骨髓间充质干细胞和内皮祖细胞移植促进血流重建的比较

目的:比较骨髓间充质细胞(Bone Marrow Mesenchymal Stem Cells,BM/MSC)和骨髓源内皮祖细胞(Bone Marrow Endothelialprogenitor cells,BM/EPC)移植促进血流重建的效果,为进一步优化骨髓干细胞移植治疗肢体缺血提供理论基础。方法:获取Lewis大鼠骨髓单个核细胞,在体外培养分化为MSC和EPC。采用Lewis大鼠建立单侧后肢缺血模型。在模型建立后3天,将0.8mlD-Hanks液注入大鼠缺血侧后肢,为对照组(n=6);将8×106个骨髓MSC植入大鼠缺血侧后肢,为MSC组(n=6);将体外培养的8×106个EPC植入大鼠缺血侧后肢,为EPC组(n=6)。细胞移植后3周行缺血大鼠后肢动脉造影,检测缺血侧后肢侧支血管数;获取缺血侧后肢腓肠肌,分别行CD31和α-SMA免疫组化染色,计算毛细血管密度和小动脉密度。结果:MSC组与EPC组侧支血管数无显著性差异,二者均高于对照组;EPC组毛细血管密度明显高于MSC组,二者均高于对照组;MSC组与EPC组小动脉密度无显著性差异,二者均高于对照组。结论:骨髓间充质干细胞移植和内皮祖细胞移植均能够明显促进血流重建,而且骨髓间充质干细胞在治疗肢体缺血性疾病中的优势应该受到重视。     

内皮祖细胞分离培养及应用技术新进展

内皮祖细胞(Endothelial progenitor cells,EPCs)是能够增殖、迁移、粘附并分化为血管内皮细胞潜能的一种原始细胞,在修复血管内皮和促进血管生成中具有重要的作用。EPC来源于骨髓,存在于骨髓、外周血、脐带血,新研究表明在脂肪组织、心肌中也能发现EPC的存在。EPC与干细胞的细胞表面标志物相似,功能上亦接近干细胞,但不具有自我更新的功能。近年来EPC已成为热点问题,对疾病诊断,预后判断和靶向治疗方面发挥重要作用,在冠状动脉粥样硬化性疾病、糖尿病血管病变、恶性肿瘤等治疗中全身或局部注射EPC具有更广泛的前景和应用价值。但关于分离培养EPC的方法及细胞表面标志物不完全相同,报道较少,至今尚没有形成统一的标准,本文就对于内皮祖细胞基本现状、分离培养技术、分选鉴定及临床应用方面做一综述。     

内皮祖细胞对急性肺损伤保护作用的研究进展

内皮祖细胞（EPC）是一种多潜能细胞,主要来源于骨髓。外周血EPC可以参与修复多种血管内皮细胞损伤的疾病。目前研究证实EPC通过动员、迁移、归巢和分化等步骤在受损的肺组织处参与内皮细胞修复,调节失控的炎症反应,增强抗氧化能力,对修复和维持肺泡毛细血管屏障的完整性起着重要作用。EPC在心血管疾病和组织工程领域应用研究的成功,为EPC在急性肺损伤的治疗提供了新的思路。     

内皮祖细胞及其在组织工程中的应用

目前,组织工程化血管的构建和工程化组织器官的血管化因内皮种子细胞的扩增能力不足和生物活性不强而受到限制。内皮祖细胞(EPC)是内皮细胞的前体细胞。出生后,EPC主要存在于骨髓,可向外周血液缓慢释放,参与机体缺血组织的血管重建和损伤血管的重新内皮化。现对EPC的来源、分布、表型特征、动员、分化、归巢、分离、培养与鉴定等生物学特性和EPC在组织工程中的应用进行了全面的综述,并指出目前存在的问题和研究方向。     

成人外周血内皮祖细胞的分离、扩增及鉴定

目的: 从健康成人外周血中分离获取内皮祖细胞(endothelial progenitor cell ,EPC),并探索EPC体外扩增所需的条件.方法: 密度梯度离心法获取外周血单个核细胞,接种在人纤维连接蛋白包被培养板,用含VEGF的培养液培养7 d,收集贴壁细胞,激光共聚焦显微镜和流式细胞仪鉴定EPC.结果: 从成人外周血可分离获得EPC; 激光共聚焦显微镜可成功鉴定EPC;VEGF和人纤维连接蛋白对该细胞的生长有促进作用.结论: 外周血EPC的分离获取及其体外扩增条件的初步认识,为EPC的进一步研究及临床应用奠定了基础.     

不同氧浓度对骨髓源内皮祖细胞分泌促血管新生相关生长因子的影响

目的:观察培养环境不同氧浓度对骨髓源内皮祖细胞分泌促血管新生相关生长因子的影响。方法:利用密度梯度离心技术分离SD大鼠骨髓单个核细胞,向内皮祖细胞进行诱导分化、扩增、培养和鉴定。然后在不同氧浓度(1%,5%,21%)的环境中培养,于第3天,7天,10天采用酶联免疫吸附试验检测内皮祖细胞分泌血管内皮生长因子(Vascular endothelial growth factor,VEGF)、基质细胞衍生因子-1α(Stromal-derived factor-1α, SDF-1α)、胰岛素样生长因子I(Insulin-like growth factor-I, IGF-I)等生长因子的水平。结果:第3天,不同氧浓度下各组EPC分泌VEGF、SDF-1α、IGF-I无明显差异(P均0.05)。第7天和第10天,各组EPC分泌IGF-I无明显差异(P均0.05);但是和21%氧浓度相比,相对低氧浓度(1%和5%)能够明显增强EPC分泌VEGF和SDF-1α(P均0.001)。结论:适当时间的低氧环境培养能够显著刺激内皮祖细胞分泌VEGF和SDF-1α,进而增强其促血管新生能力。     

脐血源EPC联合造血干细胞移植在造血重建和GVHD中作用研究

针对脐血源内皮祖细胞(endothelial progenitor cell,EPC)与造血干细胞(hematopoietic stem cell,HSC)联合移植方式进行研究,并探讨移植后的造血细胞重建及移植物抗宿主病(graft versus host disease,GVHD)的影响。构建人-鼠移植EPC与HSC模型,采用移植HSC的小鼠为参考对比,分析移植后的体内植入及造血重建状况,并分析小鼠有无GVHD相关症状,最后采用病理对GVHD进行实验验证。EPC与HSC联合移植小鼠组(n=10)的实验组,生存的平均时间约54 d,长时间生存率接近70%;单纯移植小鼠组(n=10)的实验组中,生存的平均时间约51 d,长时间生存率达到60%;EPC与HSC联合移植小鼠组与单纯移植小鼠组进行比较,生存时间有显著的增高趋势,但经统计学分析两者比较无显著差异性(p=0.314)。在血细胞恢复实验中,联合移植组相比较单纯组,随着时间的增加,HBG、WBC及PLT的恢复值均有增高趋势。联合移植组小鼠骨髓中人CD45比例为(18.21±10.57)%,单纯组骨髓中人CD45嵌合比例为(13.27±6.36)%,无明显统计学差异性(p=0.308);在联合移植组脾脏中的嵌合比例为(5.14±2.97)%,在单纯移植组比为(4.91±7.56)%,无显著统计学差异(p=0.840)。在联合移植组中检查到2只小鼠带有GVHD病症,表现是轻中度活动度明显减少和体重有下降趋势;单纯移植组检查到3只小鼠带有GVHD病症,表现为活动度下降、体重下降、弓背姿势及耸毛现象;共同点为带有GVHD症状的小鼠均有骨髓与小肠不同损伤情况。本研究初步认为脐血源内皮祖细胞与造血干细胞联合移植的方式,能够对移植后的造血系统重建及移植物抗宿主病产生一定的积极影响,但是效果并不显著。     

TLR4在四氯化碳诱导的肝硬化大鼠骨髓血窦内皮细胞的表达

目的：检测Toll样受体4（TLR4）在四氯化碳诱导的肝硬化大鼠骨髓血窦内皮细胞的表达,为进一步研究肝硬化时骨髓损伤的发生机制提供实验依据。方法：选择Wistar大鼠给予腹腔注射CCl4,一周两次,建立肝硬化大鼠模型。分别于建模8周和12周检测大鼠血浆内毒素的水平,免疫组化检测大鼠骨髓血窦内皮细胞上TLR4的表达情况,RT-PCR测定骨髓组织中TLR4mRNA的表达,分析TLR4的表达与内毒素血症间的关系。结果：给予CCl4 8周和12周时,对照组大鼠血浆内毒素水平分别为（0．216±0．024）Eu／ml和（0．133±0．022）Eu／ml,模型组大鼠血浆内毒素水平分别为（0．626±0．021）Eu／ml和（0．725±0．031）Eu／ml,分别较对照组显著升高,差异均有统计学意义（P〈0．001）;骨髓血窦内皮细胞TLR4蛋白表达及骨髓组织中TLR4mRNA的表达均显著高于对照组,差异均有统计学意义（P〈0．05）。大鼠骨髓TLR4蛋白和mRNA表达与血浆内毒素水平均呈显著正相关（r=0．841,0．803,P均〈0．001）。结论：CCl4诱导的肝硬化大鼠骨髓血窦内皮细胞TLR4表达升高,并伴随大鼠内毒素血症的发生,提示肝硬化时肠源性内毒素血症可能参与了骨髓的造血功能的损害和病变。     

内皮祖细胞在血管组织工程研究中的应用现状

组织工程血管是修复紫绀型先心病右心室流出道的潜在替代材料,实验研究表明外周血内皮祖细胞(endothelial progenitor cell,EPC)可作为构建组织工程血管的良好种子细胞。现阶段国内外对组织工程血管的研究方兴未艾,EPC在血管组织工程研究中的应用还处在体外培养或动物实验阶段,尚无临床应用报道。近来某些基础研究的成果对于新生血管的形成和EPC的自动募集机制有了合理的解释,其中缺氧被认为是重要的始动因素,这些研究成果也为EPC作为种子细胞应用于血管组织工程提供了理论基础。所以,阐明EPC的低氧诱导机制及其在血管组织工程的应用必将有助于复杂紫绀型先心病的外科治疗。本文主要介绍了目前该领域研究现状及相关研究基础的进展,并总结提出了在EPC研究和未来临床应用中需解决的问题。     

小鼠外胎盘锥细胞与层粘连蛋白相互作用机制的研究

本文用LN及含其活性位点序列的合成肽段cYIGSR和RGDS对小鼠EPC细胞与LN的相互作用机制进行研究。结果表明:合成肽段cYIGSR和RGDS能促进EPC粘附并具协同效应。cYIGSR还能促进EPC扩展与次生TGCs迁移。LNA链上RGD和B_1链上YIGSR两个活性位点协同地参与了LN对EPC的粘附、扩展以及次生TGCs的迁移的促进作用。cY R合用不能完全竞争性抑制EPC与LN的结合,说明还有其他作用位点存在。     

粒细胞集落刺激因子对失代偿期肝硬化患者骨髓干细胞的动员效果及安全性观察

目的观察失代偿期肝硬化患者行自体骨髓干细胞移植前粒细胞集落刺激因子（G-CSF）对骨髓干细胞的动员效果及安全性。方法在51例失代偿期肝硬化患者行自体骨髓干细胞经肝动脉移植术前,连续2 d给予G-CSF 4μg/（kg·d）动员骨髓干细胞。抽取骨髓的当日化验血常规、肝肾功等指标;从患者髂后上棘抽取骨髓150-200 ml,分离收集骨髓单个核细胞并计数,应用流式细胞仪检测CD34＋细胞并计数,观察应用G-CSF期间不良反应的类型和发生率。患者治疗前后比较采用配对t检验进行统计学分析。结果G-CSF皮下注射后,外周血白细胞由术前（3.31±0.96）×10^9/L升至（11.35±1.92）×10^9/L（P〈0.01）,骨髓单个核细胞数（1.91±0.83）×10^9/kg,CD34＋细胞为（2.02±1.29）×10^7/kg;患者皮下注射后,发热率17.6﹪,体温最高38℃,停药后降至正常;腹部胀痛3例,四肢皮肤散发皮疹2例,均未给予特殊处理,2-3 d后恢复正常。结论给予G-CSF皮下注射后提取骨髓干细胞移植治疗失代偿期肝硬化的是一种临床确切有效的、安全的干细胞动员方法。     

Purification and growth of endothelial progenitor cells from murine bone marrow mononuclear cells

This study reports the culture and purification of murine bone marrow endothelial progenitor cells (EPCs) using endothelial cell-conditioned medium (EC-CM). Endothelial-like cells appeared at day 5 in culture of bone marrow mononuclear cells in the presence of EC-CM in the culture system, and these cells incorporated acetylated low-density lipoproteins (Ac-LDL) and reacted with endothelial-specific Ulex Europaeus Lectin. Continued incubation of these cells at low density with EC-CM for longer than 10 days resulted in the formation of endothelial cell colonies which gave rise to colonies of endothelial progeny and can be passed for many generations in the EC-CM culture system. Cells derived from these colonies expressed endothelial cell markers such as vWF and CD31, incorporated Dil-Ac-LDL, stained positive for Ulex Europaeus Lectin, formed capillary-like structures on Matrigel, and demonstrated a high proliferative capacity in culture. These bone marrow-derived adherent cells were identified as EPCs. The purification and the formation of EPC colonies by using EC-CM were associated with the cytokines secreted in the EC-CM. VEGF, bFGF, and GM-CSF in the EC-CM stimulated the proliferation and growth of EPCs, whereas AcSDKP (tetrapeptide NAc-Ser-Asp-Lys-Pro) in EC-CM suppressed the growth of mesenchymal stem cells (MSC) and fibroblasts. This approach is efficient for isolation/purification and outgrowth of bone marrow EPCs in vitro, a very important cell source in angiogenic therapies and regenerative medicine.     

狗骨髓长期培养的贴壁层细胞及条件培养液对粒系祖细胞的影响

本文报告狗骨髓长期培养的初步研究结果。采用马血清与狗血清混合加入培养体系中,可建立起比较稳定的贴壁细胞层,可以维持19周以上。但是,这种贴壁层不能有效地维持造血干细胞的增殖与分化,上清液中血细胞与GM-CFU_C的数目,在第二次接种骨髓后2—3周即迅速下降。狗骨髓培养的条件液中存在着抑制GM-CFU_C生长的物质,但它对小鼠骨髓GM-CFU_C和CFU-S无影响。如以贴壁细胞作为底层,用双层琼脂平皿培养法与正常狗骨髓共同培养,发现贴壁细胞不仅没有抑制反而有加强CSF刺激GM-CFU_C生长的作用。因此对狗骨髓长期培养体系不能长期支持造血干细胞生长和繁殖的可能原因尚须作进一步探索。     

TF-1细胞增殖法测定神经生长因子生物学活性的建立与应用

目的：建立并优化用于检测神经生长因子（NGF）生物学活性的TF-1细胞增殖法,并应用于重组人NGF和鼠NGF制品的活性检测。方法：将梯度稀释的人NGF国际参考品与TF-1细胞相作用,通过MTT法测定细胞增殖情况,建立测定NGF活性的TF-1细胞增殖法;从粒细胞巨噬细胞集落刺激因子（CM-CSF）残留、NGF加样稀释率、TF-1细胞接种浓度、培养时间等多个环节对实验条件进行优化;将建立的TF-1细胞增殖法应用于重组人NGF和鼠NGF的活性检测。结果：建立了用于NGF活性检测的TF-1细胞增殖法;实验条件优化结果表明,在无GM-CSF残留的情况下,将稀释至1／4的NGF样品与4×10^5／mL的细胞相互作用,培养48h,可以得到更为典型的的四参数曲线;利用条件优化后建立的检测方法对重组入NGF和鼠NGF各2批样品分别进行4次测定,结果平均值分别为10．01×10^5、10．81×10^5和3．55×10^5、3．30×10^5U／mg,变异系数分别为7．5％、7．2％和7．2％、9．1％,检测结果稳定均一,表明检测方法具有很好的重复性。结论：建立并优化了用于NGF活性检测的TF-1细胞增殖法,该方法操作简便、定量准确、重复性好、稳定可靠,可有效应用于人NGF和鼠NGF制品的活性检测。     

Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers

Fibroblast colonies (clones) were obtained by explantation of bone marrow single-cell suspensions and were used to establish multicolony and single-colony derived fibroblast cultures by successive passaging of either pooled or individual colonies. When transplanted in diffusion chambers after 20-30 cell doublings in vitro, the descendants of fibroblast colony-forming cells (FCFC), whether grown from single or pooled colonies, retained the ability for bone and cartilage formation. The content of osteogenic precursors in the cultured progeny significantly outnumbered the initiating FCFC. Thus the high proliferative potential of bone marrow FCFC and their ability to serve as common precursors of bone and cartilage-forming cells makes them probable candidates for the role of osteogenic stem cells.     

Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers

Abstract. Fibroblast colonies (clones) were obtained by explantation of bone marrow single-cell suspensions and were used to establish multicolony and single-colony derived fibroblast cultures by successive passaging of either pooled or individual colonies. When transplanted in diffusion chambers after 20–30 cell doublings in vitro , the descendants of fibroblast colony-forming cells (FCFC), whether grown from single or pooled colonies, retained the ability for bone and cartilage formation. The content of osteogenic precursors in the cultured progeny significantly outnumbered the initiating FCFC. Thus the high proliferative potential of bone marrow FCFC and their ability to serve as common precursors of bone and cartilage-forming cells makes them probable candidates for the role of osteogenic stem cells.     

大鼠异基因骨髓移植急性移植物抗宿主病模型的建立

目的建立较稳定的异基因骨髓移植急性移植物抗宿主病动物模型,为异基因骨髓移植后的急性移植物抗宿主病（aGVHD）的相关研究提供实验参照。方法以雄性SD大鼠为供鼠,雌性Wistar大鼠为受鼠,受体大鼠随机分成A、B、C、D、E 5组,移植当天所有受鼠均接受8.5 GY的全身照射（TBI）,于照射后4～6 h内,A组回输等量培养液,B组经尾静脉输注供鼠骨髓细胞（2×10^8个/kg）,C、D、E组分别回输供鼠骨髓细胞（2×10^8个/kg）＋不同比例的脾细胞。观察各组大鼠生存期、外周白细胞计数、及有无aGVHD的临床及病理表现。结果A组大鼠于15d内全部死亡,外周血白细胞计数明显减低,骨髓病理示造血组织减少,提示死于造血衰竭。B、C、D、E组大鼠外周血白细胞计数均有明显恢复,B组大鼠8只存活超过50 d,C、D、E组大鼠均于50 d观察期内死亡,并有aGVHD的临床表现及病理表现,但C组大鼠aGVHD的程度较轻且时间不集中,其中D、E组大鼠可于相对集中的时间内观察到典型aGVHD临床及病理。结论TBI预处理的方式是可行的,单纯输入异基因骨髓细胞不能引起明显的aGVHD,骨髓细胞与脾细胞1∶1及1∶1.5混合组均可作为异基因骨髓移植后理想的aGVHD动物模型。     

Interactions of tumor necrosis factor with granulocyte-macrophage colony-stimulating factor and other cytokines in the regulation of dendritic cell growth in vitro from early bipotent CD34+ progenitors in human bone marrow.

Colonies of CD1a+ HLA-DR+/DQ+ CD4+ cells with the functional and some of the structural attributes of Langerhans cells are observed in human bone marrow cultures in semi-solid media and are assumed to be the progeny of an early progenitor, the dendritic/Langerhans cell CFU (CFU-DL). The cytokine-regulated growth of these cells has been studied using a chemically defined serum-free system to culture both unfractionated and highly enriched bone marrow progenitor cell populations. Although unfractionated cell growth was optimal in serum replete cultures with PHA-stimulated leukocyte-conditioned medium (PHA-LCM) suboptimal proliferation of CFU-DL was observed in serum even in the absence of PHA-LCM. No colonies were observed under serum-free conditions when granulocyte-macrophage CSF (GM-CSF), IL-3, granulocyte CSF (G-CSF), and macrophage CSF (M-CSF) were present at levels optimal for granulocyte colony-forming unit (CFU-G) and macrophage colony-forming unit (CFU-M) growth. Addition of IL-1 alpha to these cytokines stimulated a small number of CFU-DL. However, in the presence of GM-CSF and IL-3, TNF-alpha or TNF-beta (5 U/ml) were both highly effective in promoting growth up to 82% of optimal and CFU-G growth was also enhanced at these concentrations. TNF was only active during the first 3 days of culture and higher concentrations of TNF-alpha but not TNF-beta were inhibitory for both CFU-DL and CFU-G. CD34+ cell-enriched populations were also enriched for both myeloid progenitors (CFU-G + CFU-M) and CFU-DL to 36- and 48-fold, respectively, and single cell cultures of CD34+ cells yielded single colonies containing both CD1a+ dendritic cells and CD1a- macrophages. Thus dendritic/Langerhans progenitors in the bone marrow expresses CD34, have a capacity for both macrophage and dendritic cell differentiation, and depend on hemopoietic growth factors and TNF for their further development in vitro.     

GROWTH OF MOUSE AND HUMAN BONE MARROW IN DIFFUSION CHAMBERS IN MICE

Both murine and human bone marrow cells were cultured in plasma clots which were formed inside diffusion chambers implanted into cyclophosphamide- and saline-treated mice. After an initial fall, the number of mouse bone marrow cells and numbers of mouse myeloid stem cells (CFU-C) and agar cluster-forming units rose faster in the cyclophosphamide-treated animals. These hosts also favored formation of myeloid (CFU-D-G) and erythroid (CFU-D-E) colonies and myeloid clusters in the plasma clot. The number and growth rate of mouse CFU-D-G were higher than those of CFU-C from the same marrow population. These observations suggest the existence of humoral factors stimulating granulocyte progenitor cell replication and differentiation. At its best the increment of CFU-D-E number was equivalent to that caused by a single 0·1 unit erythropoietin dose. Culture of normal human marrow cells resulted in colonies in the plasma clot containing only granulocytes and macrophages. Cyclophosphamide-treated host animals were essential for human CFU-D-G development. Plating efficiency for human marrow myeloid colonies was better in the conventional in vitro agar cultures than in diffusion chambers.     

Proliferative and differentiation potentials of skeletogenic bone marrow colony-forming cells

The clonal nature of bone marrow fibroblast colonies derived from clonogenic bone marrow osteogenic cells (CFUf) was proved by the chromosome analysis. During subsequent passages of multi-colony derived bone marrow fibroblast strains there occurs a pronounced increase in the cell number and in the number of osteogenic units (tested by transplantation in diffusion chambers). Single colony-derived strains are capable of forming bone and cartilage simultaneously. It follows that CFUf or part of them are clonogenic cells with high proliferative potentials and are common precursors for bone and cartilage tissue. Thus, CFUf may be regarded as osteogenic stem cells.