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.

Role of calcium in differentiation of murine erythroleukemia cells

Calcium plays a crucial role in the normal and abnomal 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[Ca^2+]i concentration and DNA contents simultaneously,by employing the fluorescent probe,fluo-3 acetoxymethyl ester and DNA dye Hoechst 33342.During MELC differentiation.[Ca^2+]i concentration incresed.We also demonstrated that calcium ionophore,A23187,enhanced the HMB-induced MELC differentiation,while verapamil,an inhibitor of calcuim uptake,slightly reduced differentiation.These results suggested that an increase in the [Ca^2+]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 Ca^2+-activated CaM contents. Intensity of CaM immunoflurescence gave total CaM level,and Ca^2+-activated CaM was measured by fluorescence intensity of CaM antagonist trifluoperazine (TFP).In mouse erythroleukemia (MEL) cells,total CaM level increased from G1 through S to G2M,reaching a maximum of 2-fold increase,then reduced to half amount after cell division.Meanwhile,Ca^2+-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,Ca^2+-dependent activation of CaM.Ca^2+-activated CaM decreased after cell division.The results suggested that CaM gene expression and C^2+-modulated CaM activation act synergistically to accomplish the cell cycle progression.

DIFFERENTIATION AND MALIGNANT SUPPRESSION INDUCED BY MOUSE ERYTHROID DIFFERENTIATION AND DENUCLEATION FACTOR ON MOUSE ERYTHROLEUKEMIA CELLS

Objective.To investigate the roles of mouse erythroid differentiation and denucleation factor(MEDDF),a novel factor cloned in our laboratory recently ,in erythroid terminal differentiation.Methods.Mouse erythroleukemia(MEL) cells were transfected with eukaryotic expression plasmid pcDNA-MEDDF.Then we investigated the changes on characteristics of cell growth by analyzing cells growth rate,mitotic index and colony-forming rate in semi-soid medium.The expressions of c-myc and β-globin genes were analysed by semi-quantitative RT-PCR。Results.MEL cells transfected with pcDNA-MEDDF showed significant lower growth rate,mitotic index,and colony-forming rate in semi-solid medium(P<0.01).The percentage of benzidine-positive cells was 32.8% after transfection.The expression of β-globin in cells transfected with pcDNA-MEDDF was 3.43 times higher than that of control(MEL transfected with blank vector,pcDNA3.1) ,and the expression of c-myc decreased by 66.3%.Conclusions.MEDDF can induce differentiation of MEL cell and suppress its malignancy.

Vagal-mAChR4 signaling promotes Friend virus complex(FV)-induced acute erythroleukemia

Erythroleukemia belongs to acute myeloid leukemia(AML)type 6(M6),and treatment remains difficult due to the poor prognosis of the disease.Friend virus(FV)is a complex of two viruses:Friend murine leukemia virus(F-MuLV)strain along with a defective spleen focus-forming virus(SFFV),which can induce acute eryth-roleukemia in mice.We have previously reported that activation of vagalα7 nicotinic acetylcholine receptor(nAChR)signaling promotes HIV-1 transcription.Whether vagal muscarinic signaling mediates FV-induced erythroleukemia and the underlying mechanisms remain unclear.In this study,sham and vagotomized mice were intraperitoneally injected with FV.FV infection caused anemia in sham mice,and vagotomy reversed this change.FV infection increased erythroblasts ProE,EryA,and EryB cells in the spleen,and these changes were blocked by vagotomy.In bone marrow,FV infection reduced EryC cells in sham mice,an effect that was coun-teracted by vagotomy.FV infection increased choline acetyltransferase(ChAT)expression in splenic CD4^(+)and CD8þT cells,and this change was reversed by vagotomy.Furthermore,the increase of EryA and EryB cells in spleen of FV-infected wild-type mice was reversed after deletion of ChAT in CD4^(+)T cells.In bone marrow,FV infection reduced EryB and EryC cells in sham mice,whereas lack of ChAT in CD4^(+)T cells did not affect this change.Activation of muscarinic acetylcholine receptor 4(mAChR4)by clozapine N-oxide(CNO)significantly increased EryB in the spleen but decreased the EryC cell population in the bone marrow of FV-infected mice.Thus,vagal-mAChR4 signaling in the spleen and bone marrow synergistically promotes the pathogenesis of acute erythroleukemia.We uncover an unrecognized mechanism of neuromodulation in erythroleukemia.

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.

Transcriptomic profile of human erythroleukemia cells in response to Sargassum fusiforme polysaccharide and its structure analysis

Sargassum fusiforme(S.fusiforme)has been used as an ingredient in Chinese herbal medicine for thousands of years.However,there are a limited number of studies concerning its therapeutic mechanism.High performance gel permeation chromatography(HPGPC)analysis showed that the average molecular weight of the S.fusiforme polysaccharide,SFPS 191212,is 43 kDa.SFPS 191212 is composed of mannose,rhamnose,galactose,xylose,glucose,and fucose(at a molar ratio:2.1:2.9:1.8:15.5:4.6:62.5)withα-andβ-configurations.The present research evaluated the anti-tumor potential of the S.fusiforme polysaccharide in human erythroleukemia(HEL)cells in vitro.To explore the SFPS 191212’s apoptosis mechanism in HEL cells,transcriptome analysis was performed on HEL cells that were incubated with SFPS 191212.The inhibitory effect of SFPS 191212 on HEL cell growth was also analyzed.It was found that SFPS 191212 inhibited HEL cell proliferation,reduced cell viability in a concentration-dependent manner,and induced an insignificant toxic effect on normal human embryonic lung(MRC-5)cells.Compared with the control group,transcriptome analysis identified a total of 598 differentially expressed genes(DEGs),including 243 up-regulated genes and 355 downregulated genes.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were performed on all DEGs,and 900 GO terms and 52 pathways were found to be significantly enriched.Finally,23 DEGs were randomly selected and confirmed by quantitative real-time polymerase chain reaction(qRT-PCR).Moreover,SFPS 191212 down-regulated the PI3K/Akt signal transduction pathway.Our results provide a framework for understanding the effect of SFPS 191212 on cancer cells and can serve as a resource for delineating the anti-tumor mechanisms of S.fusiforme.

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.

MALDI-TOF-TOF鉴定IPG IEF分离的蛋白质

K562细胞是一株分化差、恶性程度高的人白血病细胞.研究表明,在一些分化诱导剂的作用下,细胞可以向红细胞系、粒细胞系和巨K562核细胞系方向分化成熟,并表现出相应血细胞类型的成熟标志.用一维固相pH梯度等电聚焦电泳(LPG IEF)分离K562细胞总蛋白质,其每一条带常包含多个蛋白质,难以用肽指纹谱技术来鉴定.……

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 。

雷公藤内酯醇对红白血病K562细胞株增殖和凋亡的影响

目的探讨雷公藤内酯醇对人类红白血病K562细胞株增殖和凋亡的影响。方法运用四甲基偶氮唑盐(MTT)法、流式细胞术(FCM)等检测雷公藤内酯醇对K562细胞株增殖和凋亡的影响。结果雷公藤内酯醇能明显抑制K562细胞的增殖,呈时间与剂量依赖性,作用24h时其半数抑制浓度IC50为25nmol/L。且经雷公藤内酯醇处理后K562细胞G1/S期所占的百分比较对照组上升(P<0.05)。雷公藤内酯醇作用48h,其浓度的变化与K562细胞凋亡率具有相关性(r=0.97,P<0.05),而作用24h时,则无明显诱导凋亡的作用。结论雷公藤内酯醇有明确的抗白血病细胞增殖的能力,干扰K562细胞周期,上调细胞G/S期检测点,并具有促进细胞凋亡的作用。

骨髓增生异常综合征大鼠模型的建立及其鉴定

用化学诱变剂二甲基苯蒽(DMBA)诱发大鼠(TR_1)骨髓病变。以35mg/kgDMBA给出生30—45天的大鼠尾静脉注射,分4组,每周分别给药1—4次,其中连续给药2—4次组均可发生明显的骨髓红系和巨核系病态造血,并可进一步发展为白血病,且绝大多数为红白血病。实验结果证明,大鼠2—4次给药后3个月内骨髓象和血象变化与人骨髓增生异常综合征(MDS)相似,其严重程度与DMBA的剂量积累相关,即1次给药组13只大鼠均不发生MDS;2次给药组2/6发生MDS,其中1只发展为白血病;3次给药组9/14发生MDS,其中1只发展为白血病;4次给药组15/16发生MDS,其中8只发展为白血病。3—4次给药组MDS病期为3—4个月,足以满足MDS实验研究。应用v-erbB探针和Southern印迹杂交法,结果显示大鼠MDS及其发展的红白血病与人MDS、红白血病、白血病及恶性肿瘤有相同的c-erbB基因重排和扩增,表明此大鼠MDS模型适合于研究人MDS发病机理与人MDS基因治疗的动物模型。

红葱抗稻瘟霉活性成分研究

目的阐明红葱抗稻瘟霉活性的物质基础。方法利用稻瘟霉筛选体系对红葱的60%(φ)乙醇提取物及其氯仿、正丁醇和水的萃取部分进行活性筛选,并对活性部位的化学成分进行分离鉴定。结果与结论从活性部位中分离得到10个化合物,通过理化性质和波谱数据鉴定它们的结构分别为:异红葱甲素(1)、异红葱乙素(2)、红葱乙素(3)、β-谷甾醇(4)、8-羟基-3,4-二甲氧基-1甲-基蒽醌-2-羧酸甲酯(5)、4羟基红葱乙素(6)、hongconin(7)、4,8二羟基3甲氧基1甲基蒽醌2羧酸甲酯(8)、dihydroeleutherinol(9)、1,3,6-三羟基-8-甲基蒽醌(10)。其中,9和10为首次从该属植物中分得。化合物2、3、5、6、7、9、10具有较好的诱导稻瘟霉菌丝变形的活性,它们的最小变形浓度(minimummorphologicaldeformationconcentrate,MMDC)分别为30、30、170、130、55、60、150μmol·L-1。用MTT法测定10个化合物的抗肿瘤活性,发现化合物2、3、5、6、7、9有较强的抑制人类红白血病细胞株K562细胞生长的活性,它们的IC50值分别为33、49、266、35、174、154μmol·L-1。

红葱中的新化学成分

通过正相和反相硅胶柱色以及高效液相色谱,从红葱60%乙醇提取物中分离得到3个新化合物,利用物理化学和光谱学方法鉴定了其结构,分别为9,9′-二羟基-8,8′-二甲氧基-1,1′-二甲基-1H,1H-[′4,4′]双[萘并(2,3-c)呋喃]-3,3′-二酮(1),6,8-二羟基-3,4-二甲氧基-1-甲基-蒽醌-2-羧酸甲酯(2),2-乙酰基-3,6,8-三羟基-1-甲基蒽醌(3).这3个化合物均能不同程度地诱导稻瘟霉菌丝变形,其最小变形质量浓度(M in i-mum morphological deform ation m ass concentration,MMDC)分别为145.8,50.2和124.8μg/mL.利用MTT法测定了化合物抑制人类红白血细胞株K562细胞生长的活性,化合物2的IC50为49.1μg/mL.

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。

103例急性红白血病患者生物学特征与疗效观察

目的:本研究总结分析急性红白血病(AEL)的生物学特征与疗效。方法:回顾性分析2009年6月至2016年5月收治的103例急性红白血病患者肝功能、乳酸脱氢酶、凝血、形态学、免疫学、细胞遗传学及分子生物学特征,通过缓解率、复发率、无复发生存及总生存等指标观察疗效。结果:白细胞、粒细胞、血红蛋白及血小板中位数分别为3.04×10~9/L、0.67×10~9/L、66 g/L及45.0×10~9/L,71.1%的患者外周血涂片可以发现有核红细胞,无1例患者出现凝血功能异常,患者主要表达:CD13(93.5%)、CD117(89.1%)、HLA-DR(87.0%)、CD34(80.0%),部分患者表达淋系抗原CD4(42.9%)及CD7(28.9%)。82例患者核型分析显示,正常核型52.4%(43/82),异常核型41.5%(34/82),检查失败6.1%(5/82)。34例异常核型中,简单异常41.2%(14/34),复杂核型58.8%(20/34)。对60例患者检测融合基因显示,阳性率为16.7%(10/60)。27例患者检测预后基因突变显示,阳性率为77.8%(21/27)。初诊急性红白血病103例中接受化疗81例,可进行疗效分析66例。2个疗程累积缓解率45.5%(30/66),复发率36.7%(11/30),中位复发时间15.5(6.2-50)个月。可进行疗效分析66例患者中位生存时间为29个月。CR(30例)患者截至随访结束未达中位生存时间,明显优于未获CR(36例)患者12个月(P=0.001)。CR患者5年生存率65%,无复发生存(RFS)中位时间46.2个月,3年RFS为58%。结论:AEL患者具有高表达CD34抗原、多为复杂核型等生物学特点。虽然AEL患者缓解率低,生存期短,但缓解患者人群具有较好的长生存及良好的生存质量。

红白血病——骨髓增生异常综合征的一种亚型?

为了研究红白血病是否为急性髓细胞白血病一个独立亚型,从临床实验及病程进展方面对21例红白血病患者进行了分析。结果显示,诊断时患者合并白细胞减少、贫血和血小板减少者分别为42.9%、81%、81%。外周血分类显示85.7%患者有幼稚粒细胞和幼稚单核细胞,52.4%患者有幼稚红细胞和有核红细胞;骨髓涂片显示骨髓增生活跃或明显活跃;红系细胞占58.3±8.0%,原始粒细胞占NEC58.0±18.4%;66.7%患者有病态造血,与典型AML不同。在病程中52.4%患者发生疾病转型,转为RAEB/RAEB-T和AML-M2,19例在确诊M6后接受化疗,11例有效(57.9%),其中CR10例、PR1例,CR中位维持6个月、PR2个月,但CR患者绝大多数伴有骨髓病态造血,外周血细胞减少。中数生存期在初诊M6和MDS转化为M6组分别为13.0±13.3和2.3±1.3月。结论:临床诊断急性红白血病与典型AML有许多差异,可能大部分是以红系过度增生为表现的MDSRAEB和RAEB-T型。

Fr. MuLV小鼠红白血病模型的建立及叠氮胸苷抗病毒作用的研究

目的应用Friend鼠白血病病毒(Friend murine leuke-mia virus,Fr. MuLV)建立小鼠红白血病模型,并在此模型上观察抗逆转录病毒药物叠氮胸苷(Zidovudine,AZT)的治疗作用。方法用Fr.MuLV感染BALB/c小鼠。以RT-PCR法测定脾脏病毒载量;血常规分析白细胞(WBC)、血红蛋白(Hbg)及血小板(PLT)变化;镜检计数外周血及骨髓有核细胞。结果与正常对照组相比,模型组小鼠脾脏病毒载量、WBC和Hbg明显升高;外周血及骨髓中红系和非红系均出现大量原始和幼稚细胞,骨髓中非红系原始细胞(NEC)计数≥0.30,有核红细胞≥0.50;AZT治疗组上述指标明显改善。结论用Fr.MuLV感染BALB/c小鼠成功建立小鼠红白血病模型,AZT在此模型上显示出良好的抗病毒作用。