非清髓性造血干细胞移植及嵌合体检测

目的评价非清髓性造血干细胞移植(NMSCT)的疗效及供受者嵌合体的检测。方法用氟达拉宾25mg/m2,-5d~-1d、马法兰140mg/m2?1d、抗胸腺细胞球蛋白15mg/(kg.d)-4d~ 5d作为预处理方案,环胞霉素3mg/(kg.d),麦考芬酸脂15mg/(kg.d)用于GVHD的预防。为17例患者做了,并对嵌合体进行检测。结果17例患者达到了早期植入,中性粒细胞恢复至0.5×109/L,中位时间为13d,血小板恢复至30×109/L,中位时间为17d。全部供者粒细胞嵌合体形成中位时间为4周,全部供者T细胞形成中位时间为8周。结论应用氟达拉宾、马法兰、抗胸腺细胞球蛋白作为非清髓性造血干细胞移植预处理方案,可获得供者早期植入,不良反应少。     

白血病异基因造血干细胞移植并发真菌性前色素膜炎1例

男性,42岁.1年前因患慢性粒细胞白血病2年,在我院干细胞移植中心行血缘相关性异基因造血干细胞移植,供者系其胞姐,45岁,血型为AB型,患者血型为B型,HLA配型主要A、B、DR位点全相合,经Bu-CY方案,Bu4 mg/公斤体质量×4 d,CY60 mg/公斤体质量×2 d.预处理后第2天回输CD34+造血干细胞5.6×106/L公斤体质量,回输后第17天,患者外周血中性粒细胞达1.0×109/L之上.     

自体骨髓间充质干细胞联合造血干细胞移植治疗系统性红斑狼疮

目的 探讨间充质干细胞(MSC)联合自体造血干细胞移植(HSCT)治疗系统性红斑狼疮(SLE)的安全性、疗效和对造血重建的影响.方法 采用无血清体系培养扩增骨髓MSC,联合APBSCT治疗1例SLE.干细胞动员应用异环磷酰胺(IF0)7g/m2,分两天应用和粒细胞集落刺激因子(G-CSF)5～10 μ g/kg·d-1;预处理方案包括氟达拉滨(30 mg/m2·d-1,-6、-5、-4、-3、-2d),抗胸腺细胞球蛋白(ATG 15 mg/kg·d-1,-3、-2、-1、0、 1d).结果 患者获得造血重建,中性粒细胞＞0.5×109/L的时间是 4d,血小板未＜20 × 109/L;SLE的临床表现明显减轻,尿蛋白消失,自身抗体转阴,泼尼松用量＜10mg/d.结论 MSC联合APBSCT治疗SLE近期疗效显著,造血重建恢复迅速,安全有效,远期疗效尚需进一步观察.     

异基因造血干细胞移植治疗成人B细胞性急性淋巴细胞白血病的疗效

目的 探讨异基因造血干细胞移植(Allo-HSCT)治疗B细胞性急性淋巴细胞白血病(B-ALL)的疗效.方法 外周血Allo-HSCT治疗B-ALL 15例.预处理方案:6例为改良马利兰(BU)联合环磷酰胺(CY),9例为环磷酰胺/全身照射(TBI).预防急性移植物抗宿主病(GVHD)采用环孢霉素A、短程甲氨喋呤及霉酚酸酯(骁悉).结果 输注单个核细胞5.36-12.30×108/kg,中性粒细胞大于0.5×109/L的为10-15 d.血小板大于20×109/L的为11-16 d.6例(40.0％)发生急性GVHD,6例(40.0％)发生慢性GVHD,皆为广泛性.中位随访时间14个月(2.5-65个月),9例患者无病生存.结论 Allo-HSCT可以作为治疗成人B-ALL的有效手段.     

异基因间充质干细胞和造血干细胞联合移植治疗难治白血病的初步临床研究

目的　初步探讨异基因间充质干细胞 (MSC)联合造血干细胞移植 (HSCT)治疗难治白血病的安全性、疗效 ,以及对移植物抗宿主病 (GVHD)和造血重建的影响。方法　采用无血清体系培养扩增供者骨髓MSC ,联合移植 3例难治白血病 ,其中 1例联合HLA匹配、2例联合HLA半匹配HSCT。植活证据采用短串联重复序列 聚合酶链反应 (STR PCR)、染色体核型或荧光原位杂交技术(FISH)检测。结果　联合移植无明显不良反应。中性粒细胞≥ 0 5× 10 9/L和血小板≥ 2 0× 10 9/L的时间分别为移植后 13、16、15天和 2 5、30、39天。分别于移植后 16、2 3、2 8天骨髓检查显示完全供者型植入。发生Ⅰ、Ⅲ度急性GVHD各 1例 ,经甲基泼尼松龙治疗后有效控制。 3例患者移植后均获完全缓解 (CR) ,2例持续CR 2 4 8和 15 6天 ,1例移植后 94天复发 ,经供者淋巴细胞输注 (DLI)治疗后再获CR。结论　MSC联合HLA匹配和半匹配HSCT治疗难治白血病安全、有效 ,MSC对异基因造血干细胞移植 (Allo HSCH)造血重建和GVHD的影响有待进一步研究     

外周血造血干细胞移植治疗恶性血液病103例临床研究

目的 观察外周血造血干细胞移植(peripheral blood stem cell transplantation,PBSCT)治疗恶性血液病的临床效果.方法 选择2004年7月～2010年10月恶性血液病103例,其中80例行异基因PBSCT治疗,23例行自体PBSCT治疗.异基因PBSCT采用粒细胞集落刺激因子(colony-stimulating factor,G-CSF)、自体PBSCT采用化疗联合G-CSF动员外周血造血干细胞.白血病和骨髓增生异常综合征、多发性骨髓瘤、淋巴瘤的治疗先预处理,并预防异基因PBSCT移植物抗宿主病(graft versus host disease,GVHD)的发生.结果 本组均成功植入并快速重建造血,中性粒细胞恢复至≥0.5×109/L、血小板恢复至≥20×109/L的中位天数分别为19 d和21 d.随访至今,死亡21例,病死率为20.4%.发生出血性膀胱炎4例,巨细胞病毒性肺炎1例.结论 PBSCT能很快重建造血,移植相关病发生较少,病死率较低,安全可行,是治疗恶性血液病有效的手段之一.     

非清髓异基因外周造血干细胞移植治疗白血病疗效观察

目的 探讨非清髓性异基因外周血造血干细胞移植(NAPBSCT)治疗年龄较大的白血病患者安全性和疗效.方法 采用NAPBSCT治疗6例45～65 岁白血病患者,其中急性淋巴细胞白血病1例,淋巴瘤细胞白血病2例,急性粒细胞白血病1例,慢性粒白血病2例.以FAC为预处理方案,氟拉达宾(Flud)50mg/d(-6d～-2d)(以造血干细胞输注当日为0d,输注前为"-",输注后为" "),抗淋巴细胞免疫球蛋白(ALG)15mg/(kg·d)(-6～-2d),环磷酰胺(CTX)30mg/(kg·d)(-4～-3d).于 30 d进行第1次供者淋巴细胞输注,每4周1次,共6次.输注的供体淋巴细胞数由5×104 /kg逐渐增加至1.5×108 /kg.结果 1例患者于移植后56天死于急性移植物抗宿主病(aGVHD),1例患者于移植后81天死于肺部真菌感染,其余4例患者目前均存活,最长的1例已存活达35个月.6例患者在移植早期均形成混合性嵌合体和血液学部分缓解,供体淋巴细胞输注后有3例患者形成供体型完全嵌合体,并达到血液学完全缓解.结论 非清髓性外周血造血干细胞移植治疗年龄较大的白血病患者较为安全且疗效较好.     

HLA半相合异基因造血干细胞移植并发症的预防

异基因造血干细胞移植是治疗白血病的有效方法，在缺乏HLA配型完全相合供者的情况下只得用HLA半相合异基因造血干细胞移植，但易发生移植物抗宿主病(GVHD)、出血性膀胱炎(HC)、肝静脉闭塞病(VOD)等严重并发症而导致移植失败。本文报告HLA半相合异基因造血干     

非清髓异基因外周血造血干细胞移植治疗一例慢性粒细胞白血病

异基因造血干细胞移植仍是目前唯一公认能够治愈慢性粒细胞白血病(CML)的方法,在慢性期移植效果最佳,50%～60%患者可获得长期存活和长期缓解。我们采用非清髓性HLA相合同胞供者外周血造血干细胞移植成功治疗CML 1例,现报告如下。     

半相合移植移植物中CD34+细胞数量与临床结果相关性

目的 探讨半相合移植移植物中CD34 细胞数量对临床结果的影响.方法 对26例半相合移植患者移植物中CD34 细胞、T、B淋巴细胞数量与临床造血重建时间、急慢性移植物抗宿主病(GVHD)及存活率进行相关性分析.结果 所有患者输入CD34 细胞是(3.66～9.29)×106/kg,其中细胞数量高组(＞5.60×106/kg,A组)和低组(＜5.60×106/kg,B组)患者移植物中T、B淋巴细胞数量均无显著性差异;两组中性粒细胞造血重建时间均为11d,血小板重建时间均为18d;急性和慢性GVHD发生率A组分别为46.2%和38.5%,B组分别为46.2%和46.2%.患者总生存率A组为61.5%,B组为69.2%.结论 半相合干细胞移植输入(3.66～9.29)×106/kg的CD34 细胞是安全的,不增加GVHD的发生率,也不降低生存率.     

Embryonic stem cells and the next generation of developmental toxicity testing

Introduction: The advent of stem cell technology has seen the establishment of embryonic stem cells (ESCs) as molecular model systems and screening tools. Although ESCs are nowadays widely used in research, regulatory implementation for developmental toxicity testing is pending.

Areas Covered: This review evaluates the performance of current ESC, including human (h)ESC testing systems, trying to elucidate their potential for developmental toxicity testing. It shall discuss defining parameters and mechanisms, their relevance and contemplate what can realistically be expected. Crucially this includes the question of how to ascertain the quality of currently employed cell lines and tests based thereon. Finally, the use of hESCs will raise ethical concerns which should be addressed early on.

Expert Opinion: While the suitability of (h)ESCs as tools for research and development goes undisputed, any routine use for developmental toxicity testing currently still seems premature. The reasons for this comprise inherent biological deficiencies as well as cell line quality and system validation. Overcoming these issues will require collaboration of scientists, test developers and regulators. Also, validation needs to be made worthwhile for academia. Finally we have to continuously rethink existing strategies, making room for improved testing and innovative approaches.     

Targeting survival pathways in chronic myeloid leukaemia stem cells

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder characterized by the presence of a fusion oncogene BCR-ABL, which encodes a protein with constitutive TK activity. The implementation of tyrosine kinase inhibitors (TKIs) marked a major advance in CML therapy; however, there are problems with current treatment. For example, relapse occurs when these drugs are discontinued in the majority of patients who have achieved a complete molecular response on TKI and these agents are less effective in patients with mutations in the BCR-ABL kinase domain. Importantly, TKI can effectively target proliferating mature cells, but do not eradicate quiescent leukaemic stem cells (LSCs), therefore allowing disease persistence despite treatment. It is essential that alternative strategies are used to target the LSC population. BCR-ABL activation is responsible for the modulation of different signalling pathways, which allows the LSC fraction to evade cell death. Several pathways have been shown to be modulated by BCR-ABL, including PI3K/AKT/mTOR, JAK-STAT and autophagy signalling pathways. Targeting components of these survival pathways, alone or in combination with TKI, therefore represents an attractive potential therapeutic approach for targeting the LSC. However, many pathways are also active in normal stem cells. Therefore, potential targets must be validated to effectively eradicate CML stem cells while sparing normal counterparts. This review summarizes the main pathways modulated in CML stem cells, the recent developments and the use of novel drugs to target components in these pathways which may be used to target the LSC population.

Linked Articles

This article is part of a themed section on Emerging Therapeutic Aspects in Oncology. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-8     

Embryonic signature in breast cancers; Pluripotency roots of cancer stem cells

Drug discovery programs for preclinical oncology typically select compounds which have a predilection for inducing cytotoxic effects in cancer cell lines and subsequently, for inhibiting the growth of the transplanted cancer cells in vivo (Winquist et al., 2010). Unfortunately, the cytotoxic effect in vitro and inhibition of tumor growth in animal models are not the end story for curing cancer in preclinical models. The reason behind that is the exciting of small sub type of cells that are relatively resistance to therapy and able to repopulate in vivo, called cancer stem cells (CSCs). O leis et al. recently reported that the pluripotency gene Sox2 but not Oct4 or Nanog is expressed in early stage of breast tumor. Furthermore, the authors demonstrated that Sox2 downregulation, inhibited mammosphere formation and delayed tumor formation in xenograft tumor initiation models (Leis et al., 2012). In this review, we will shed the light on the importance of Sox2 in breast and other tissue tumorigenesis and associated aggressiveness.     

碱性成纤维细胞因子对SD大鼠表皮干细胞分化为神经干细胞的影响

目的 研究碱性成纤维细胞因子(bFGF)对大鼠表皮干细胞分化为神经细胞的影响。 方法 分离获得饲养了1~3天的新生SD大鼠表皮基底层组织,利用10 min快速贴壁法消化、分离获得表皮干细胞,于倒置显微镜下观察表皮干细胞的形态。培养表皮干细胞的培养基为K-SFM,然后按照不同密度表皮干细胞进行分组处理(0.1×10⁷/mL,0.3×10⁷/mL,0.5×10⁷/mL,0.1×10⁶/mL),每组加入20 ng/mL的bFGF,利用细胞免疫组织化学法检测细胞标志物Nestin和NSE的变化,以及观察细胞形态发生的变化。 结果 成功分离得到SD大鼠表皮干细胞;bFGF诱导后,0.3×10⁷/mL组和0.1×10⁷/mL组细胞第3天即可见细胞开始伸展生长,在约1周时细胞开始分化成神经细胞;且在细胞形态发生双极化改变的数目趋势上也具有一致性;Nestin和NSE检测均呈阳性表达。     

Disruption of cardiogenesis in human embryonic stem cells exposed to trichloroethylene

Trichloroethylene (TCE) is ubiquitous in our living environment, and prenatal exposure to TCE is reported to cause congenital heart disease in humans. Although multiple studies have been performed using animal models, they have limited value in predicting effects on humans due to the unknown species‐specific toxicological effects. To test whether exposure to low doses of TCE induces developmental toxicity in humans, we investigated the effect of TCE on human embryonic stem cells (hESCs) and cardiomyocytes (derived from the hESCs). In the current study, hESCs cardiac differentiation was achieved by using differentiation medium consisting of StemPro‐34. We examined the effects of TCE on cell viability by cell growth assay and cardiac inhibition by analysis of spontaneously beating cluster. The expression levels of genes associated with cardiac differentiation and Ca²⁺ channel pathways were measured by immunofluorescence and qPCR. The overall data indicated the following: (1) significant cardiac inhibition, which was characterized by decreased beating clusters and beating rates, following treatment with low doses of TCE; (2) significant up‐regulation of the Nkx2.5/Hand1 gene in cardiac progenitors and down regulation of the Mhc‐7/cTnT gene in cardiac cells; and (3) significant interference with Ca²⁺ channel pathways in cardiomyocytes, which contributes to the adverse effect of TCE on cardiac differentiation during early embryo development. Our results confirmed the involvement of Ca²⁺ turnover network in TCE cardiotoxicity as reported in animal models, while the inhibition effect of TCE on the transition of cardiac progenitors to cardiomyocytes is unique to hESCs, indicating a species‐specific effect of TCE on heart development. This study provides new insight into TCE biology in humans, which may help explain the development of congenital heart defects after TCE exposure. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1372–1380, 2016.     

Protein profiles of cardiomyocyte differentiation in murine embryonic stem cells exposed to perfluorooctane sulfonate

Perfluorooctane sulfonate (PFOS) is a persistent organic contaminant that may affect diverse systems in animals and humans, including the cardiovascular system. However, little is known about the mechanism by which it affects the biological systems. Herein, we used embryonic stem cell test procedure as a tool to assess the developmental cardiotoxicity of PFOS. The differentially expressed proteins were identified by quantitative proteomics that combines the stable isotope labeling of amino acids with high‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry. Results of the embryonic stem cell test procedure suggested that PFOS was a weak embryotoxic chemical. Nevertheless, a few marker proteins related to cardiovascular development (Brachyury, GATA4, MEF2C, α‐actinin) were significantly reduced by exposure to PFOS. In total, 176 differential proteins were identified by proteomics analysis, of which 67 were upregulated and 109 were downregulated. Gene ontology annotation classified these proteins into 13 groups by molecular functions, 12 groups by cellular locations and 10 groups by biological processes. Most proteins were mainly relevant to either catalytic activity (25.6%), nucleus localization (28.9%) or to cellular component organization (19.8%). Pathway analysis revealed that 32 signaling pathways were affected, particularly these involved in metabolism. Changes in five proteins, including L ‐threonine dehydrogenase, X‐ray repair cross‐complementing 5, superoxide dismutase 2, and DNA methyltransferase 3b and 3a were confirmed by Western blotting, suggesting the reliability of the technique. These results revealed potential new targets of PFOS on the developmental cardiovascular system. Copyright © 2015 John Wiley & Sons, Ltd.     

间充质干细胞治疗特发性肺纤维化药理机制研究进展

间充质干细胞（MSCs）具有多种分化能力,可以直接迁移到损伤组织中并分化为肺泡上皮细胞,还可以分泌并释放多种细胞因子和外泌体,调节炎症和免疫反应。特发性肺纤维化（IPF）是与年龄相关、发病机制尚不明确的慢性进行性肺部疾病,临床上使用的吡非尼酮和尼达尼布仅能缓解症状。MSCs在治疗IPF方面具有广阔的应用前景,就目前关于MSCs治疗特发性肺纤维化的药理机制以及该疗法所存在的局限性进行综述和探讨。     

Intrapleural delivery of mesenchymal stem cells: a novel potential treatment for pleural diseases

Aim:

To develop a method to deliver mesenchymal stem cells (MSCs) into the pleural cavity for the treatment of pleural diseases.

Methods:

MSCs were isolated from rat bone marrow of rats and labeled with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) or green fluorescent protein (GFP) using a lentiviral vector. Eighteen Sprague-Dawley (SD) rats were inoculated intrapleurally with 1×10⁶ MSCs-DAPI. The distribution of the fluorescent cells was observed using fluorescent microscopy for the following 30 d. Another 12 rats inoculated intrapleurally with 1×10⁶ MSCs-GFP were observed for 14 d.

Results:

The isolated cells were typical MSC phenotypes and could differentiate into adipocytes, osteoblasts, and chondroblasts in vitro. Microscopic analysis revealed that the labeled cells adhered to the surface of the pleural cavity. The highest number of the labeled cells was found to be adhered to all specimens from the mediastinal pleura, but no labeled cells were detected in the lung parenchyma or other tissues/organs, such as the liver, kidney, spleen, and mesenterium. Incidentally, stomas were found in the mediastinal pleura. The recovered MSCs-GFP from the pleural cavity retained their ability to adhere and proliferate.

Conclusion:

We have established a novel method for intrapleural delivery of MSCs. The distribution of intrapleurally delivered MSCs was found to be limited to the pleurae and the pleural cavity, thereby providing us with a new approach to further investigation of the therapeutic roles of MSCs in pleural diseases.     

海风藤挥发油抗风湿作用的药效学

目的：通过药理实验确定海风藤挥发油抗风湿作用。方法：采用热板法、棉球肉芽法、耳肿胀法。结果：海风藤挥发油各剂量组均表现出明显的抗炎镇痛作用。结论：实验结果提示海风藤挥发油具有抗风湿作用。