骨髓间质干细胞诱导为肌样细胞分化相关基因的表达

目的：研究骨髓间质干细胞向肌样细胞分化前后骨骼肌特异性基因的表达。方法:体外分离成年SD大鼠骨髓干细胞，培养增殖，用5-氮杂胞苷诱导分化，RT-PCR检测分化前和分化后1 d、2 d、5 d和7 d骨骼肌特异性转录因子生肌决定因子MyoD、肌细胞生成素myogenin、MRF4以及肌肉特异性肌酸磷酸激酶MCK的表达 。结果:MyoD在诱导前后均有表达，在诱导后1 d表达显著上调（P＜0.05）；myogenin、MRF4和MCK在诱导前无表达，诱导后2 d开始出现myogenin和MRF4的表达，诱导后7 d开始出现MCK的表达。结论：骨髓间质干细胞能表达一定水平的骨骼肌细胞的分化调控基因MyoD，它向肌样细胞的分化可能与MyoD、myogenin和MRF4有关。     

Blastic plasmacytoid dendritic cell neoplasm: the first report of two cases treated by 5‐Azacytidine

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy which was first included as an independent cutaneous lymphoma in the 2008 World Health Organisation (WHO) classification (1). BPDCN usually has an extremely poor prognosis, with quick relapses after chemotherapy (2; 3). Here, we report two cases of patients diagnosed in 2011 with BPDCN and myelodysplasia, and who were treated for the first time with 5‐azacytidine (5‐Aza); a drug approved by the Food and Drug Administration (FDA) and mainly used in the treatment of myelodysplastic syndrome (Kaminskas E, et al. 2005 Clin Cancer Res, 11, 3604–8). The first case was an 81‐year‐old man who presented with unusual CD10+, CD56‐ immunohistochemistry and 45X, ‐Y abnormality using fluorescent in situ hybridization (FISH) analysis. The second case was a 78‐year‐old woman who manifested monosomy 13 and chromosome instability due to D13S319 locus deletion in 13q14 as determined by FISH. Both patients showed excellent responses of their skin lesions after one cycle of chemotherapy, and their hematological disease was stabilized; however, pulmonary sepsis set in, followed by neutropenia after the fourth and the fifth cycle of treatment, that is, eight and 9 months postdiagnosis, respectively, leading to patient death.     

Characterization of azacytidine/poly(l-lactic) acid particles prepared by supercritical antisolvent precipitation

A controlled azacytidine release system based on drug encapsulation with a polymer material has been prepared and characterized. The drug systems were prepared by precipitation from solutions, using supercritical CO₂ antisolvent technique operating in a semi-continuous mode. Azacytidine was dissolved in dimethylsulfoxide and poly-lactic acid in methylene chloride. The two solutions were mixed before being sprayed into the supercritical reactor. Experimental conditions were 40 °C for temperature, 11 MPa for pressure, and a CO₂ flow rate of 30 ml min⁻¹. The precipitated drug–polymer particles were further characterized to determine the percentage of encapsulated drug and establish the delivery kinetics under various release conditions. A sustained delivery of the drug over a period of various hours was obtained. Besides, an improved stability of the coated drug with respect to the pure azacytidine was found, thus proving the suitability of this approach for dealing with unstable compounds.     

Has introduction of azacytidine in everyday clinical practice improved survival in late-stage Myelodysplastic syndrome? A single center experience

Data derived from clinical trials consistently show a prolongation of overall survival of late-stage MDS patients with the introduction of azacytidine. Nevertheless, the applicability of the above results to real-world clinical settings may be questionable due to the strict design, the controlled medical environment, and the limited patient sample of explanatory studies. We retrospectively compared the outcome of two well-balanced groups of late-stage MDS patients. The first consisted of 46 patients treated with azacytidine (AZA cohort) and the second of 41 patients treated with other agents (non-AZA cohort). Patients in the AZA cohort displayed superior survival compared to the non-AZA ones. However, subgroup analysis revealed that azacytidine conferred a significant survival advantage only in patients with AML–MDS and those who attained a CR at any time after treatment initiation, while all other patients displayed comparable outcome with the non-AZA cohort. Larger series are needed to determine which patients benefit most from azacytidine therapy.     

5-氮胞苷诱导自体骨髓单个核细胞移植对心功能的影响

目的　观察骨髓单个核细胞 (BM MNC)经化学诱导剂 5 氮胞苷诱导后 ,移植到梗死心肌边缘 ,能否进一步改善心功能。方法　 2 6只雄性家兔 ,随机选取 2 1只结扎冠状动脉左前降支 ,建立急性心肌梗死 (AMI)模型 ,随机分为BM MNC 5 氮胞苷组、BM MNC组和AMI未治疗组 3组 ,每组 7只 ;其余 5只为假手术对照组。AMI后第 3d抽取自体股骨骨髓 1 5ml,分离单个核细胞培养 ;AMI后 14d将培养的单个核细胞移植至梗死心肌边缘 ;移植 2 8d后 ,采用超声心动图 ,并结合左心导管、血流动力学等参数评价心功能情况。结果　AMI后兔心功能明显受损 ,与假手术对照组相比 ,AMI后各组左室舒张末期压明显升高 (P <0 0 5 ) ,左室短轴缩短率、左室壁厚度、射血分数、左室收缩压、压力变化率最大值显著降低(P <0 0 5 )。细胞移植后 ,与AMI未治疗组相比 ,细胞移植组左室舒张末期压显著降低 ,左室短轴缩短率、左室壁厚度、射血分数 ,以及左室收缩压、压力变化率最大值明显升高。但 5 氮胞苷诱导的骨髓单个核细胞移植组与未经诱导组间各项指标差异无显著性。结论　 5 氮胞苷诱导的骨髓单个核细胞移植能明显改善心功能 ,并防止AMI后左室重构发生 ;但与单纯骨髓单个核细胞移植相比差异无显著性。     

去甲基化药物治疗骨髓增生异常综合征效果及安全性的Meta分析

目的系统评价去甲基化药物治疗骨髓增生异常综合征(MDS)的效果和安全性,为该类药物的临床应用提供依据和指导。方法计算机检索PubMed、Web of Science、Embase、Cochrane Library、中国期刊全文数据库(CNKI)、中国生物医学文献数据库(CBM)、维普(VIP)数据库2000年1月至2019年12月发表的去甲基化药物治疗MDS的随机对照试验(RCT)。经过筛选,采用RevMan 5.3软件进行疗效及完全性的Meta分析。结果共纳入7项RCT研究的1 172例患者。Meta分析显示,去甲基化药物治疗组在完全缓解率(OR=6.26,95%CI 1.74~22.49,P=0.005)、部分缓解率(OR=4.65,95%CI 1.51~14.29,P=0.007)、总有效率(OR=14.14,95%CI 7.27~27.51,P<0.01)、血液学改善(OR=3.47,95%CI 1.44~8.32,P=0.005)方面均优于支持治疗组。阿扎胞苷治疗组患者总生存得到改善(HR=0.62,95%CI 0.50~0.77,P<0.01)。在不良反应方面,去甲基化药物增加了粒细胞减少性发热的发生(OR=4.19,95%CI 2.26~7.76,P<0.01),但是在粒细胞减少、血小板减少、贫血、感染、恶心、肝损害、疲劳的发生率方面,两组间差异均无统计学意义(均P>0.05)。结论去甲基化药物治疗MDS效果明显,可提高缓解率,阿扎胞苷可延长患者生存时间,但去甲基化药物增加了粒细胞减少性发热的发生。在临床应用该类药物时,需进一步仔细评估其临床安全性。     

体外定向化学诱导成人脂肪源性间充质干细胞分化为心肌样细胞

目的：研究在体外不同浓度、不同作用时间诱导条件下5-氮胞苷（5-aza）对脂肪间充质干细胞（ADMSCs）诱导分化为心肌细胞的影响。方法：酶消化筛选法体外分离ADMSCs并传代培养，流式细胞仪鉴定细胞CD44、CD34的表达，不同浓度5-aza（1、5、10、15、20μmol／L）分别诱导12、24、48、72h，相差显微镜下观察细胞形态变化；诱导后4周时免疫细胞化学鉴定心肌特异性肌钙蛋白Ⅰ（cTn—Ⅰ）的表达，分析细胞转化率。结果：体外分离培养的ADMSCs表达CD44，不表达CD34；5-aza诱导后4周时免疫细胞化学显示cTn—Ⅰ表达阳性，以10μmol／L5-aza诱导24h为适宜诱导条件；结论：体外5-aza化学诱导下成人脂肪组织间充质干细胞具有向心肌细胞分化的潜能。     

氮杂胞苷对SLE患者外周血T细胞Th2细胞因子基因表达的影响

目的探讨DNA甲基化抑制剂氮杂胞苷对SLE患者和正常人外周血T细胞DNA甲基转移酶1和细胞因子IL-4，IL-6，IL-10表达的影响。方法分离活动期SLE患者（n=15）、非活动期SLE患者（n=13）以及正常人对照（n=14）外周血T细胞，经PHA刺激1天后，分为甲基化抑制组和非抑制组进行培养，分别加入或不加氮杂胞苷继续培养3天；用RT—PCR方法测定各组外周血T细胞IL-4，IL-6，IL-10和DNA甲基转移酶1（Dnmt1）mRNA的表达水平。结果①非抑制组内，活动期、非活动期SLE患者的IL-4，IL-6，IL-10mRNA表达均高于正常人对照，Dnmt1mRNA表达显著下降，差异有统计学意义（P值均〈0．05）；并且，活动期、非活动期SLE患者DNA甲基转移酶1的表达与IL-4，IL-6，IL-10mRNA水平呈负相关（P值均〈0．05）。②与非抑制组比较，甲基化抑制组中的正常人T细胞IL-4，IL-6，IL-10mRNA表达增加，而Dnmt1表达则明显降低，差异均有统计学意义（P值均〈0．05）。甲基化抑制组中的活动期、非活动期SLE患者与非抑制组比较，T细胞表达Th2细胞因子、DNA甲基转移酶1表达差异无统计学意义（P值均〉0．05）。结论T细胞表达Th2细胞因子与基因组的DNA甲基化程度相关。     

Synergistic interactions between differentiation-inducing agents in inhibiting the proliferation of HL-60 human myeloid leukaemia cells in clonogenic micro assays

Summary All-trans-retinoic acid, hexamethylene bisacetamide and 5-azacytidine are inducers of granulocytic differentiation of HL-60 human myeloid leukaemic cells, which eventually leads to inhibition of cell proliferation. The effect of graded concentrations of all-trans-retinoic acid (RA) (1 nM-1 M), hexamethylene bisacetamide (HMBA) (0.5–4 mM) and/or 5-azacytidine (5azaC) (1 nM-1 mM), alone and in combination with each other on colony formation and growth of HL-60 cells was studied in agar capillary clonogenic micro assays in order to identify new potential therapeutic regimens for elderly patients with acute myeloid leukaemia. ED₉₀ concentrations, inducing 90% inhibition of colony formation for RA, HMBA and 5azaC, were 128 nM, 2.7 mM and 40 M, respectively. The drug interactions between these differentiating agents were analysed by Berenbaum's general algebraic solution. The combinations: RA + HMBA, 5azaC + HMBA and RA + 5azaC were significantly synergistic in inhibiting HL-60 colony formation. Their interaction indices were 0.62, 0.83, and 0.97, respectively, at a specific effect level of 15%. The addition of 1 mM HMBA to 100 nM 5azaC- and 1 nM RA-treated cultures significantly increased the colony-formation inhibition from only 2.6% and 7.0% to 46.4%, and 43.1 %, respectively. Also, HMBA showed marked synergism with RA and 5azaC in inhibiting colony growth. The interaction indices (I) of HMBA + RA and HMBA + 5azaC were 0.013 and 0.009, respectively, at the same specific level of 15%. Moreover, the triple combination of RA + HMBA + 5azaC showed synergism in inhibiting both the colony formation (I=0.7) and colony growth (I=0.4) at the same specific level of 15%. Since RA, HMBA and 5azaC were effective when administered alone in phase I clinical trials of myeloid leukaemic patients, their synergistic combinations could provide shorter and less toxic courses of treatment in elderly myeloid leukaemic patients.I is < 1, =1 or > 1 in synergistic, additive or antagonistic interactions, respectively.Abbreviations AML acute myeloid leukaemia - 5-azaC 5-azacytidine - HMBA hexamethylene bisacetamide - RA all-trans-retinoic acid     

维奈克拉联合阿扎胞苷治疗伴播散性镰刀菌感染的AML一例

急性髓系白血病（AML）是起源于造血干/祖细胞的恶性克隆性疾病。化学治疗是其主要的治疗方法，若部分初治患者应用标准方案治疗2个疗程仍无效，即为难治性AML。该文报道1例经维奈克拉联合阿扎胞苷治疗的伴播散性镰刀菌感染的难治性AML患者，其被诊断为AML（M5a），存在DNMT3A、IDH2、BCOR基因突变，中危组。经标准剂量IA方案（去甲氧柔红霉素+阿糖胞苷）治疗无效，追加高三尖杉酯碱再次诱导治疗仍无效。其后改用维奈克拉联合阿扎胞苷方案治疗，患者疾病缓解，并再次用原方案巩固治疗3次，达完全缓解。患者第1次化学治疗后出现反复发热，四肢紫红色硬结伴中央坏死，皮下可见液性包块，活组织病理学检查等提示镰刀菌感染，针对镰刀菌感染采用两性霉素B联合伏立康唑抗真菌治疗，经治疗后患者播散性镰刀菌感染得到控制。该例提示，维奈克拉联合阿扎胞苷方案治疗难治性AML缓解率高，播散性镰刀菌感染是AML化学治疗后少见的致死性并发症，早期明确诊断并给予有效的抗真菌治疗是提高患者生存率的有效手段。