慢性粒细胞白血病患者骨髓间充质干细胞的生物学特性及其造血调控

背景:由于慢性粒细胞白血病存在造血微环境异常和免疫异常,推测间充质干细胞可能在慢性粒细胞白血病的病理过程中扮演了一个重要角色.目的:观察慢性粒细胞白血病骨髓间充质干细胞的生物学特征和对造血调控的影响,比较其与正常人骨髓来源的间充质干细胞的异常.方法:分离正常人和慢性粒细胞白血病患者的骨髓间充质干细胞,分别检测它们的形态、表型、生长曲线和对T细胞活化的能力;将它们分别与脐带血干细胞共培养,检测共培养体系中对集落形成的影响和相关分子表达的差异.结果与结论:慢性粒细胞白血病患者和正常志愿者骨髓来源的间充质干细胞的细胞形态、表型和干细胞的倍增时间均没有差异,慢性粒细胞白血病患者的骨髓间充质干细胞抑制T细胞活化的能力减弱;共培养体系中,悬浮细胞簇增加,说明造血干细胞与间充质干细胞之间的黏附性减弱,同时蛋白和RNA水平显示慢粒组中基质金属蛋白酶9升高,同时使ICAM-1的KitL从膜形式转变为可溶性形式.结果提示,慢性粒细胞白血病骨髓间充质干细胞是细胞分化发育的微环境,其基础的机制在于基质金属蛋白酶9对细胞外基质的降解和黏附分子的裂解,改变骨髓微环境对造血干细胞黏附,增殖和分化的支持作用,导致造血干细胞异常增殖和动员,降低了恶变细胞对免疫细胞的敏感性导致免疫逃逸.     

脐血单个核细胞与骨髓间充质干细胞滋养层的共培养

背景:骨髓间充质干细胞具有维持骨髓正常造血功能,在造血调控中发挥重要的作用。目的:观察骨髓间充质干细胞的生物学性状和多向分化能力,并检测其在体外支持造血的能力。方法:利用密度梯度培养法分离骨髓单个核细胞进行培养,流式细胞仪检测骨髓间充质干细胞表型;接种脐血单个核细胞于骨髓间充质干细胞滋养层培养板上共培养,观察粒-单系祖细胞集落变化。结果与结论:骨髓间充质干细胞呈典型的成纤维样细胞形态,强表达CD44,CD29,不表达CD34和CD106。可促进脐血单个核细胞扩增并形成造血祖细胞集落。提示骨髓间充质干细具有造血支持作用。     

人羊膜间充质干细胞支持造血的体外实验

背景:骨髓是目前间充质干细胞的主要来源,但由于取材不便、细胞数量受年龄限制等原因,其应用具有一定局限性.近年来,羊膜作为间充质干细胞的新来源受到越来越广泛的关注.目的:探讨人羊膜来源间充质干细胞在体外对造血干细胞的扩增是否有支持作用,以及怎样提高羊膜间充质干细胞和造血干细胞共移植成功率.方法:利用组织块培养法,从足月分娩的人羊膜中分离培养羊膜间充质干细胞.密度梯度离心法分离脐血单个核细胞,采用免疫磁珠分选技术分选其中 CD34+细胞.分别用脐血单个核细胞和 CD34+细胞与羊膜间充质干细胞共培养,连续 4 周,每周计悬浮细胞浓度,并以骨髓间充质干细胞组及无滋养层组作为对照.采用集落形成实验,把扩增的脐血单个核细胞和CD34+细胞分别接种至甲基纤维素半固体培养基中,14 d 后根据典型形态特征计数造血集落数.结果与结论:羊膜间充质干细胞可促进人脐血单个核细胞和 CD34+细胞扩增,扩增后两者在甲基纤维素半固体培养基中能够形成造血祖细胞集落,其造血支持作用与骨髓间充质干细胞相似,两者对比无明显统计学差异.提示,羊膜间充质干细胞体外具有与骨髓间充质干细胞相似的造血支持作用,有可能为造血干细胞体外扩增及临床间充质干细胞与造血干细胞联合移植、提高造血干细胞移植成功率提供一种更加理想的间充质干细胞新来源.     

体外培养慢性粒细胞白血病患者骨髓间充质干细胞的生物学特性

背景:课题组从胎儿骨髓间充质干细胞的培养体系中鉴定出一类贴壁细胞,已证实此类细胞在单细胞水平可以向造血及内皮细胞分化.目的:检测骨髓间充质干细胞的生物学特性,为慢性粒细胞白血病的治疗提供相关的依据.方法:以慢性粒细胞白血病患者骨髓为研究对象,体外培养并扩增原始间充质干细胞,检测其BCR/ABL融合基因的表达、免疫学特性和生长曲线,使用RT-PCR和FISH的方法检测其BCR/ABL融合基因的表达情况.结果与结论:慢性粒细胞白血病患者骨髓来源的间充质干细胞呈成纤维样生长,大部分细胞处于G0/G1期,并且高表达Flk1,CD13,CD29,CD44,用RT-PCR和FISH的方法能够检测出BCR/ABL融合基因的表达.提示慢性粒细胞白血病的白血病基因转化可能发生在比造血干细胞更高的骨髓间充质干细胞水平上,对慢性粒细胞白血病的疾病起源及干细胞移植治疗有重要的意义.     

体外培养慢性粒细胞白血病患者骨髓间充质干细胞的生物学特性

背景：课题组从胎儿骨髓间充质干细胞的培养体系中鉴定出一类贴壁细胞,已证实此类细胞在单细胞水平可以向造血及内皮细胞分化。目的：检测骨髓间充质干细胞的生物学特性,为慢性粒细胞白血病的治疗提供相关的依据。方法：以慢性粒细胞白血病患者骨髓为研究对象,体外培养并扩增原始间充质干细胞,检测其BCR/ABL融合基因的表达、免疫学特性和生长曲线,使用RT-PCR和FISH的方法检测其BCR/ABL融合基因的表达情况。结果与结论：慢性粒细胞白血病患者骨髓来源的间充质干细胞呈成纤维样生长,大部分细胞处于G0/G1期,并且高表达Flk1,CD13,CD29,CD44,用RT-PCR和FISH的方法能够检测出BCR/ABL融合基因的表达。提示慢性粒细胞白血病的白血病基因转化可能发生在比造血干细胞更高的骨髓间充质干细胞水平上,对慢性粒细胞白血病的疾病起源及干细胞移植治疗有重要的意义。     

慢性粒细胞白血病肿瘤干细胞的免疫功能

背景:研究认为间充质干细胞可能是骨髓造血微环境的免疫保护位点。由于慢性粒细胞白血病存在造血微环境异常和免疫异常,所以推测间充质干细胞可能在慢性粒细胞白血病的病理过程中扮演了一个重要角色。目的:观察慢性粒细胞白血病骨髓来源的肿瘤干细胞的免疫学特征,比较其与正常人来源的间充质干细胞是否存在免疫功能的异常。方法:分离正常人和慢性粒细胞白血病患者的骨髓间充质干细胞,分别检测它们对T细胞周期、活化、抑制和增殖的作用。结果与结论:慢性粒细胞白血病和正常志愿者骨髓来源的间充质干细胞形态和表型没有差异,慢性粒细胞白血病患者来源的间充质干细胞抑制T细胞增殖的作用减弱,抑制T细胞周期及活化的能力减弱,慢性粒细胞白血病患者抑制T细胞凋亡的作用增强。提示慢性粒细胞白血病患者骨髓来源的间充质干细胞存在明显的免疫调节功能缺陷,如果使用慢性粒细胞白血病患者自体的间充质干细胞移植治疗可能不是一种很好的选择,对于骨髓增生异常综合征患者最好是选用异基因的间充质干细胞移植。     

转化生长因子β1促进骨髓间充质干细胞迁移与上调Snail表达有关(英文)

背景:转化生长因子β1可促进骨髓间充质干细胞迁移及增殖,但机制尚不清楚。目的:观察转化生长因子β1对体外培养的骨髓间充质干细胞侵袭力的影响,以及对Snail、基质金属蛋白酶2表达的调节作用。方法:采用密度梯度离心结合贴壁法分离、培养大鼠骨髓间充质干细胞。用改良的Transwell小室检测0,0.5,1,2,5,10μg/L的转化生长因子β1对细胞迁移的影响。用脂质体转染针对Snail基因的特异性小分子干扰RNA至骨髓间充质干细胞,观察转染前后Snail和基质金属蛋白酶2的表达。结果与结论:外源性转化生长因子β1对骨髓间充质干细胞迁移的促进作用具有剂量依赖性,在2μg/L时达到最高。在此浓度下,Snail、基质金属蛋白酶2mRNA表达明显增高。Snail基因沉默可以明显抑制转化生长因子β1对基质金属蛋白酶2表达的促进作用。提示转化生长因子β1可通过促进骨髓间充质干细胞基质金属蛋白酶2的表达提高其迁移能力,此作用与上调Snail表达有关。     

大鼠骨髓间充质干细胞血管细胞黏附分子1及细胞间黏附分子1的表达(英文)

背景:骨髓间充质干细胞系统性输注后,何种因素促使其迁移到正确部位尤为关键,目前认为黏附分子在介导骨髓间充质干细胞向缺血或损伤组织迁移过程中起重要作用.目的:观察血管细胞黏附分子1与细胞间黏附分子1在大鼠骨髓间充质干细胞中的表达.方法:采用直接贴壁法体外分离培养大鼠骨髓间充质干细胞,免疫细胞化学染色检测血管细胞黏附分子1及细胞间黏附分子1蛋白的表达,应用免疫荧光直标法在流式细胞仪上检测血管细胞黏附分子1及细胞间黏附分子1抗原的表达率,RT-PCR半定量分析血管细胞黏附分子1及细胞间黏附分子1 mRNA的表达.结果与结论:免疫细胞化学染色结果显示,骨髓间充质干细胞血管细胞黏附分子1呈弱阳性表达,细胞间黏附分子1呈强阳性表达.流式细胞仪检测结果显示,血管细胞黏附分子1表达率为6%,细胞间黏附分子1表达率为100%.RT-PCR检测结果显示,血管细胞黏附分子1 mRNA呈微弱表达,细胞间黏附分子1 mRNA呈高度表达.提示在生理状态下,体外培养的大鼠骨髓间充质干细胞低表达血管细胞黏附分子1,高表达细胞间黏附分子1.     

骨髓与脐带血间充质干细胞的生物学特性比较

目的:为寻找最佳的间充质干细胞来源,比较脐带血来源间充质干细胞与骨髓来源间充质干细胞在生长、细胞表型及多项分化功能方面的差别.方法:实验于2006-06/2007-03在南昌大学医学院第二附属医院分子实验室、江西省医学分子重点实验室,省级重点实验室完成.[1]实验材料:脐带血30份及正常骨髓30份均取自正常自愿捐献者,实验经医院伦理委员会批准.[2]实验方法:无菌条件下采集脐血及骨髓,分离单个核细胞.将脐血及骨髓单个核细胞以1×109L-1接种于培养瓶中,加入含体积分数0.20自体血清,1mmol/L氢化可的松的IMDM培养液,7d后换液,去除悬浮细胞,以后每三四天换液1次,待细胞90%融合后,经胰蛋白酶 乙二胺四乙酸混合消化,传代培养,取第3代细胞,采用流式细胞术鉴定脐带血来源间充质干细胞表面分子,并比较两种不同来源间充质干细胞的生长特点及向脂肪细胞分化能力的差别.结果:[1]骨髓来源间充质干细胞在较早阶段形态即成长梭形,第3代后与脐带血来源间充质干细胞形态无明显差别.[2]培养至第3代的间充质干细胞表面分子检测与脐血单个核细胞比较结果显示,CD166、CD29、CD54表达增高,CD13、CD34、CD45表达降低.[3]脐带血来源间充质干细胞培养的成功率低于骨髓来源间充质干细胞,在较早阶段脐带血来源间充质干细胞增殖快于骨髓来源间充质干细胞,但后期培养过程中脐带血来源问充质干细胞增殖慢于骨髓来源间充质干细胞,两种来源间充质干细胞均能分化为脂肪细胞,且分化能力无明显差别.结论:脐血来源间充质干细胞作为一种新来源的间充质干细胞在生长速度、培养成功率上虽然低于骨髓来源间充质干细胞,但在细胞表型及分化功能方面无明显差别.     

慢性粒细胞白血病患者骨髓来源Flk1~+CD31~-CD34~-间充质干细胞中BCR/ABL融合基因的检测

背景:免疫表型为Flk1+CD31-CD34-的间充质干细胞体内外实验研究均证实在单细胞水平可以向造血及内皮细胞分化。目的:观察慢性粒细胞白血病患者骨髓BCR/ABL融合基因的表达情况。方法:体外培养扩增慢性粒细胞白血病患者骨髓来源原始间充质干细胞,测定其生长曲线,分析其细胞周期及免疫表型,使用RT-PCR和FISH的方法检测其BCR/ABL融合基因的表达情况。结果与结论:慢性粒细胞白血病患者骨髓来源的原始间充质干细胞呈成纤维样生长,大部分细胞处于G0/G1期,并且高表达Flk1,CD13,CD29,CD44,用RT-PCR和FISH的方法能够检测出BCR/ABL融合基因的表达。提出慢性粒细胞白血病的白血病基因转化可能发生在比造血干细胞更高的血液血管干细胞水平上。     

神经干细胞与脑胶质瘤干细胞

所有的肿瘤组织并不是由均一的肿瘤细胞所组成的,不同的细胞具有不同的增殖、浸润和转移能力,亦即肿瘤的异质性.其中存在少数担当着干细胞角色的肿瘤细胞,具有干细胞的基本特性,包括自我更新能力、无限的增殖能力和多向分化潜能,为肿瘤干细胞.神经干细胞具有很强的自我更新机制,获得较少突变即有可能恶性转化,而且干细胞存活时间较长,这意味着干细胞比成熟细胞发生细胞复制的错误几率更大.因外界环境的刺激而发生突变的机会更多,最终形成脑胶质瘤干细胞,同时调节神经干细胞增殖和自我更新的基因在脑胶质瘤的脑胶质瘤干细胞中也表达,这也是支持神经干细胞是脑胶质瘤干细胞来源的;也有推测认为它可能起源于已分化的细胞,由这些细胞突变发生去分化得来,并通过基因突变而获得了干细胞自我更新的特性,从而形成脑胶质瘤干细胞.通过探讨神经干细胞与脑胶质瘤干细胞,为脑胶质瘤的治疗提供依据.     

胃肠道干细胞及胃肠疾病的干细胞移植治疗

目的:对胃肠道干细胞的特性、分布及其调控机制等进行总结归纳,分析胃肠道干细胞、造血干细胞、骨髓干细胞移植在胃肠疾病临床治疗中的应用情况及其存在的问题。资料来源:应用计算机检索CNKI2004/2006有关胃肠道干细胞及干细胞移植在胃肠道疾病治疗中应用的相关文章,检索词“胃肠道干细胞,胃干细胞,肠道干细胞,胃肠疾病”,限定文章语言种类为中文。应用计算机检索PUBMED1999/2004相关文章,检索词:gastrointestinal stem cell,gastric stem cells,intestinal stem cell,限定文章语言种类为English。资料选择:对资料进行初审,纳入标准:①有关胃肠道干细胞、胃干细胞、肠道干细胞增殖分化特性及其调控机制方面的相关文章;②有关胃肠疾病的干细胞移植治疗方面的相关文章。排除标准:①重复性研究;②陈旧性文献。资料提炼:共搜集到上述文章41篇,按上述标准纳入30篇,其中有关胃肠道干细胞及其在胃肠道损伤修复中应用的相关研究文章8篇,胃干细胞及其在胃肠疾病治疗中应用的相关研究文章3篇,肠干细胞及其在胃肠疾病治疗中应用的相关研究文章7篇,造血干细胞、骨髓干细胞移植在胃肠疾病及其他相关疾病治疗中应用的相关文章12篇。资料综合:胃肠道干细胞具有自我更新与增殖能力,一般认为,胃的干细胞定位于腺体峡部区域,肠道干细胞位于肠黏膜隐窝的基底部,胃肠道干细胞能根据组织需要调解细胞增生分化的速度,对维持胃肠黏膜更新及保持组织内环境稳定起着重要作用。肿瘤干细胞理论认为胃肠道肿瘤的发生与干细胞的突变有关,胃肠道干细胞可能是基因突变和积累的靶细胞。造血干细胞、骨髓干细胞移植在胃癌、炎症性肠病、胃肠道上皮损伤等胃肠道疾病治疗中得到广泛应用,并取得较好疗效,但胃肠干细胞的特异性标志及干细胞移植治疗胃肠道疾病的作用机制有待于深入研究。结论:胃肠道干细胞在胃肠道组织及细胞损伤修复中发挥着关键作用,胃肠干细胞突变以及生长调控机制异常可能导致胃肠肿瘤,在胃肠疾病治疗中造血干细胞、骨髓干细胞移植具有广阔的应用前景。     

表皮干细胞与毛囊干细胞生物学特性的比较

背景:获取更合适的组织工程皮肤种子细胞可使皮肤功能得到更好的修复。目的:分离、培养表皮干细胞和毛囊干细胞,并比较两种细胞的生物学特性。方法:体外分离培养2月龄新西兰兔表皮干细胞和毛囊干细胞,取生长良好的第2,3,6代细胞观察其生物学特性。结果与结论:毛囊干细胞较表皮干细胞贴壁快,具有更高的增殖活性。免疫组化及荧光定量PCR检测显示毛囊干细胞β1整合素、角蛋白19蛋白及mRNA表达均明显高于表皮干细胞。提示作为皮肤组织工程的种子细胞,毛囊干细胞较表皮干细胞更具优势。     

间充质干细胞在造血干细胞移植中的应用

应用计算机检索PubMed数据库2000—01／2008-01期间有关间充质干细胞与造血干细胞移植相关的文章，检索词为“MSC，HSCT”，限定文章语言种类为English。同时计算机检索维普数据库2000-01／2008-01期间相关文章，检索词为“间充质干细胞，造血干细胞移植”，限定文章语言种类为中文。并查阅与干细胞实验研究有关的书籍。对资料进行初审，选取符合要求的有关文章查找全文。纳入标准：①有关间充质干细胞对造血干细胞移植影响的研究。②造血干细胞移植机制的相关性研究。排除标准：重复性文章。共收集到66篇相关文献，28篇文献符合纳入标准。临床资料显示，两者共移植安全可行，可提高移植效果，降低近期死亡率。但其确切效果还需前瞻性随机对照临床实验进一步证实。     

Hematopoietic stem cell

Hematopoietic stem cells (HSCs) maintain themselves over cell divisions (self-renewal) and produce all kinds of blood cells (multi-potency). Depletion of these cells eventually causes hematopoietic failure, while deregulated HSC division causes development of myeloproliferative disorders and leukemias. HSCs can be prospectively purified to nearly homogeneity in mice, but such a high-level purification has not been achieved in humans. HSCs are localized to an anatomical place called 'niche'. Specialized osteoblasts arrayed on the endosteum of cavernous bone and sinusoidal endothelial cells located at the distant position from the endosteum are the two representative candidates of such an HSC niche. A number of adhesion molecules and signaling molecules are thought to comprise the niche-HSC synapse. HSCs divide only once in 1-2 months. Both environmental signaling from the niche and HSC-autonomous molecular programs contribute to the quiescent state of HSCs, which is essential for the maintenance of HSC self-renewal capacity and homeostasis of blood production.     

Mesenchymal stem cells

About 40 years ago Friedenstein described stromal cells in the bone marrow that were spindle shaped and proliferate to form colonies. These cells attach to plastic and are able to differentiate under defined in vitro conditions into multiple cell types present in many different tissues, e.g. osteoblasts, chondroblasts, adipocytes, etc. Later on these cells, obtained from postnatal bone marrow, were called mesenchymal stem cells (MSC) or stromal stem cells. Recently the presence of somewhat similar cells has been demonstrated in many other tissues too. In spite of extensive attempts to characterize these cells we are still lacking definitive in vivo markers of MSC although retrospective functional data strongly support the existence of common adult stem cells that have the capacity to differentiate along various specific differentiation lineages. Since MSC can be rather easily isolated from the bone marrow and can also be expanded in vitro they have become a prime target for researchers of tissue regeneration. These cells have now been extensively used for transplantation experiments in animals and also for some therapeutic trials in humans. However, much new research is needed to learn enough on the molecular mechanisms of MSC differentiation to evaluate their full capacity for tissue regeneration.     

Cancer stem cells

The cancer stem cell (CSC) hypothesis has had a major effect on the fields of cancer cell biology and clinical oncology. CSCs were originally described in hematologic malignancies, and subsequently in a variety of solid tumors. Their unique biological characteristics, including self-renewal capability, stem cell signaling pathways, relative quiescence and resistance to standard chemotherapy and radiotherapy are providing researchers and clinicians with new challenges. One important outcome of this new perspective on tumors is the recognition that effective treatment approaches will need to target both the rapidly proliferating bulk tumor cells, and the quiescent CSCs, which contain the ability to reestablish the malignancy when treatment is withdrawn. The clinical laboratory will undoubtedly see an influx of new molecular and histopathological tests to augment initial diagnosis, treatment decisions, and prognostic monitoring of cancer patients related to identifying and quantifying these as CSCs.     

Muscle-derived stem cells

The existence of cells with stem cell-like abilities derived from various tissues can now be extended to include the skeletal muscle compartment. Although researchers have focused on the utilization of these cells with regard to their myogenic capacity, initially exploring more efficient cellular therapy treatments for muscular dystrophy, it is becoming increasingly apparent that such cells may one day be used in the treatment of non-myogenic disorders. Evidence regarding the existence and differentiation capacity of muscle-derived stem cells is discussed, along with current theories regarding their proposed position within the myogenic hierarchy.     

Leukaemic stem cells

All haemopoietic cell lineages arise from multipotential self-renewing stem cells that give rise to committed progenitor cells. These progenitor cells subsequently differentiate into more lineage-committed cells with a restricted range of plasticity. A hierarchical order is considered to exist, where lineage commitment and differentiation are thought to be irreversible. As cells differentiate, they gradually lose the ability to self-renew. The most primitive haemopoietic progenitor cells have the ability to reconstitute long-term haemopoiesis in myeloablated recipients. However, as cells differentiate, there is an orchestrated silencing of some genes and activation of others, resulting in lineage commitment and generally a reduction in proliferative ability. Here, we discuss potential differences between normal and leukaemic stem cells, some of which may have therapeutic implications.     

Mesenchymal stem cells

About 40 years ago Friedenstein described stromal cells in the bone marrow that were spindle shaped and proliferate to form colonies. These cells attach to plastic and are able to differentiate under defined in vitro conditions into multiple cell types present in many different tissues, e.g. osteoblasts, chondroblasts, adipocytes, etc. Later on these cells, obtained from postnatal bone marrow, were called mesenchymal stem cells (MSC) or stromal stem cells. Recently the presence of somewhat similar cells has been demonstrated in many other tissues too. In spite of extensive attempts to characterize these cells we are still lacking definitive in vivo markers of MSC although retrospective functional data strongly support the existence of common adult stem cells that have the capacity to differentiate along various specific differentiation lineages. Since MSC can be rather easily isolated from the bone marrow and can also be expanded in vitro they have become a prime target for researchers of tissue regeneration. These cells have now been extensively used for transplantation experiments in animals and also for some therapeutic trials in humans. However, much new research is needed to learn enough on the molecular mechanisms of MSC differentiation to evaluate their full capacity for tissue regeneration.