miR-382-5p通过靶基因PTEN阻滞NB4细胞分化

该文主要研究在急性早幼粒细胞白血病NB4细胞中, miR-382-5p通过调控靶基因PTEN(phosphatase and tensin homologue)抑制了全反式维甲酸(all-trans retinoic acid, ATRA)诱导的急性早幼粒细胞分化。我们运用ATRA(1μmol/L)诱导细胞分化; Western blot检测PTEN及髓系分化标志物CD11b的蛋白质水平;实时荧光定量PCR检测miR-382-5p的表达水平;过表达PTEN的慢病毒载体分别感染NB4细胞和HL-60、THP-1细胞;脂质体转染miR-382-5p的模拟剂(mimics)和特异性抑制剂(inhibitors)NB4细胞。结果显示, PTEN促进ATRA诱导的NB4细胞分化,而在HL-60和THP-1细胞中并无明显促分化效应。NB4细胞中,脂质体转染miR-382-5p mimics在mRNA和蛋白水平均抑制了PTEN的表达,并且抑制了ATRA诱导的分化;转染miR-382-5p inhibitors则恢复了PTEN表达,同时促进了ATRA诱导的急性早幼粒细胞NB4细胞的分化。该文结果提示, miR-382-5p靶向抑制了PTEN的表达从而抑制ATRA诱导的NB4细胞分化。     

EGCG通过PTEN增强ATRA对早幼粒白血病细胞株的分化作用

探讨PTEN在表没食子儿茶素没食子酸酯(EGCG)增强维甲酸(ATRA)诱导的急性早幼粒细胞白血病(APL)细胞分化中的作用。我们分别将ATRA、EGCG及两种药物联合应用于NB4与HL-60细胞72 h,从形态学上观察细胞核的变化;Western blotting检测PTEN及髓系分化标志CD11b的表达;免疫荧光检测PTEN的入核情况;CCK-8检测细胞增殖率。结果显示,ATRA与EGCG联合作用于NB4与HL-60细胞后,PTEN与CD11b的表达较单独应用ATRA时增加。PTEN特异性抑制剂SF1670作用于NB4后,细胞分化明显减少;PI3K抑制剂LY294002作用后,PTEN及CD11b表达增加。这些均表明EGCG能够增强ATRA诱导APL细胞株的分化作用,且这种促进作用与PTEN的增加是相关的。     

长链非编码RNA AK043773在脂肪细胞分化中的作用

探索长链非编码RNA(lnc RNA)AK043773在脂肪细胞分化过程中的表达变化和调控作用。将小鼠前脂肪细胞成脂诱导,利用实时定量PCR技术检测成脂分化调控基因KLF7及AK043773的表达,构建AK043773过表达载体检测其对成脂分化的影响。经UCSC数据库检索发现AK043773位于KLF7基因4号外显子的3'端下游,骨髓基质细胞系ST2和前脂肪细胞系3T3-L1成脂诱导分化后,实时定量PCR检测结果显示AK043773及KLF7表达协同下调;构建p CDNA3.1-AK043773过表达载体,转染ST2细胞后能显著提高AK043773的表达,进行脂肪细胞分化条件诱导,通过油红O染色及OD520nm吸光值检测可见AK043773过表达显著抑制ST2细胞中脂滴产生。AK043773及KLF7在脂肪细胞分化过程中协同下调,上调AK043773的表达可显著抑制脂肪细胞分化。     

CD11b/DNA双参数流式细胞术检测HL-60细胞分化的细胞周期

目的采用双参数流式细胞术研究全反式维甲酸(alltransretinoidacid,ATRA)诱导人类急性早幼粒白血病细胞HL-60细胞分化的细胞周期。方法HL-60细胞经分化诱导剂ATRA(终浓度为1μmol/L)诱导不同时间点后,利用CD11b/DNA双参数流式细胞术同时检测分化细胞表面抗原CD11b的表达及分化细胞DNA含量。结果HL-60细胞经ATRA诱导后,细胞表面分化抗原CD11b表达明显升高,细胞阻滞于G0/G1期,且CD11b阳性细胞主要位于G0/G1期。结论CD11b/DNA双参数流式细胞术能简便,快速,直观地检测细胞分化的细胞周期。     

转谷氨酰胺酶2通过mTOR通路和自噬调控全反式维甲酸诱导的白血病细胞HL60和U937的髓系分化

全反式维甲酸(ATRA)是具有融合基因PML-RARα的急性早幼粒细胞白血病(APL)特异的靶向治疗药物。此外，ATRA在无PML-RARα融合的急性髓系白血病及其它一些肿瘤中也有一定治疗效果。但ATRA治疗也会引起一些并发症或发生愈后复发。因此，对ATRA诱导分化调控机制的研究非常重要。转谷氨酰胺酶2(TGM2)是一种多功能酶，能调控mTOR信号通路和自噬等。ATRA能诱导APL细胞中TGM2表达上调，TGM2敲低抑制ATRA诱导的细胞分化。但其调控机制及涉及的信号通路尚不明确。本研究发现，在HL60和U937细胞中，ATRA能够上调CD11b和TGM2的表达(P<0.05),抑制mTOR信号通路，并增强自噬；与对照相比，敲低TGM2,mTOR信号通路增强，自噬被抑制，而ATRA诱导的CD11b表达被抑制(P<0.05),分化减弱，被ATRA抑制的mTOR信号通路得到部分恢复，而被ATRA增强的自噬适当减弱。这表明ATRA使HL60和U937细胞发生髓系分化，并诱导TGM2表达升高；而TGM2通过mTOR信号通路和自噬途径调控ATRA诱导的髓系分化。该研究将有利于更深入地...     

MiR-15b促进急性早幼粒细胞白血病细胞分化并抑制增殖

该文探讨了miR-15b在全反式维甲酸(all-trans retinoic acid, ATRA)治疗急性早幼粒细胞白血病(acute promyelocytic leukemia, APL)过程中发挥的作用。采用实时荧光定量PCR检测miR-15b的表达;流式细胞术检测细胞的分化情况; CCK-8实验检测细胞增殖;双荧光素酶报告实验检测miR-15b与CCNE1 3′UTR端的结合能力; Western blot检测下游靶基因CCNE1的表达。结果显示,ATRA促进miR-15b的表达;过表达miR-15b增强了ATRA对APL细胞的分化作用,而抑制miR-15b表达后则出现相反结果; miR-15b抑制了APL细胞的增殖能力;双荧光素酶报告实验显示miR-15b与CCNE1的3′UTR端结合; Western blot显示mi R-15b可以抑制下游靶基因CCNE1的表达。这些结果表明, miR-15b通过抑制CCNE1的表达促进APL细胞分化,抑制细胞增殖。     

敲低ACTL6A通过Notch1信号通路促进早幼粒细胞分化

探讨ACTL6A在人类白血病NB4细胞分化中的作用及其相关机制。我们用ATRA人为诱导NB4细胞分化,Western blotting检测ACTL6A和CD11b的表达水平变化;敲低ACTL6A,通过瑞氏染色观察NB4细胞的形态学改变,Western blotting检测ACTL6A和CD11b的表达水平变化及其相关蛋白的表达水平;敲低同时用ATRA处理NB4细胞,用流式细胞术检测分化标志物CD11b的阳性率;免疫荧光检测ACTL6A在NB4细胞中的空间定位;结果显示NB4经敲低ACTL6A后,CD11b的蛋白水平表达升高;瑞氏染色观察到分化改变;免疫荧光检测到ACTL6A主要分布于细胞核; Western blotting检测到Notch1,Hes1,Sox2蛋白表达水平明显下调。研究表明,敲低ACTL6A可以促进人类白血病NB4细胞分化;其机制涉及Notch1信号通路的抑制。     

TGFβ1参与视黄酸诱导的HL-60细胞分化

为研究内源性TGF-β１对全反式视黄酸（ATRA）作用HL60细胞的影响,应用定量RT-PCR和ELISA方法,研究ATRA诱导HL-60细胞分化过程中TGF-β１ 表达的变化.并构建TGF-β１ RNA干扰表达质粒,抑制HL-60细胞内源性TGF-β１表达,进而研究ATRA诱导内源性TGF-β１表达下降的HL-60细胞分化的情况．结果发现,ATRA诱导HL-60细胞分化过程中,TGF-β１ 表达明显增高．得到4个针对不同靶位点的RNA干扰表达质粒.其中,针对起始编码区的质粒转染48 h后对HL-60细胞的TGF-β１蛋白抑制率为73~2％．内源性TGF-β１表达下降后,ATRA作用的HL-60细胞NBT还原试验的光密度值降低,CD33抗原阳性的细胞比例较对照组升高,CD11b抗原阳性的细胞比例较对照组降低.表明内源性TGF-β１表达下降后,ATRA诱导HL-60细胞分化的作用有所减弱,提示内源性TGF-β１在ATRA诱导HL-60细胞分化中起一定的作用．     

核仁磷酸蛋白基因突变对K562白血病细胞分化的影响

核仁磷酸蛋白基因（nucleophosmin,NPM1）突变在急性髓系白血病的发生发展中发挥着重要作用,而与白血病分化阻滞的关系尚未完全阐明。为探讨NPM1基因突变对白血病细胞体外分化的影响,将携带NPM1 A型突变（NPM1-mA）的表达质粒载体pEGFPC1-NPM1-mA转染白血病K562细胞系,构建稳定表达NPM1-mA蛋白的细胞株（K562 mA）,同时设立野生型NPM1转染组（K562 wt）、空载体转染组（K562 C1）和未处理组（K562）为对照。利用豆蔻酰佛波醇乙酯（PMA）诱导各组细胞分化,瑞氏–吉姆萨染色观察细胞分化的形态改变,计算诱导分化率;相差显微镜计数贴壁细胞数量;流式细胞术分析细胞表面分化抗原CD41的表达。结果显示,PMA作用72 h后,与对照组相比,K562 mA组细胞的诱导分化率及贴壁细胞数明显降低（P〈0.05）;同时,CD41的表达受到显著抑制（P〈0.01）。提示NPM1基因突变能够阻滞白血病细胞系K562的体外分化。     

NLS-RARα对人白血病细胞NB4分化抑制及其机制的研究

该文旨在探讨带核定位信号的维甲酸受体α(nuclear localization signal retinoic acid receptor alpha,NLS-RARα)对人急性早幼粒白血病(acute promyelocytic leukemia,APL)细胞株NB4分化的影响及其机制。免疫印迹实验检测全反式维甲酸(all-trans retinoic acid,ATRA)诱导的NB4细胞分化标志物C/EBPβ、CD11b和p38α蛋白质水平;利用慢病毒介导的NLS-RARα基因过表达,进一步用免疫印迹实验验证过表达效率并检测NLS-RARα对NB4细胞分化标志物C/EBPβ、CD11b和p38α蛋白质水平的影响;间接免疫荧光实验分析NLS-RARα与p38α的空间共定位;免疫共沉淀实验分析NLS-RARα与p38α的相互作用。结果显示,生理浓度和药理浓度的ATRA促进NB4细胞分化的同时也激活了p38α,且p38α的活性变化与髓系分化标志物C/EBPβ变化一致;髓系分化表面标志物CD11b表达量在药理浓度ATRA(1μmol/L)处理下达到最高;NLS-RARα抑制NB4细胞的分化,且只有在ATRA存在的条件下,NLS-RARα抑制NB4细胞的分化与下调p38α活性相关;NLSRARα与p38α存在空间共定位且NLS-RARα与p38α直接相互作用。该研究结果提示,当存在ATRA诱导时,NLS-RARα与p38α直接相互作用后下调p38α的活性进而抑制NB4细胞的分化。     

On the origin of the Hirudinea and the demise of the Oligochaeta

The phylogenetic relationships of the Clitellata were investigated with a data set of published and new complete 18S rRNA gene sequences of 51 species representing 41 families. Sequences were aligned on the basis of a secondary structure model and analysed with maximum parsimony and maximum likelihood. In contrast to the latter method, parsimony did not recover the monophyly of Clitellata. However, a close scrutiny of the data suggested a spurious attraction between some polychaetes and clitellates. As a rule, molecular trees are closely aligned with morphology-based phylogenies. Acanthobdellida and Euhirudinea were reconciled in their traditional Hirudinea clade and were included in the Oligochaeta with the Branchiobdellida via the Lumbriculidae as a possible link between the two assemblages. While the 18S gene yielded a meaningful historical signal for determining relationships within clitellates, the exact position of Hirudinea and Branchiobdellida within oligochaetes remained unresolved. The lack of phylogenetic signal is interpreted as evidence for a rapid radiation of these taxa. The placement of Clitellata within the Polychaeta remained unresolved. The biological reality of polytomies within annelids is suggested and supports the hypothesis of an extremely ancient radiation of polychaetes and emergence of clitellates.     

On the ontogeny of the haptor and the evolution of the Monogenea

Data on the ontogeny of the posterior haptor of monogeneans were obtained from more than 150 publications and summarised. These data were plotted into diagrams showing evolutionary capacity levels based on the theory of a progressive evolution of marginal hooks, anchors and other attachment components of the posterior haptor in the Monogenea (Malmberg, 1986). 5 + 5 unhinged marginal hooks are assumed to be the most primitive monogenean haptoral condition. Thus the diagrams were founded on a 5 + 5 unhinged marginal hook evolutionary capacity level, and the evolutionary capacity levels of anchors and other haptoral attachement components were arranged according to haptoral ontogenetical sequences. In the final plotting diagram data on hosts, type of spermatozoa, oncomiracidial ciliation, sensilla pattern and protonephridial systems were also included. In this way a number of correlations were revealed. Thus, for example, the number of 5 + 5 marginal hooks correlates with the most primitive monogenean type of spermatozoon and with few sensillae, many ciliated cells and a simple protonephridial system in the oncomiracidium. On the basis of the reviewed data it is concluded that the ancient monogeneans with 5 + 5 unhinged marginal hooks were divided into two main lines, one retaining unhinged marginal hooks and the other evolving hinged marginal hooks. Both main lines have recent representatives at different marginal hook evolutionary capacity levels, i.e. monogeneans retaining a haptor with only marginal hooks. For the main line with hinged marginal hooks the name Articulon-choinea n. subclass is proposed. Members with 8 + 8 hinged marginal hooks only are here called Proanchorea n. superord. Monogeneans with unhinged marginal hooks only are here called Ananchorea n. superord. and three new families are erected for its recent members: Anonchohapteridae n. fam., Acolpentronidae n. fam. and Anacanthoridae n. fam. (with 7 + 7, 8 + 8 and 9 + 9 unhinged marginal hooks, respectively). Except for the families of Articulonchoinea (e.g. Acanthocotylidae, Gyrodactylidae, Tetraonchoididae) Bychowsky's (1957) division of the Monogenea into the Oligonchoinea and Polyonchoinea fits the proposed scheme, i.e. monogeneans with unhinged marginal hooks form one old group, the Oligonchoinea, which have 5 + 5 unhinged marginal hooks, and the other group form the Polyonchoinea, which (with the exception of the Hexabothriidae) has a greater number (7 + 7, 8 + 8 or 9 + 9) of unhinged marginal hooks. It is proposed that both these names, Oligonchoinea (sensu mihi) and Polyonchoinea (sensu mihi), will be retained on one side and Articulonchoinea placed on the other side, which reflects the early monogenean evolution. Except for the members of Ananchorea [Polyonchoinea], all members of the Oligonchoinea and Polyonchoinea have anchors, which imply that they are further evolved, i.e. have passed the 5 + 5 marginal hook evolutionary capacity level (Malmberg, 1986). There are two main types of anchors in the Monogenea: haptoral anchors, with anlages appearing in the haptor, and peduncular anchors, with anlages in the peduncle. There are two types of haptoral anchors: peripheral haptoral anchors, ontogenetically the oldest, and central haptoral anchors. Peduncular anchors, in turn, are ontogenetically younger than peripheral haptoral anchors. There may be two pairs of peduncular anchors: medial peduncular anchors, ontogentically the oldest, and lateral peduncular anchors. Only peduncular (not haptoral) anchors have anchor bars. Monogeneans with haptoral anchors are here called Mediohaptanchorea n. superord. and Laterohaptanchorea n. superord. or haptanchoreans. All oligonchoineans and the oldest polyonchoineans are haptanchoreans. Certain members of Calceostomatidae [Polyonchoinea] are the only monogeneans with both (peripheral) haptoral and peduncular anchors (one pair). These monogeneans are here called Mixanchorea n. superord. Polyonchoineans with peduncular anchors and unhinged marginal hooks are here called the Pedunculanchorea n. superord. The most primitive pedunculanchoreans have only one pair of peduncular anchors with an anchor bar, while the most advanced have both medial and lateral peduncular anchors; each pair having an anchor bar. Certain families of the Articulonchoinea, the Anchorea n. superord., also have peduncular anchors (parallel evolution): only one family, the Sundanonchidae n. fam., has both medial and lateral peduncular anchors, each anchor pair with an anchor bar. Evolutionary lines from different monogenean evolutionary capacity levels are discussed and a new system of classification for the Monogenea is proposed.In agreeing to publish this article, I recognise that its contents are controversial and contrary to generally accepted views on monogenean systematics and evolution. I have anticipated a reaction to the article by inviting senior workers in the field to comment upon it: their views will be reported in a future issue of this journal. EditorIn agreeing to publish this article, I recognise that its contents are controversial and contrary to generally accepted views on monogenean systematics and evolution. I have anticipated a reaction to the article by inviting senior workers in the field to comment upon it: their views will be reported in a future issue of this journal. Editor