人脐血CD34+造血干细胞的分离和体外扩增培养问题

目的通过细胞形态学观察以及计数法对分离得到的脐血CD34+造血干细胞进行研究,掌握脐血CD34+造血干细胞的生长规律和体外培养方法。方法采用Isolex50免疫磁珠法分离纯化脐血CD34+造血干细胞。在体外扩增培养中,将细胞因子SCF、IL-3、IL-6、G-CSF、EPO组合成不同实验组进行诱导,以检测单个核细胞数和集落形成单位(CFU)的形成率来找到细胞培养较好的细胞因子组合。结果本研究中使用细胞因子的细胞培养孔的单个核细胞数明显大于无细胞因子的细胞培养孔,使用此5种细胞因子联合对脐带造血干细胞培养效果最好。结论研究结果表明,不同的细胞生长因子组合会对培养CD34+造血干细胞产生不同的作用效果,对单个核细胞的增殖与扩增有明显的促进作用。     

人骨髓间充质干细胞联合细胞因子的无血清培养体系体外扩增脐血干细胞研究

目的 探讨以人骨髓间充质干细胞(BM-MSC)为基质的无血清培养方法，体外扩增脐血造血干细胞(CBSC)，研究BM-MSC支持造血的功能和体外扩增时间的选择。方法 用含血小板生成素(TPO)、干细胞因子(SCF)、flt3／flk2配体(FL)和粒细胞-集落刺激因子(G-CSF)的无血清培养液，以人BM-MSC为基质，比较体外扩增脐血CD34^ 细胞7d及14d后总细胞(TC)、CD34^ 细胞和集落形成单位(CFU)数增加倍数。结果在以人BM-MSC为基质及TPO、FL、SCF和G-CSF这四种细胞因子作用下，经体外扩增7d后TC、CD34^ 细胞、CFU-GM和CFU-C数较起始分别增加了87、16、15和26倍；14d后较起始分别增加了427、38、125和104倍，与7d扩增倍数有显著性差异。结论以人BM-MSC为基质的无血清体外培养体系可以有效扩增CBSC，有潜在的临床应用价值，扩增时间应以14d为宜。     

转HOXB4基因人骨髓MSC促进脐血CD34~+细胞体外扩增

目的 研究转HOXB4基因的人骨髓间充质干细胞(MSC)对脐血CD34~+细胞体外扩增的支持作用.方法 用病毒载体质粒转染包装细胞293T,收获病毒上清(VCM)感染MSC后,用潮霉素筛选获得转HOXB4的MSC(MSC-HOX).分别将MSC(对照组)与MSC-HOX细胞(实验组)作为CD34~+细胞体外扩增的饲养层细胞,结合细胞因子Fh3/Flk3配体(FL)、促血小板生成素(TPO)、干细胞因子(SCF)和粒细胞-集落生长因子(G-CSF)体外扩增脐血CD34~+细胞10 d,收集所有脐血细胞,检测细胞总数、CD34~+细胞总数,集落细胞形成单位(CFU)数.结果 生产重组逆转录病毒可有效将HOXB4基因转入靶细胞内并表达.脐血CD34~+细胞经体外扩增10 d后,细胞总数和CFU数两组间差异无统计学意义(P>0.05),脐血CD34~+细胞总数和比例高于对照组(P<0.05).结论 转HOXB4基因的人骨髓MSC在脐血CD34~+细胞体外扩增中,可保持CD34~+细胞未分化状态,有潜在的应用价值.     

大鼠骨髓间充质干细胞的培养、扩增与鉴定

目的:建立大鼠骨髓间充质干细胞(MSCs)分离及培养的方法,探讨体外培养MSCs的生物学特性.方法:采用密度梯度离心法和贴壁筛选法纯化MSCs,镜下连续观察细胞的形态变化.流式细胞仪鉴定其表面抗原CD29、CD34、CD44和CD45的表达情况.结果:原代MSCs呈集落状生长,细胞呈梭形、纺锤形,呈放射状排列.传代后大都均匀分布生长,细胞生长迅速,倍增时间约45.5 h.流式细胞术鉴定表明,培养的第3代和第5代MSCs CD44、CD29阳性,CD45和CD34阴性.结论:采用密度梯度离心法结合贴壁筛选法可成功分离和培养大鼠的骨髓MSCs,MSCs可以作为组织工程中种子细胞的来源.     

骨髓造血干细胞分离 保存的研究

目的探讨骨髓造血干细胞分离及保存的方法。方法应用羟乙基淀粉(HES)或percoll液分离骨髓造血干细胞;联合应用二甲基亚砜(DMSO)和HES对造血干细胞进行液氮保存。应用血细胞计数法、锥虫蓝拒染实验、粒-巨噬细胞集落生成单位(CFU-GM)的体外培养等方法对造血干细胞冷冻前后的有核细胞(NC)数、存活率、体外分化能力进行检测;应用流式分析法计数CD34+细胞数。结果利用HES沉降法分离的单个核细胞数、CD34+细胞数、CFU-GM集落数均比percoll液离心法明显增多;骨髓造血干细胞冷冻保存1年后的有核细胞数、CD34+细胞数、锥虫蓝活率、CFU-GM集落计数与保存前差异无统计学意义。结论HES法分离骨髓造血干细胞方法安全、有效;通过程序降温,联合使用DMSO及HES的低温冻存方法对骨髓造血干细胞的长期保存是适合的。     

人脐血源性间充质干细胞的体外培养及生物学鉴定

目的 建立体外培养、扩增人脐带血间充质干细胞(MSCs)的方法．初步鉴定其生物学特性。方法 无菌条件下取正常足月剖宫产的脐带血,用淋巴细胞分离液分离脐血的单个核细胞．以偏酸性的Mesencult^TM作为培养基进行培养和纯化获得贴壁细咆层,测定MSCs的生长曲线．用流式细胞技术分析细胞的表面抗原。结果 来源于脐血的单个核细胞存体外用合适的培养基培养,细胞贴壁后出现破骨样及间充质样的细胞。间充质细胞为成纤维样的细胞形态,并表达MSCs相关的抗原CD29、CD44、CD105。但不表达CD34、CD45、CD106和HLA-DR。这与源于骨髓的MSCs一致。结论 脐带血中含有的MSCs可在体外培养、扩增,能够为实验和临床的应用提供一种新的间充质干细胞来源。     

骨髓间充质干细胞旁分泌HGF体外调控肝星状细胞

目的 探讨大鼠骨髓间充质干细胞（BMSCs）与肝星状细胞（HSCs）体外共培养体系中,BMSCs旁分泌肝细 胞生长因子（HGF）对 HSCs 增殖、凋亡、活化的影响。方法 全骨髓贴壁法分离、培养、纯化大鼠 BMSCs,另培养 HSCs。6 孔板半透膜建立上下两层细胞非直接接触共培养体系,实验设 H 组（HSCs 单独培养）、H-H 组（HSCs 与 HSCs共培养）、M-H组（BMSCs与HSCs共培养）、M-H-C组（BMSCs与HSCs共培养并加c-met抑制剂）,各组细胞培 养48 h后,流式细胞仪鉴定BMSCs,检测HSCs凋亡率,MTT法检测HSCs的增殖,免疫荧光共聚焦定量检测HSCs中 α-肌动蛋白（α-SMA）的表达量,ELISA法检测共培养体系上清液中HGF的浓度。结果 MSCs高表达阳性表面分子 CD29、CD90,低表达造血细胞表面标记CD45;BMSCs能明显抑制HSCs的增殖、活化并促进其凋亡,且M-H组上清液 中HGF的浓度明显高于其他组。结论 BMSCs与HSCs共培养过程中,BMSCs通过旁分泌HGF促进HSCs的凋亡,抑 制HSCs的增殖、活化。     

六种重组细胞因子对人骨髓造血细胞的体外扩增效应

目的 :观察造血生长因子对骨髓CD34 +细胞的扩增效应及扩增细胞的分化特性。方法 :采用重组人白细胞介素 (rhIL) 1 +rhIL 3+rhIL6 +重组人粒细胞集落刺激因子 +重组人粒 巨噬细胞集落刺激因子和重组人干细胞因子对人骨髓单个核细胞 (MNC)进行刺激 ,动态观察了经上述细胞因子作用后MNC的增殖效应及液态扩增培养后粒 巨噬集落形成率 (CFU GM ) ,利用流式细胞技术 (FACS)分析了扩增前后CD34 +细胞及其亚群的动态变化。结果 :经上述 6种细胞因子作用 2 4d ,骨髓MNC总数是原代MNC的 38.33倍 ,液态扩增 1 0d后 ,CFU GM的数量达高峰 ,是原代MNC的 1 9.0 4倍 ,2 4d时CFU GM总数降至 3.94倍 ,且集落明显小于扩增初期。扩增至 6～ 1 0d ,CD34 +细胞总数是原代MNC的 1 36 .40～ 90 .40倍 ,1 9d时降为 6 1 .40倍。结论 :外源性造血生长因子对CD34 +骨髓细胞具有较强的扩增效应 ,选择适宜的扩增时间是保证骨髓移植后尽快重建造血功能的重要环节。     

907例复温脐血造血干细胞质量的分析

目的:探讨深低温冻存和复温的过程对脐血造血干细胞(HSCs)生物学功能的影响。方法:应用SPSS23软件,分析907例经-196℃冻存脐血复温后有核细胞活率、HSCs活率,有核细胞总数(TNC)、CD34⁺细胞总数、集落形成单位(CFU)回收率,对各检测数据进行冻存前后的差异性分析。结果:脐血复温后TNC回收率为(86.86±5.99)%,冻存前后有显著差异(Z=26.09,P<0.01);CD34⁺细胞总数回收率为(120.74±36.32)%,冻存前后有显著差异(Z=12.63,P<0.01);CFU回收率为(96.31±40.82)%,冻存前后有显著差异(Z=6.34,P<0.01)。有核细胞活率为(91.33±4.02)%;HSCs活率为(98.86±0.13)%。结论:现阶段深低温冻存和复温的技术,对HSCs活率影响很小,对HSCs生物学功能存能在一定程度影响。各检测数据与冻前相比均有显著差异。临床应用前对脐血的质量评估应以复温后的检测数据为主要依据。     

脐带血血清培养体系扩增脐带间充质干细胞及其生物学特征的实验研究

目的:通过观察以人脐带血血清为主体的培养体系对脐带间充质干细胞(Mesncllymal stem Ce]ls,MSCs)体外培养扩增的影响,探讨人脐带血血清替代动物血清用于培养临床组织工程用干细胞的可行性.建立从足月脐带分离间充质干细胞和体外传代培养技术,并对其生物学特性进行研究.方法:采用双酶法分离脐带间充质干细胞.并通过传代进行纯化和扩增培养.绘制生长曲线:用流式细胞仪检测脐带表面抗原及细胞周期.结果:成功建立了以人脐带血血清为主体的培养体系的脐带间充质干细胞培养扩增的方法:流式细胞仪检测结果显示,贴壁细胞均表CD44、CD29,低表达CD106,不表达追血细胞表型CD14、CD34、CD45和内皮细胞表型CD31,也不表达HLADR:细胞倍增时间为30h,细胞周期分析表明,G0-G1期和+C2+M期所占比例分别为78.84%和11.16%.结论:应用灭活脐带血清培养体系可成功扩增人脐带间充质干细胞.培养的细胞具有阃充质干细胞的基本特性.为建立间充质干细胞库和临床应用提供了理论依据.     

Cardiac-derived stem cells

Current scientific dogma holds that cardiomyocyte stem cells do not exist in the adult mammalian heart, and furthermore, that there is little, if any, potential for the regeneration of damaged myocardium. In order to approach this topic, I have begun with a brief overview of advances in stem cell research in other organ systems, such as the bone marrow and the brain. Very recent progress in cardiac stem cell research is then discussed, which indicates that a cardiomyocyte progenitor cell contributes to cardiomyocyte replacement throughout life. This progenitor cell may reside in the heart itself, or derive from a circulating marrow stem cell.     

Targeting cancer stem cells

Recent evidence has demonstrated the existence of a small subset of the tumour mass that is wholly responsible for the sustained growth and propagation of the tumour. This cancer stem cell (CSC) compartment is also likely to be responsible both for disease relapse and the resistance to therapy that often accompanies relapse. The evidence for CSCs in various malignancies is presented. The failure of existing therapeutics to eradicate CSCs suggests that they are relatively resistant to present cancer treatments. This resistance may reflect the preservation of normal stem cell protective mechanisms, such as an increased expression of drug efflux pumps or alterations in apoptotic, cell cycle and DNA repair mechanisms. Targeting these mechanisms, and taking advantage of potential differences in the biology of normal stem cells and CSCs, such as differences in surface phenotype, self renewal/quiescence and stem cell-niche interactions are discussed and preliminary preclinical or clinical data are presented. Finally, the authors give their opinion of the direction in which one must travel to successfully target the CSC and improve treatment outcomes in malignant disease.     

Resident cardiac stem cells

The introduction of stem cells in cardiology provides new tools in understanding the regenerative processes of the normal and pathologic heart and opens new options for the treatment of cardiovascular diseases. The feasibility of adult bone marrow autologous and allogenic cell therapy of ischemic cardiomyopathies has been demonstrated in humans. However, many unresolved questions remain to link experimental with clinical observations. The demonstration that the heart is a self-renewing organ and that its cell turnover is regulated by myocardial progenitor cells offers novel pathogenetic mechanisms underlying cardiac diseases and raises the possibility to regenerate the damaged heart. Indeed, cardiac stem progenitor cells (CSPCs) have recently been isolated from the human heart by several laboratories although differences in methodology and phenotypic profile have been described. The present review points to the potential role of CSPCs in the onset and development of congestive heart failure and its reversal by regenerative approaches aimed at the preservation and expansion of the resident pool of progenitors.     

Human muscle stem cells

Stem cells are unspecialized cells that have been defined in many different ways but they have two important characteristics that distinguish them from other cells in the body. First, they can replenish their numbers for long periods through cell division. Second, after receiving certain chemical signals, they can produce, through asymmetric cell division, a progeny that can differentiate or transform into specialized cells with specific functions, such as heart, nerve or muscle. In recent years, stem cells have received much attention owing to their potential use in cell-based therapies for human neurodegenerative diseases such as Parkinson's disease, stroke and muscular dystrophies. However, many questions need to be resolved before stem cells with myogenic potential are used in clinical standard protocols.     

Targeting cancer stem cells

Recent evidence has demonstrated the existence of a small subset of the tumour mass that is wholly responsible for the sustained growth and propagation of the tumour. This cancer stem cell (CSC) compartment is also likely to be responsible both for disease relapse and the resistance to therapy that often accompanies relapse. The evidence for CSCs in various malignancies is presented. The failure of existing therapeutics to eradicate CSCs suggests that they are relatively resistant to present cancer treatments. This resistance may reflect the preservation of normal stem cell protective mechanisms, such as an increased expression of drug efflux pumps or alterations in apoptotic, cell cycle and DNA repair mechanisms. Targeting these mechanisms, and taking advantage of potential differences in the biology of normal stem cells and CSCs, such as differences in surface phenotype, self renewal/quiescence and stem cell–niche interactions are discussed and preliminary preclinical or clinical data are presented. Finally, the authors give their opinion of the direction in which one must travel to successfully target the CSC and improve treatment outcomes in malignant disease.     

Controlled differentiation of stem cells

The extracellular microenvironment plays a significant role in controlling cellular behavior. Identification of appropriate biomaterials that support cellular attachment, proliferation and, most importantly in the case of human embryonic stem cells, lineage-specific differentiation is critical for tissue engineering and cellular therapy. In addition to growth factors and morphogenetic factors known to induce lineage commitment of stem cells, a number of scaffolding materials, including synthetic and naturally-derived biomaterials, have been utilized in tissue engineering approaches to direct differentiation. This review focuses on recent emerging findings and well-characterized differentiation models of human embryonic stem cells. Additionally, we also discuss about various strategies that have been used in stem cell expansion.     

自体脑脊液定向诱导骨髓间充质干细胞分化为神经干细胞

目的 探讨自体脑脊液定向诱导骨髓间充质干细胞分化为神经干细胞的可行性.方法 采用自体脑脊液为诱导剂,体外诱导骨髓源性间充质干细胞.从形态学、免疫组化法、反转录聚合酶链反应(RT-PCR)方法对诱导后细胞进行鉴定.结果 自体脑脊液诱导后的间充质干细胞呈现神经干细胞改变,可分化为神经元、星形胶质细胞和少突胶质细胞,并表现相应的特征结构和生物学特性.结论 白体脑脊液能定向诱导骨髓间充质干细胞向神经样细胞转化.