but rather a substantial delay in HMBA-induced differentiation. The studies provide more clues as well as experimental models for further study on the mechanism of induced differentiation of MEL cells.

Protein kinase clk/STY is differentially regulated during erythroleukemia cell differentiation: a bias toward the skipped splice variant characterizes postcommitment stages

Clk/STY is a LAMMER protein kinase capable to phosphorylate serine/arginine-rich (SR) proteins that modulate pre-mRNA splicing. Clk/STY alternative splicing generates transcripts encoding a full-length kinase and a truncated catalyti-cally inactive protein. Here we showed that clk/STY, as well as other members of the family (e.g. clk2, clk3 and clk4),are up-regulated during HMBA-induced erythroleukemia cell differentiation. mRNAs coding for the full-length and thetruncated forms were responsible for the overall increased expression. In clk/STY, however, a switch was observed forthe ratio of the two alternative spliced products. In undifferentiated cells the full-length transcript was more abundantwhereas the transcript encoding for the truncated form predominated at latter stages of differentiation. Surprisingly,overexpression of clk/STY did not alter the splicing switch upon differentiation in MEL cells. These results suggest thatclk/STY might contribute to control erythroid differentiation by a mechanism that implicates a balance between thesetwo isoforms.

Effects of TFAR19 gene on the in vivo biorheological properties and pathogenicity of mouse erythroleukemia cell line MEL

After injecting VP16, MEL cells and MEL-TF19 cells into the body of mice, with those injected with the same dose of saline as the control group, we observed the mice for their blood pictures, histological changes of the liver and spleen, and the hemorhelogical indexes within 4 weeks. The results indicated that after injecting MEL cells, the mice entered into a pathological status similar to erythroleukemia, which had the following exhibitions: the tissue structures of the liver and spleen were damaged, a mass of proerythroblasts, basophil erythroblasts and polychromatophilic erythroblasts could be observed on the smears of the bone marrow and spleen, and the deformability and orientation ability of erythrocytes were both depressed. The pathogenicity of MEL-TF19 cells carrying TFAR19 gene was obviously lower than that of MEL cells, and the MEL-TF19 cells even lost their faintish pathogenicity under the apop-tosis-inducing effect of the chemotherapeutic reagent. The outcome from the animal experiments suggests that the TFAR19 gene suppresses the pathogenicity of MEL cells to the mice, and the effect may be better exerted with the synergy of the chemotherapeutic reagent.

Effect of Sargassum fusiforme polysaccharide on apoptosis and its possible mechanism in human erythroleukemia cells

This study aimed to investigate the effects of Sargassum fusiforme polysaccharide(SFPS I,II,and III)on the apoptosis and regulation of human erythroleukemia(HEL)cells.The effect of different doses of SFPS on HEL cell growth was detected using the Cell Counting Kit-8 method,and apoptosis was detected by Hoechst staining.Cell cycle distribution and apoptosis were detected using flow cytometry.Expression of the cell cycle gene,p53,antiapoptotic genes,Bcl-xL and Bcl-2,and pro-apoptotic genes,Bax,Bad,and Caspase-3,as well as the expression of the corresponding proteins,were detected using real-time quantitative polymerase chain reaction(qPCR)and Western blot.The results showed that SFPS Ⅱ and Ⅲ decreased HEL cell viability and induced HEL cell apoptosis.Different concentrations of SFPS(Ⅰ,Ⅱ,and Ⅲ)were detected that induced much less toxic effect in normal human embryonic lung(MRC-5)cells,and SFPS Ⅰ increased cell proliferation,indicating its favorable selectivity towards cancer cells.The mechanism by which SFPS induced apoptosis was also found to be related to the induction of cell cycle arrest in the G0/G1 phase and the increased expression of apoptosis-related genes and proteins.We concluded that SFPS induces HEL cell apoptosis,possibly via activation of the Caspase pathway,providing the theoretical basis for the development of SFPS-based anti-tumor drug products.

芦可替尼对人红白血病细胞免疫检查点分子表达的影响

目的:芦可替尼通过抑制酪氨酸蛋白激酶2(tyrosine protein kinase 2,JAK2)通路来抑制人红白血病(human erythroleukemia,HEL)细胞的血管新生及迁移能力,而HEL细胞中程序性死亡受体1(programmed death-1,PD-1)、程序性死亡受体配体1(programmed death-ligand 1,PD-L1)的表达及芦可替尼对其表达的影响尚无相关报道。本研究探讨芦可替尼对JAK2 V617F突变阳性HEL细胞增殖、凋亡以及免疫检查点分子PD-1、PD-L1表达的影响。方法:用不同浓度的芦可替尼处理HEL细胞,细胞增殖检测试剂盒(cell counting kit-8,CCK-8)检测细胞活力,流式细胞术检测HEL细胞凋亡率,荧光定量PCR检测JAK2、PD-1及PD-L1 mRNA表达水平,蛋白质印迹法检测p-JAK2、PD-1、PD-L1蛋白质表达水平。流式细胞术分析淋巴细胞与HEL细胞共培养48 h后HEL细胞PD-1、PD-L1的表达和淋巴细胞PD-1的表达及调节性T细胞比例。结果:不同浓度芦可替尼能够抑制HEL细胞增殖,并与时间和剂量呈正相关。芦可替尼能够降低JAK2、PD-1、PD-L1 mRNA及蛋白质表达水平,并与时间和剂量呈正相关。结论:芦可替尼可能通过特异性抑制JAK2信号通路降低HEL细胞PD-1、PD-L1的表达。

绞股蓝总皂甙对小鼠S_(180)肉瘤及K_(562)细胞的抑制作用

目的 绞股蓝总皂甙 (GP)对小鼠S180 肉瘤及K562 细胞的抑制作用。方法 通过动物实验观察绞股蓝总皂甙对小鼠S180 肉瘤生长状况、肿瘤坏死面积 (TNA)与肿瘤总面积 (TTA)的比率、瘤周瘤内免疫活性细胞浸润状况及荷瘤小鼠脾脏的影响 ;通过细胞培养观察绞股蓝总皂甙对K562 细胞生长的抑制作用。结果 经重复实验证实 ,GP能显著抑制小鼠S180 肉瘤的生长 ,TNA与TTA的比率显著增加 ,瘤周尤其是瘤内淋巴细胞、巨噬细胞浸润数量明显增加 ,荷瘤小鼠脾重增加、脾白髓数目增多、体积增大。同时证实 ,GP对K562 细胞株具有明显的生长抑制作用。结论 GP的抑瘤作用主要是直接杀伤瘤细胞 。

PIC-BE诱导K562/ADM细胞凋亡及逆转其MDR的研究

β榄香烯吗素（ＰＩＣ－ＢＥ）是抗癌新药β榄香烯的水溶性衍生物．采用人红白血病的多药耐药性（ＭＤＲ）细胞株Ｋ５６２／ＡＤＭ作为实验模型，观察ＰＩＣ－ＢＥ对Ｋ５６２／ＡＤＭ细胞的生长抑制和凋亡诱导作用，并进而研究其对该细胞ＭＤＲ的可能影响．结果显示：（１）Ｋ５６２／ＡＤＭ细胞对ＡＤＭ具有明显的抗性，与Ｋ５６２细胞相比，抗性倍数约为４０倍，而两者对ＰＩＣ－ＢＥ的ＩＣ５０接近，无显著差异；（２）ＰＩＣ－ＢＥ（１０．０～３０．０μｇ／ｍｌ）对Ｋ５６２／ＡＤＭ细胞具有明显的生长抑制和凋亡诱导作用，两种作用的强度在一定的范围内均具药物浓度和作用时间依赖性；（３）低毒剂量ＰＩＣ－ＢＥ（１０．０μｇ／ｍｌ）与ＡＤＭ（４．０μｇ／ｍｌ）联合应用，可显著增强ＡＤＭ对该细胞的生长抑制和凋亡诱导作用，升高细胞内ＡＤＭ的浓度，降低该细胞对ＡＤＭ的ＩＣ５０，使该细胞对ＡＤＭ的抗性有数倍逆转．上述结果提示，ＰＩＣ－ＢＥ不仅是一种有效的广谱抗肿瘤剂。

β-榄香烯吗素抗肿瘤作用的实验研究

β-榄香烯吗素 (PIC- BE)是抗癌新药β-榄香烯的水溶性衍生物 ,本文观察了 PIC- BE对多药耐药 K5 6 2 / ADM细胞系及其敏感细胞 K5 6 2 的生长抑制和凋亡诱导作用。结果显示 ,(1 ) K5 6 2 / ADM细胞对 ADM具有明显的抗性 ,与 K5 6 2 细胞相比 ,抗性倍数约为 40倍 ,而两者对 PIC- BE的 IC5 0 接近 ,无显著差异 ;(2 ) PIC- BE (1 0 .0 - 30 .0μg/ ml)对 K5 6 2 和 K5 6 2 / ADM细胞不仅具有明显的生长抑制作用 ,而且显著地诱导细胞凋亡 ,其作用强度在一定的范围内呈相对浓度和时间的依赖性。以上结果提示 ,PIC- BE是一种有效的抗肿瘤化合物 ,且已产生 MDR的 K5 6 2 / ADM细胞对它不具耐药性。

蝌蚪提取液对小鼠红白血病细胞分化的诱导作用

蝌蚪提取液（Ｔ－８７１）与ＤＭＥＭ培养基按１：５０（ｖ／ｖ）混合制成Ｔ－８７１培养基。作为研究分化模型的小鼠红白血病细胞（ＭＥＬ）培养于Ｔ－８７１培养基中。结果发现Ｔ－８７１诱导ＭＥＬ发生终末分化，即在其胞质中有血红蛋白合成。用流式细胞仪（ＦＡＣＳ－４２０）分析经Ｔ－８７１培养的ＭＥＬ，证明其Ｐ（５３）蛋白含量降低。由于Ｐ（５３）蛋白是细胞由Ｇ１期转变为Ｓ期所必需的，所以本研究认为ＭＥＬ中Ｐ（５３）蛋白含量降低可能导致细胞周期中Ｇ０＋Ｇ１期细胞的积累和Ｇ２＋Ｍ期细胞的减少。

三氧化二砷对K_(562)细胞凋亡的诱导及生长抑制作用的研究

目的 :研究三氧化二砷 ( As2 O3)对人红白血病细胞株 K56 2 的生长抑制和凋亡诱导作用。方法 :以As2 O3作为耐药逆转剂 ,用台盼兰排染法 ,噻唑兰 ( MTT)还原法 ,Hoechst33342和 PI荧光染色法 ,流式细胞仪技术和荧光分光光度法 ,观察了不同浓度的 As2 O3( 0 .2～ 5 .0 μm ol/L)对人红白血病细胞株 K56 2 的生长抑制和凋亡诱导作用。结果 :As2 O3对 K56 2 细胞具有明显生长抑制和凋亡诱导作用 ,其作用强度在一定范围内均具药物浓度和时间依赖性。结论 :As2 O3主要以诱导肿瘤细胞凋亡而表现其毒性作用。

FTIR对TFAR19基因促红白血病MEL细胞凋亡作用的研究

采用 FTIR方法 ,研究了 TFAR19基因转染前后 ,小鼠红白血病 MEL 细胞内重要组分的变化。经脂质体介导基因转染 ,获得了稳定携带 TFAR19基因的 MEL- TF19细胞 ,RT- PCR检测表明 ,该基因在 m RNA水平有表达 ;撤除血清诱导凋亡 ,在此过程中进行了 FTIR检测 ,结果显示 :转染 TFAR19基因后 ,细胞内蛋白质相对核酸的含量增高 ,细胞转录活性增强 ,细胞内磷脂的相对含量降低。所有这些变化都反映了 TFAR19基因的促凋亡作用 。

用单倍体相合小鼠FBL-3红白血病细胞株移植建立CB6F1小鼠红白血病模型

本研究探讨建立F1代单倍体相合小鼠FBL-3红白血病模型的可行性,并观察FBL-3细胞在小鼠体内的生物学特性。将FBL-3H2-d小鼠红白血病细胞经尾静脉分别接种给小鼠C57BL/6和CB6F1H-2b/d(C57BL/6×BALB/c),观察两种小鼠的生存时间和染色体变化,并对濒死小鼠的肝、脾、肺和肾进行病理检查,部分进行电子显微镜检查。分析骨髓和脾脏细胞染色体核型及MHC分子表达情况。结果表明:静脉接种103-107个白血病细胞的情况下,C57BL/6小鼠和CB6F1小鼠发病率分别为100%和92.5%;接种的细胞数量与存活时间呈线性关系;接种相同数量FBL-3细胞的CB6F1小鼠平均生存时间较C57BL/6小鼠延长。白血病细胞主要侵及肝、脾、骨髓、肺和肾组织,糖原染色阳性、氯醋酸染色部分阳性,过氧化物酶、碱性磷酸酶、丁酸染色均为阴性。电子显微镜观察到细胞内病毒样颗粒。脾脏和骨髓细胞染色体多数为非二倍体,且H-2b表达率升高,H-2d表达率降低。结论:经尾静脉接种FBL-3细胞可以建立CB6F1小鼠红白血病模型。

黑木耳多糖对人红白血病K562细胞周期和凋亡的影响

目的:观察黑木耳多糖(Auricularia auricula polysaccharide,AAP)体外对人红白血病K562细胞的抗肿瘤作用及其机制。方法:将K562细胞与AAP以及AAP作用于体外培养人外周血单个核细胞(PBMCs)的上清液培养,用MTT法检测K562细胞增殖和流式细胞仪检测细胞周期和凋亡情况。结果:AAP对K562细胞没有显示直接的细胞毒作用,但AAP作用的PBMC上清液能显著抑制K562细胞的增殖,抑制率最大可达49.68%;AAP作用的PBMCs上清液能使G0/G1期细胞显著增加(P<0.05,P<0.01);200μg/mL到800μg/mL浓度AAP刺激的PBMCs上清液能显著诱导K562细胞凋亡(P<0.05,P<0.01),其凋亡率可达24.70%。结论:AAP通过激活免疫细胞抑制肿瘤细胞生长,诱导其凋亡并使细胞阻滞于G0/G1期。

二甲基亚砜诱导小鼠红白血病细胞分化的差异蛋白质组学

目的利用二甲基亚砜(DMSO)诱导小鼠红白血病(MEL)细胞分化,系统地分析和鉴定诱导分化不同时间的差异蛋白质组学。方法 DMSO诱导MEL细胞分化,通过联苯胺染色、MTT比色法和Ter119免疫荧光等实验检测细胞分化的比率和细胞的活力,并利用双向电泳耦联质谱技术,结合生物信息学分析诱导MEL细胞分化过程中差异表达的蛋白质。结果 1.2%DMSO诱导MEL细胞分化,分别培养0h、6h、12h、24h、36h、48h、72h、96h、120h,利用双向电泳和质谱分析,共鉴定出87种蛋白质。1.2%DMSO作用MEL细胞120h后,细胞诱导分化率达到67%。MTT实验显示,1.0%、1.2%、1.4%的DMSO对MEL细胞的活力无明显的抑制作用。结论 DMSO对MEL细胞有诱导分化作用,但无明显地抑制细胞增殖。87种双向电泳差异蛋白质按功能可分为12类,比例最大的前3类分别是:占41%的酶类蛋白、15%的结构蛋白、13%的调节蛋白。

MTT法测定稀土离子对BEL-7402及K562细胞的毒性及增殖毒性

用MTT法测定稀土离子在不同浓度、不同培养液中,与BEL 7402和K562细胞作用不同时间,对细胞的毒性和增殖毒性。结果表明,在含10%小牛血清培养液中,仅个别稀土离子在较高浓度时对BEL 7402细胞增殖有较弱的抑制作用;对于K562细胞,稀土离子在低浓度时对细胞增殖即表现出较强的抑制作用(P<0.05)。当培养液不含小牛血清时,较低浓度的稀土离子即可抑制BEL 7402细胞的增殖(P<0 05)。

益气养阴方对白血病细胞Caspase-3及Bcl-2表达的影响

目的 研究益气养阴方对白血病细胞Caspase-3及Bcl-2蛋白表达的影响,探讨复方中药诱导白血病细胞凋亡的机制。方法 给EL9611红白血病小鼠灌服益气养阴方水煎剂13 d后处死,取股骨骨髓细胞,用免疫组化法测细胞Caspase-3及Bcl-2蛋白表达。结果 益气养阴方组与对照组比较,Caspase-3表达阳性率增加(P<0.01);Bcl-2表达积分下降(P<0.01)。结论 益气养阴方上调白血病细胞Caspase-3表达、下调Bcl-2表达是其诱导白血病细胞凋亡的重要途径。

髓清丸对HEL细胞系作用的血清药理学研究

目的 :研究中药复方髓清丸对白血病细胞的作用。方法 :采用血清药理学方法体外培养观察人红白血病细胞系 (HEL)的增殖、凋亡、细胞周期、细胞形态等细胞学指标。结果 :活细胞百分率各含药血清组均较对照组低 ,MTT法测定髓清丸中剂量组和高剂量组OD值明显低于对照组 (p<0 0 1) ,中剂量组和高剂量组细胞毒活性均大于 2 0 %,高剂量含药血清组G1期DNA含量明显低于对照组 (P <0 0 5 ) ,中剂量组凋亡细胞百分率较高 (P <0 0 5 )。结论 :髓清丸能抑制HEL细胞的增殖 ,并有一定的诱导HEL细胞分化、凋亡的作用。

COX-2抑制剂对人红白血病HEL细胞增殖、凋亡及迁移的影响

目的 :探讨环氧化酶(cyclooxygenase-2,COX-2)抑制剂塞来昔布对人红白血病HEL细胞增殖、凋亡及迁移的影响及其作用机制。方法:不同浓度的塞来昔布处理HEL细胞,CCK-8法检测细胞增殖抑制率;Hoechst33342荧光染色检测细胞凋亡;Transwell小室检测细胞迁移率;RT-PCR检测COX-2及JAK2 mRNA水平;Western blot检测COX-2及p-JAK2蛋白表达。结果:塞来昔布能够时间和剂量依赖性抑制HEL细胞增殖,不同浓度(25、75、125μmol/L)的塞来昔布在48 h时对细胞生长抑制率分别为(9.96±0.82)%、(18.46±2.01)%、(21.36±2.48)%(P<0.05);Hoechst33342凋亡细胞染色显示125μmol/L塞来昔布处理HEL细胞后,凋亡细胞明显增多;细胞迁移实验结果显示75μmol/L塞来昔布处理细胞24 h后漏出细胞为(22.13±7.51)个,明显低于对照组(77.89±6.94)个,P<0.05;RT-PCR结果显示不同浓度塞来昔布处理HEL细胞48 h后COX-2 mRNA呈剂量依赖性减低,而对JAK2 m RNA无明显影响;Western blot结果显示塞来昔布处理HEL细胞COX-2蛋白表达明显减低(P<0.05),而对p-JAK2无明显影响。结论:塞来昔布能够抑制HEL细胞增殖,可能与抑制COX-2表达有关,而对JAK2信号通路无明显影响。