改良组织块贴壁法制备人脐带间充质干细胞

目的采用改良组织块贴壁法制备人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)。方法无菌条件下取足月婴儿脐带,采用改良组织块贴壁法分离间充质干细胞,台盼蓝染色法计数单个核细胞数,倒置相差显微镜下观察细胞形态,流式细胞仪检测细胞周期及免疫表型。结果原代hUCMSCs的数量为(1~5)×105个。培养至8 d,组织块边缘处有单细胞爬出;培养至2周,贴壁生长的细胞散在分布或形成小克隆;培养至20 d,细胞呈梭型、三角形或多角形。培养第20天的原代hUCMSCs的G0/G1期细胞占88.12%,S期细胞占1.23%,G2/M期细胞占10.65%,大多数细胞处于细胞增殖的潜伏期,CD105阳性率为97.65%,CD34阳性率为2.54%。结论采用改良组织块贴壁法分离了hUCMSCs,其生物学特性与骨髓间充质干细胞相似,为其临床应用奠定了基础。     

血小板裂解液大规模扩增人脐带间充质干细胞

目的用血小板裂解液(platelet lysate,PL)大规模扩增人脐带间充质干细胞(umbilical cord mesenchymal stem cell,UCMSC),并检测其生物学特性,为临床应用提供实验依据。方法分别采用PL和胎牛血清(FBS)低密度扩增人UCMSC,比较两组UCMSC的细胞形态、大小、克隆形成率、增殖能力、细胞表型和分化能力。结果 PL扩增的UCMSC形态细长;直径明显小于FBS扩增的UCMSC(P<0.05);克隆形成率与FBS扩增的UCMSC差异无统计学意义(P>0.05);有更高的细胞累积群倍数;与FBS扩增的UCMSC有相似的细胞表型;与FBS扩增的UCMSC均具有成骨、成脂诱导分化能力,但PL扩增的UCMSC成骨分化能力更强。结论 PL可取代FBS用于大规模扩增UCMSC。     

脐带间充质干细胞促进烫伤创面愈合的作用机制

目的探讨人脐带间充质干细胞(umbilical cord-derived mesenchymal stem cells,UCMSCs)多点局部注射后,修复烫伤皮肤创面的潜在机制。方法机械消化法分离人UCMSCs,培养至第3代后,进行形态观察、免疫表型和多向分化潜能鉴定。将第3代UCMSCs与碱性成纤维细胞因子(basic fibroblast growth factor,b FGF)共培养14 d,免疫荧光法检测成纤维细胞标志物波形蛋白的表达;对比小鼠Ⅲ度烫伤模型多点局部注射UCMSCs后创面愈合率及创面愈合时间;Dil标记UCMSCs,局部注射后检测UCMSCs在创面局部的分布情况。结果分离培养的UCMSCs的形态、表面抗原表达及体外诱导分化能力均符合间充质干细胞特性;经b FGF诱导后可显著提高UCMSCs中成纤维细胞标志物波形蛋白的表达量;局部注射UCMSCs可显著提高烫伤小鼠的创面愈合率,UCMSCs可定植于烫伤创面下。结论 UCMSCs具有较强的成纤维样细胞分化能力,经局部注射后,可通过定植于烫伤创面而显著提高创面愈合率。     

人脐带间充质干细胞来源工程化纳米囊泡的制备与鉴定

目的：探索来源于人脐带间充质干细胞（hUC-MSCs）的工程化纳米囊泡（Nanovesicle,NVs）的制备与鉴定方法,为深入了解工程化NVs的生物学功能提供数据基础。方法：采用物理方式破碎hUC-MSCs以获取NVs,并采用改良的超速离心法进行分离和纯化;通过纳米颗粒跟踪分析（NTA）、透射电镜（TEM）观察、蛋白质免疫印迹（WB）及生物学活性分析方法鉴定NVs的特性。结果：建立的方法成功分离出NVs,TEM下观察到NVs呈中间凹陷的茶杯样结构;NTA结果显示NVs直径在100 nm左右;Western Blot实验证实NVs几乎不表达外泌体特异性蛋白TSG101;生物学活性实验表明NVs不能促进人皮肤成纤维（HSF）细胞增殖。结论：本研究建立的hUC-MSCs来源的NVs提取及鉴定方法可行,为NVs在未来的临床研究提供了数据基础。     

一种分离培养人脐带Wharton's jelly间充质干细胞的新方法

目的探讨一种分离培养人脐带Wharton′s jelly间充质干细胞(mesenchymal stem cell,MSC)的新方法。方法取人脐带组织,除去动静脉后剪碎成2～5 mm3,将组织碎片浸入4 g/LⅠ型胶原酶和1 g/L透明质酸酶的混合液中,于37℃处理1 h,再用0.25%胰蛋白酶在相同条件下消化30 min,得到的消化液经70μm细胞滤网过滤后,制备单个细胞悬液,培养并传代。取P1、P3、P7代脐带Wharton′s jelly MSC,绘制细胞生长曲线;取P3代细胞,采用流式细胞术检测细胞表面标记分子,并分别采用成骨和成脂诱导培养基进行成骨及成脂诱导分化,茜素红染色和油红O染色观察结果。结果 P1、P3代脐带Wharton′s jelly MSC的增殖能力强,且P1代细胞的增殖能力强于P3代,P7代细胞的增殖能力较P3代细胞有所减弱。P3代脐带Wharton′s jelly MSC高表达CD90(99.8%)、CD105(100%)和CD166(100%),低表达CD45(0.3%)、CD14(0.1%)、CD34(0.2%)和CD79a(0.3%),不表达HLA-DR。P3代Wharton′s jellyMSC经成骨诱导后,茜素红染色可见红色结节;经成脂诱导后,油红O染色可见脂质沉积。结论本方法获得的Wharton′s jelly MSC活性好,增殖能力强,为后续实验研究及临床应用提供了理想的种子细胞。     

干扰素γ对血小板裂解液扩增的人脐带间充质干细胞免疫调节功能的影响

目的探讨干扰素(interferonγ,IFNγ)对血小板裂解液扩增的人脐带间充质干细胞(umblical cord mesenchymal stem cells,UCMSCs)免疫调节功能的影响。方法采用血小板裂解液(platelet lysate,PL)扩增UCMSCs,通过细胞形态、细胞表型和分化能力检测IFNγ(10 ng/m L)对UCMSCs生物学特性的影响。同时采用ELISA法分析IFNγ对UCMSCs分泌免疫相关因子PGE-2、TGF-β1和IL-6的影响,CCK-8法检测IFNγ对UCMSCs抑制人外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)增殖的影响。结果 IFNγ预处理对UCMSCs在细胞形态、细胞表型、细胞成骨和成脂肪的分化能力方面无明显影响(P0.05);IFNγ预处理可显著促进UCMSCs的TGF-β1及IL-6分泌(P0.05),但对PGE-2的分泌无明显影响(P0.05);IFNγ预处理可增强UCMSCs对PBMCs增殖的抑制作用(P0.05)。结论 IFNγ预处理可增强UCMSCs的免疫调节功能。     

人脐带华通氏胶间充质干细胞的生物学特性及其向肌源性细胞分化的能力

目的探讨人脐带华通氏胶间充质干细胞(human umbilical cord Wharton′s Jelly mesenchymal stem cells,hUCMSCs)的生物学特性及其向肌源性细胞分化的能力。方法收集健康足月产胎儿脐带组织,采用酶消化法从华通氏胶中分离hUCMSCs,选取第3代对数生长期细胞,显微镜观察细胞形态,流式细胞术检测细胞免疫表型分子的表达,免疫荧光染色检测干细胞标记物及其体外向肌源性细胞的分化,荧光显微镜观察及流式细胞术检测携带绿色荧光蛋白(green fluorescent protein,GFP)的慢病毒转染hUCMSCs后GFP的表达。结果采用酶消化法获得可贴壁生长的大量hUCMSCs,其能在体外成功进行扩增培养。hUCMSCs可高表达CD29、CD44、CD73、CD90和CD105,而极低表达CD14、CD34、CD45和HLA-Ⅱ;能表达干细胞标记物Oct4和Sox2,且在体外特殊培养条件下表达骨骼肌干细胞标记物Pax7和Myo D;转染的hUCMSCs可稳定表达GFP,转染率高达50%~70%。结论人脐带华通氏胶组织存在间充质干细胞,且其具有向肌源性细胞分化的潜能。     

脐带间充质干细胞长期体外培养的生物学特性及遗传稳定性

目的观察脐带间充质干细胞(umbilical cord mesenchymal stem cells,UC-MSCs)体外长期培养的生物学特性及遗传稳定性。方法从剖宫产足月健康新生儿脐带中分离培养UC-MSCs,并连续传代,显微镜下观察细胞形态。分别检测P3和P12代UC-MSCs的增殖能力、免疫表型、向成脂肪细胞和成骨细胞的分化诱导率、衰老情况、成纤维细胞集落形成单位(CFU-F)及染色体核型。结果 P3代UC-MSCs为形态相对均一的梭形贴壁细胞,呈平行排列生长或旋涡状生长;P12代UC-MSCs宽大扁平,贴壁性减退,漂浮细胞增多,胞浆内出现黑色颗粒和空泡;P3和P12代UCMSCs均表达CD73、CD90、CD105,不表达CD34、CD45、HLA-DR;P3较P12代UC-MSCs的细胞增殖能力及向成脂肪细胞分化诱导率均明显升高(P均0.05);向成骨细胞分化诱导率及细胞衰老率均明显降低(P均0.05);成纤维细胞集落数及染色体中期分裂相明显增多(P0.05)。结论长期体外培养的UC-MSCs生物学特性发生改变,遗传学特性稳定,但染色体中期分裂相减少。     

大鼠骨髓间充质干细胞来源微颗粒的提取及鉴定

目的 分离并纯化SD大鼠骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs),在此基础上收集骨髓间充质干细胞来源的微颗粒(bone mesenchymal stem cell-derived microparticles,BMSC-MPs),并对其进行鉴定.方法 采用全骨髓贴壁法分离纯...     

人胎盘来源的造血干细胞和间充质干细胞的分离及鉴定

目的建立人胎盘来源的造血干细胞(placenta hematopoietic stem cells,hP-HSCs)和间充质干细胞(placentaderived mesenchymal stem cells,hP-MSCs)的分离方法,并进行鉴定和组分分析。方法选取10份新生健康婴儿胎盘组织,采用机械破碎法联合磁珠分选法分离h P-HSCs,胎盘绒毛膜组织块贴壁法分离hP-MSCs,利用形态学观察、集落培养、流式细胞术等进行鉴定。结果 hP-HSCs:分离后有核细胞数(total nucleated cell number,TNC)为(11. 82±2. 46)×10~8个,TNC回收率≥80%;细胞活性为(99. 7±0. 3)%;细胞表面抗体CD34~+CD45dim表达率为(8. 69±0. 36)%,CD34~+总数为(108. 0±6. 48)×10~6个;集落形成总数为(1. 88±1. 07)×10~6。hP-MSCs:冻存细胞总数为(40. 78±9. 35)×10~7个;细胞活性为(99. 0±1. 5)%;细胞表面抗体CD34~+CD45~+表达率为(0. 1±0. 1)%,CD44~+CD105~+为(99. 6±0. 2)%,CD14~+CD19~+为(0. 1±0. 1)%,CD90~+CD73+为(98. 9±0. 2)%;且具有良好成脂、成骨分化潜能。结论成功建立了hP-HSCs和hP-MSCs体外分离培养方法,为胎盘的临床应用奠定了基础,并提供了细胞种子资源。     

人脐血干细胞在局灶性脑缺血大鼠体内的迁移与分化

目的观察人脐血干细胞(human umbilical cord blood stem cells,HUCBSCs)在局灶性脑缺血大鼠体内的迁移与分化,探讨HUCBSCs移植对缺血性脑损伤大鼠神经功能恢复可能的作用机制。方法采用线栓法制备大鼠大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)再灌注模型,将造模成功的20只大鼠随机分为2组:移植组15只,造模成功后24 h,经尾静脉移植1 ml(2×106个)DAPI/CM-Dil荧光染料标记的HUCBSCs;模型组5只,造模成功后24 h,经尾静脉移植等量生理盐水。分别于移植前及移植后3、7、15 d进行神经功能缺损评分(neurological severity score,NSS),并取脑组织,制备冰冻切片,HE染色观察脑组织形态,荧光显微镜观察HUCBSCs在脑组织中的迁移和分布,免疫荧光法检测脑组织中巢蛋白(Nestin)和神经元特异性烯醇化酶(neuron specific enolase,NSE)的表达。结果移植组大鼠HUCBSCs移植后7 d起,其改良的NSS(m NSS)显著低于对照组(P<0.05)。移植后15 d,大鼠脑组织病理改变明显减轻。移植后3 d起,可见病灶及其周围部位有阳性细胞存在,随着时间的推移,脑缺血病灶部位的阳性细胞增多;移植后7 d,迁移至病灶部位的细胞数增加;移植后15 d,迁移至病灶部位的细胞数量达观察期内的高峰,各时间点阳性细胞数差异有统计学意义(P<0.05)。移植后15 d,大鼠脑组织中CM-Di I/NSE-FITC和CM-Di I/Nestin-FITC双阳性细胞率分别为85.2%和81.6%。结论 HUCBSCs经静脉移植后,能在大鼠体内存活,并向损伤部位迁移,具有向神经元细胞方向分化的潜能,对脑缺血大鼠的神经功能恢复具有促进作用。     

Computational modeling of megakaryocytic differentiation of umbilical cord blood-derived stem/progenitor cells

Quantifying the effect of exogenous parameters regulating megakaryopoiesis would enhance the design of robust and efficient protocols to produce platelets. We developed a computational model based on time-dependent ordinary differential equations (ODEs) which decoupled expansion and differentiation kinetics of cells using a subpopulation dynamic model. The model described umbilical cord blood (UCB)-derived cell's behavior in response to the external stimuli during expansion and megakaryocytic differentiation ex vivo. We observed that the rate of expansion of Mk progenitors and production of mature Mks were higher when TPO was included in the expansion stage and cytokines were added during differentiation stage. Our computational approach suggests that the Mk progenitors were an important intermediate population that their dynamic should be optimized in order to establish an efficient protocol. This model provides important insights into dynamics of cell subpopulations during megakaryopoiesis process and could potentially contribute toward the rational design of cell-based therapy bioprocesses.     

SF/COL/PLCL静电纺丝三维纳米纤维支架与人脐带血间充质干细胞的细胞相容性研究

目的研究SF/COL/PLCL静电纺丝三维纳米纤维支架与人脐带血(human umbilical cord blood,hUCB)间充质干细胞(mesenchymal stem cells,MSCs)的细胞相容性。方法分离、培养hUCBMSCs,并进行传代,取第3代hUCBMSCs,茜素红染色和Von Kossa染色检测其体外诱导成骨分化的能力;流式细胞术检测其表面相关抗原的表达。制备SF/COL/PLCL静电纺丝三维纳米纤维支架,扫描电镜观察其形貌表征,并检测其力学性能。将hUCBMSCs接种于静电纺丝三维纳米纤维支架上,观察细胞在支架上的生长及增殖情况。结果 hUCBMSCs具有成骨诱导分化能力,其表达CD44、CD29、CD90和CD105,不表达CD45和CD34。SF/COL/PLCL复合纳米纤维的纤维形貌良好,随着PLCL含量的增加,纤维的直径和力学性能均逐渐增加。hUCBMSCs能够在三维纳米纤维支架上很好地黏附,并相互连接向周围扩展,三维纳米纤维支架能很好地促进细胞黏附和增殖,与常规培养的细胞相比,差异有统计学意义(P0.05或P0.01),当SF/COL与PLCL的质量比为30∶70时,最有利于细胞的生长。结论 hUCBMSCs能够在SF/COL/PLCL静电纺丝纳米纤维支架上生长、增殖,这种支架材料具有良好的力学性能及细胞相容性,有望成为一种新型组织工程支架材料。     

Human umbilical cord blood stem cells: rational for use as a neuroprotectant in ischemic brain disease

The use of stem cells for reparative medicine was first proposed more than three decades ago. Hematopoietic stem cells from bone marrow, peripheral blood and human umbilical cord blood (CB) have gained major use for treatment of hematological indications. CB, however, is also a source of cells capable of differentiating into various non-hematopoietic cell types, including neural cells. Several animal model reports have shown that CB cells may be used for treatment of neurological injuries. This review summarizes the information available on the origin of CB-derived neuronal cells and the mechanisms proposed to explain their action. The potential use of stem/progenitor cells for treatment of ischemic brain injuries is discussed. Issues that remain to be resolved at the present stage of preclinical trials are addressed.     

Distribution of the molecular species of phospholipids in human umbilical cord blood

The composition of phosphatidylcholine (PC) and sphingomyelin (SM) was studied in cord blood lipoproteins to determine whether equilibration of the molecular species of phospholipids among lipoproteins was comparable with that reported for adults. The molecular species distributions of PC in low density lipoprotein (LDL) differed from that of high density lipoprotein (HDL). Whereas LDL PC was richer in combinations of fatty acids with 16 and 18 carbon atoms than HDL, the HDL was markedly enriched in combinations of fatty acids with 18 and 20 carbon atoms. Sphingomyelins in LDL were richer in palmitic acid than HDL while HDL had a greater proportion of long chain sphingomyelin than LDL. The molecular species of PC and SM do not equilibrate in cord blood. The results for the SM distributions were similar to other reports for adult human lipoprotein. However, the marked differential distribution of PC among lipoproteins appears unique to cord blood. The mechanisms responsible for equilibrating PC among lipoproteins are less well developed in the neonate when compared with the adult.