脾结节生成细胞(CFC-S)特性的研究

用肉眼观察和放射自显影的方法比较了9d和13d脾结节的生成。同样是能够生成脾结节的细胞,但重建照射小鼠的造血功能的潜力不同。实验结果表明,仅仅根据脾结节的生成,或者脾结节生成的先后时间,都不足以作为判定其是否为造血干细胞的唯一依据,脾结节生成细胞是在性能上不均一的细胞群体,对脾结节生成细胞的自我更新能力的实际测定,才能对其是否具有造血干细胞的基本性能做出可靠结论。     

与造血过程有关联的转录因子

多能干细胞分化过程中其子代细胞分化潜能逐渐减小,并使子代细胞获得成熟细胞的某些表型,最终达到终末分化和具有特定表型。研究这个过程的分子机理是发育生物学的一个重要课题。造血过程是从多能造血干细胞分化成各系造血祖细胞,再进一步分化成成熟血细胞(包括红细胞,白细胞和淋巴细胞)的过程。     

CD34+和CD34-细胞在NOD/SCID小鼠体内的造血重建

利用非肥胖糖尿病型重症联合免疫缺陷型(NOD/SCID)小鼠模型, 比较了新鲜及培养后的CD34+和CD34-细胞在体内植入及重建造血能力。从新鲜脐血及培养后的单个核细胞(MNC)中分离出CD34+和CD34-细胞, 经尾静脉输注入经亚致死剂量照射的NOD/SCID小鼠体内, 6周后处死存活的小鼠, 取其骨髓、脾脏和外周血细胞, 分别进行细胞表型分析、造血集落形成单位和人特异性基因的检测。经检测, 输注CD34+细胞和混合细胞的小鼠, 其体内CD45+细胞及人源各系血细胞的含量相近, 两者均远远高于输注CD34-细胞的小鼠。输注培养后CD34-细胞的小鼠饲养6周后全部死亡,输注培养后CD34+细胞的小鼠存活率约为66.7%, 而输注培养后混合细胞的小鼠全部存活, 且在两组存活的小鼠体内均能检测到CD45+细胞及人源各系血细胞。结果表明: 无论是新鲜还是培养后的CD34+细胞均具有在NOD/SCID小鼠体内植入和重建造血能力, 而CD34-细胞不具有该能力, 但CD34-细胞与CD34+细胞同时输注有助于提高小鼠的存活率, 说明其对CD34+细胞在小鼠体内发挥植入和造血重建能力有一定的辅助作用。     

人CD34^＋和CD34^-细胞在NOD／SCID小鼠体内的造血重建

利用非肥胖糖尿病型重症联合免疫缺陷型(NOD/SCID)小鼠模型,比较了新鲜及培养后的CD34 和CD34-细胞在体内植入及重建造血能力.从新鲜脐血及培养后的单个核细胞(MNC)中分离出CD34 和CD34-细胞,经尾静脉输注入经亚致死剂量照射的NOD/SCID小鼠体内,6周后处死存活的小鼠,取其骨髓、脾脏和外周血细胞,分别进行细胞表型分析、造血集落形成单位和人特异性基因的检测.经检测,输注CD34' 细胞和混合细胞的小鼠,其体内CD45 细胞及人源各系血细胞的含量相近,两者均远远高于输注CD34-细胞的小鼠.输注培养后CD34-细胞的小鼠饲养6周后全部死亡,输注培养后CD34 细胞的小鼠存活率约为66.7%,而输注培养后混合细胞的小鼠全部存活,且在两组存活的小鼠体内均能检测到CD45 细胞及人源各系血细胞.结果表明:无论是新鲜还是培养后的CD34 细胞均具有在NOD/SCID小鼠体内植入和重建造血能力,而CD34-细胞不具有该能力,但CD34-细胞与CD34 细胞同时输注有助于提高小鼠的存活率,说明其对CD34 细胞在小鼠体内发挥植入和造血重建能力有一定的辅助作用.     

小鼠骨髓细胞体外液体培养

自六十年代初相继出现了体外研究血细胞的脾结节生成的实验技术、骨髓细胞体外琼脂培养等技术,大大促进了对造血干细胞的增殖和分化特性的研究,并为实验血液学提供了重要手段。但在这些培养系统中血细胞增生只能维持几天,而且多向性干细胞很快就消失了。七十年代初,国外开始发展了骨髓细胞体外液体培养法,能在体外条件下较长时间地保持造血干细胞的增殖、分化能力,为研究造血干细胞增殖、分化过程中的各种调控因素提供了一个有用的手段,这对解决临床治疗和寻找病因等问题也具有重大的实际意义。我们参照了T.M.Dexter等的方法,根据我们实验室现有的条件及设备在1979年初也开始建立了这种培养方法。     

造血干细胞与免疫活性细胞的沉降特性

应用 Simonsen 的脾指数测定方法以及体内生成脾结节和体外琼脂培养方法,分别研究了小鼠脾脏和外周血中造血干细胞和免疫活性细胞在自然沉降条件下的沉降特性。在小鼠脾脏细胞的沉降试验中,CFU-S 的沉降速度为4.50毫米/小时,CFU-C 的沉降速度为6.69毫米/小时,脾指数阳性细胞的沉降速度为3.57毫米/小时。在小鼠外周血白细胞的沉降试验中,CFU-S 的沉降速度为4.96毫米/小时,脾指数阳性细胞的沉降速度为5.12毫米/小时。上述沉降试验表明,造血干细胞与免疫活性细胞有不同的沉降速度和分布特性,因此,有可能通过自然沉降,在一定程度上分开这两类细胞,其中,粒系定向造血干细胞(CFU-C)的沉降速度要高于多向性造血干细胞(CFU-S),因而,在沉降分离中 CFU-C 与免疫活性细胞可以得到较高程度的分离。从造血细胞中分离免疫活性 细胞是当前造血干细胞移植中的重要研究课题。本文对于应用速度沉降装置分离免疫活性细胞、减轻造血细胞移植中的移植物抗宿主反应(GVHR)的前景作了讨论。     

小鼠造血干细胞增殖和分化的分子生物学证据

本文采用Ｙ染色体特异的性别决定基因（Ｓｒｙ）作为新的细胞遗传标志,通过ＰＣＲ技术来追踪观察造血干细胞的增殖与分化性能。该方法具有简便、灵敏和特异等优点。雌性受体小鼠输注雄鼠骨髓细胞和１３天脾结节（ＣＦＵ－Ｓ１３）细胞后,Ｓｒｙ ＰＣＲ测试受体小鼠的ＣＦＵ－Ｓ结果表明,它们均为供体来源的ＸＹ细胞。用Ｓｒｙ ＰＣＲ骨髓细胞和骨髓中脾结节生成细胞（ＣＰＵ－Ｓ）的长期重建造血能力,结果表明,在存活雌性小鼠     

胚胎干细胞向造血干/祖细胞定向诱导分化的研究进展

胚胎干细胞(embryonic stem cell,ES细胞)是指由胚胎内细胞团(inner cell mass,ICM)细胞经体外抑制培养而筛选得到的细胞,具有无限增殖潜能,在体外可以向造血细胞分化,有可能为造血干细胞移植和血细胞输注开辟新的来源．此外,ES细胞向造血干/祖细胞的定向诱导分化也为阐明哺乳动物造血发育的细胞和分子机制提供了良好的体外模型．对ES细胞向造血干/祖细胞定向分化的研究进展进行了综述．     

蛋白酶3对造血干/祖细胞增殖和分化的影响

蛋白酶3(proteinase 3, PRTN3)是一种中性丝氨酸蛋白酶,与病原体清除、组织损伤和细胞凋亡有关。近期研究发现, Prtn3在造血干/祖细胞中高表达,但其生物学功能及意义仍不清楚。围绕上述问题,该文采用单细胞转录组测序方法分析Prtn3基因在野生型小鼠(WT)血细胞中的表达情况;构建Prtn3基因敲除小鼠(Prtn3–/–),并采用流式细胞术和血常规分别分析Prtn3–/–小鼠骨髓中的LT-HSC、ST-HSC、MPP、CMP、GMP、MEP及分化成熟细胞的数目和比例;体外干/祖细胞单细胞和集落培养法分析LSK细胞的增殖和分化潜能;竞争移植实验分析Prtn3–/–小鼠LSK细胞的竞争能力以及外周血、脾脏和骨髓中各种血细胞的数目和比例;免疫荧光法分析Prtn3–/–小鼠脾脏和骨髓中的供体血细胞分布。结果显示, Prtn3在小鼠造血干/祖细胞,尤其是在髓系祖细胞(CMP和GMP)中持续高表达; Prtn3–/–小鼠骨髓中LSK和LK细胞所占比例显著高于WT小鼠骨髓中LSK和LK细胞的比例且表现为LT-HSC、ST-HSC、MPP、CMP及GMP在骨髓细胞中的比例均显著增加(P...     

内皮和造血系统特异性敲除Rictor基因对胎肝造血的影响

目的:研究Rictor基因对胚胎发育过程中胎肝造血的影响。方法:利用Cre-LoxP基因敲除系统,特异性在小鼠内皮(VEC-Cre)和造血(Vav1-Cre)系统敲除Rictor基因;通过流式细胞术分析特异敲除Rictor基因后小鼠胚胎第15 d胎肝中的各系细胞比例的变化,并进一步分析造血干细胞的变化。结果:利用VEC-Cre和Vav1-Cre小鼠敲除Rictor基因后,胎肝中各系细胞比例均有所减少,B细胞比例的减少较为明显,造血干细胞比例也明显减少。结论:Rictor基因敲除损害胎肝组织中造血干细胞的产生和各系细胞的分化。     

Nucleoside diphosphate kinase Nm23-M1 involves in oligodendroglial versus neuronal cell fate decision in vitro

The adult glial progenitor cells were recently shown to be able to produce neurons in central nervous system (CNS) and to become multipotent in vitro. Although the fate decision of glial progenitors was studied extensively, the signals and factors which regulate the timing of neuronal differentiation still remain unknown. To elucidate the mechanisms underlying the neuronal differentiation from glial progenitors, we modified the gene expression profile in NG2⁺ glial progenitor cells using enhanced retroviral mutagen (ERM) technique followed by phenotype screening to identify possible gene(s) responsible for glial-neuronal cell fate determination. Among the identified molecules, we found the gene named non-metastatic cell 1 which encodes a nucleoside diphosphate kinase protein A (Nm23-M1 or NME1). So far, the Nm23 members have been shown to be involved in various molecular processes including tumor metastasis, cell proliferation, differentiation and cell fate determination. In the present study, we provide evidence suggesting the role of NME1 in glial-neuronal cell fate determination in vitro. We showed that NME1 is widely expressed in neuronal structures throughout adult mouse CNS. Our immunohistochemical results revealed that NME1 is strongly colocalized with NF200 through white matter of spinal cord and brain. Interestingly, NME1 overexpression in oligodendrocyte progenitor OLN-93 cells potently induced the acquisition of neuronal fate, while its silencing was shown to promote oligodendrocyte differentiation. Furthermore, we demonstrated that dual-functional role of NME1 is achieved through cAMP-dependent protein kinase (PKA). Our data therefore suggested that NME1 acts as a switcher or reprogramming factor which involves in oligodentrocyte versus neuron cell fate specification in vitro.     

体细胞重编程逆转为干细胞的研究进展

目前细胞和发育生物学上的研究成果为生物医学研究提供了广泛的前景.将完全分化的细胞重编程,不经过胚胎逆转为多能干细胞状态,这点燃了再生医学应用的新希望,这一成果从法律、道德、伦理等不同方面被人们所接受.通过体细胞克隆胚胎获得干细胞所面临的破坏胚胎的伦理限制,促使研究者去寻求将分化细胞重编程逆转为干细胞的新方法.主要论述了体细胞重编程的原理、过程及不经过胚胎逆转为多能干细胞的方法.     

胚胎干细胞起源的探讨

目前胚胎干细胞(ESCs)建系的取材来源包括桑椹胚的卵裂球、囊胚的内细胞团(ICM)、上胚层细胞和原始生殖细胞(PGCs),甚至从新生鼠睾丸细胞也分离得到类ES样细胞系。这就提出了一个问题,什么是ESCs最接近的体内细胞来源。传统观念常常把ESCs等同于ICM细胞,也有学者认为ESCs更象上胚层细胞,而在已知的分子标记基因方面,ESCs所具有的特征更接近体内早期生殖细胞。不清楚ESCs最接近的体内细胞来源,可能是制约许多品系小鼠和大多哺乳类动物建系成功率提高的原因之一。ESCs系与EG细胞系的分离条件不同表明,加强对ESCs多能性维持基因调控研究具有重要意义。本文从ESCs的经典概念及其发展,早期胚胎细胞和生殖细胞发育规律,早期胚胎细胞、早期生殖细胞和ESCs的关系等方面进行综合分析,认为ESCs可能有多种接近的体内细胞来源。进一步应通过对ESCs建系不同的取材细胞和不同品系的ESCs间进行比较研究,以便弄清ESCs的来源和转化机制,为提高不同物种ESCs建系效率提供理论支持。     

Fine needle aspiration (FNA) cytology of pilomatrixoma

FNA smears from five histologically confirmed cases of pilomatrixoma were reviewed to delineate the cytological features helpful in diagnosis. A combination of basaloid cells, ghost cells and foreign body giant cells appeared to be necessary in FNA smear for a confident cytodiagnosis of pilomatrixoma. Presence of naked nuclei, nucleated squamous cells and calcification were additional features in favour of the diagnosis. Another 10 cases with initial cytodiagnosis of pilomatrixoma or benign skin appendage tumour were reviewed. Using the above criteria, diagnosis of pilomatrixoma was easy in five cases. One case was problematical due to presence of atypical squamous cells. Initially the cytological features were most commonly confused with epidermal inclusion cyst, giant cell lesion or a squamous cell carcinoma. The main reasons for erroneous diagnosis were lack of awareness of cytological features, predominance of one component over the others, and non‐representative FNA smears. Atypia in nucleated squamous cells, and misinterpretation of basaloid cells as malignant can lead to diagnostic dilemma. Adequate clinical data are also necessary.     

诱导型细胞的研究进展

胚胎干细胞(ES细胞)和诱导型多能干细胞(iPS细胞)的研究进展为生物学基础研究注入了新的活力,然而免疫排斥、致瘤性以及诱导效率低等缺陷制约其进一步快速发展和临床应用．最近,科学家借鉴iPS细胞诱导技术和传统的诱导体系,将终末分化细胞直接诱导为功能性细胞,如心肌细胞、神经细胞和肝脏细胞,称为诱导型细胞．这些研究进展极大地促进了细胞分化、重编程和表观遗传学的研究,也为人类再生医学的研究提供了新的途径．     

Induction of γδT cells from HSC-enriched BMCs co-cultured with iPSC-derived thymic epithelial cells

T cells bearing γδ antigen receptors have been investigated as potential treatments for several diseases, including malignant tumours. However, the clinical application of γδT cells has been hampered by their relatively low abundance in vivo and the technical difficulty of inducing their differentiation from hematopoietic stem cells (HSCs) in vitro. Here, we describe a novel method for generating mouse γδT cells by co-culturing HSC-enriched bone marrow cells (HSC-eBMCs) with induced thymic epithelial cells (iTECs) derived from induced pluripotent stem cells (iPSCs). We used BMCs from CD45.1 congenic C57BL/6 mice to distinguish them from iPSCs, which expressed CD45.2. We showed that HSC-eBMCs and iTECs cultured with IL-2 + IL-7 for up to 21 days induced CD45.1⁺ γδT cells that expressed a broad repertoire of Vγ and Vδ T-cell receptors. Notably, the induced lymphocytes contained few or no αβT cells, NK1.1⁺ natural killer cells, or B220⁺ B cells. Adoptive transfer of the induced γδT cells to leukemia-bearing mice significantly reduced tumour growth and prolonged mouse survival with no obvious side effects, such as tumorigenesis and autoimmune diseases. This new method suggests that it could also be used to produce human γδT cells for clinical applications.     

树突状细胞与CIK细胞共培养诱生的DCCIK细胞群对肿瘤的杀伤作用

由树突状细胞(DC)与细胞因子诱导的同源杀伤细胞(CIK)的共培养诱生的细胞群(DCCIK)对肿瘤细胞的细胞毒活性的研究。DCCIK细胞体外杀伤肿瘤靶细胞A549(MTT法),效靶比为10∶1、5∶1时杀伤率分别为61%、52%。DCCIK细胞诱导培养3周后,效靶比为10∶1、5∶1时杀伤率分别为64%和56%。数据亦表明DCCIK细胞对靶细胞的杀伤优于CIK细胞。动物体内实验分荷瘤A549、BEL7404和A375三组,每组分(A)DCCIK 化疗、(B)单用化疗。治疗20天、35天后测量各组肿瘤消失率。结果显示:DCCIK 化疗的抑瘤效果明显好于单纯化疗。提示DCCIK细胞有临床应用前景。     

Bovine mammary stem cells: cell biology meets production agriculture

Mammary stem cells (MaSC) provide for net growth, renewal and turnover of mammary epithelial cells, and are therefore potential targets for strategies to increase production efficiency. Appropriate regulation of MaSC can potentially benefit milk yield, persistency, dry period management and tissue repair. Accordingly, we and others have attempted to characterize and alter the function of bovine MaSC. In this review, we provide an overview of current knowledge of MaSC gained from studies using mouse and human model systems and present research on bovine MaSC within that context. Recent data indicate that MaSC retain labeled DNA for extended periods because of their selective segregation of template DNA strands during mitosis. Relying on this long-term retention of bromodeoxyuridine-labeled DNA, we identified putative bovine MaSC. These label-retaining epithelial cells (LREC) are in low abundance within mammary epithelium (<1%). They are predominantly estrogen receptor (ER)-negative and localized in a basal or suprabasal layer of the epithelium throughout the gland. Thus, the response of MaSC to estrogen, the major mitogen in mammary gland, is likely mediated by paracrine factors released by cells that are ER-positive. This is consistent with considerable evidence for cross-talk within and between epithelial cells and surrounding stromal cells. Excision of classes of cells by laser microdissection and subsequent microarray analysis will hopefully provide markers for MaSC and insights into their regulation. Preliminary analyses of gene expression in laser-microdissected LREC and non-LREC are consistent with the concept that LREC represent populations of stem cells and progenitor cells that differ with regard to their properties and location within the epithelial layer. We have attempted to modulate the MaSC number by infusing a solution of xanthosine through the teat canal and into the ductal network of the mammary glands of prepubertal heifers. This treatment increased the number of putative stem cells, as evidenced by an increase in the percentage of LREC and increased telomerase activity within the tissue. The exciting possibility that stem cell expansion can influence milk production is currently under investigation.