早产胎儿脐血和脐带间充质干细胞分离培养的比较

目的对比早产胎儿脐血和脐带间充质于细胞（MSCs）的培养成功率,探讨早产胎儿MSCs获取的最佳途径。方法无菌条件下采集早产胎儿（不足37周）脐血,密度梯度离心法分离单个核细胞,采用贴壁培养法获得脐血MSCs;将脐带剪成1mm^3大小组织块,采用组织块贴壁法获得脐带MSCs;分别观察脐血和脐带MSCs的生物学特性,免疫荧光法检测MSCs表面标记物的表达情况。结果采用Mesencult^TM培养基,早产胎儿脐血MSCs培养成功率为100％,脐带MSCs培养成功率为67％,明显低于脐血MSCs;早产胎儿脐血和脐带MSCs均表达CD29、CD44和CD90不表达造血干细胞表面标志CD34。结论早产胎儿脐血中较易获得MSCs,为临床移植治疗提供了理想的细胞来源。     

Mid-trimester fetal blood-derived adherent cells share characteristics similar to mesenchymal stem cells but full-term umbilical cord blood does not

Stem cell transplantation is a promising treatment for many conditions. Although stem cells can be isolated from many tissues, blood is the ideal source of these cells due to the ease of collection. Mesenchymal stem cells (MSCs) have been paid increased attention because of their powerful proliferation and pluripotent differentiating ability. But whether MSCs reside in blood (newborn umbilical cord blood and fetal or adult peripheral blood) is also debatable. The present study showed that MSC-like cells could be isolated and expanded from 16-26 weeks fetal blood but were not acquired efficiently from full-term infants' umbilical cord blood (UCB). Adherent cells separated from postnatal UCB were heterogeneous in cell morphology. Their proliferation capacity was limited and they were mainly CD45+, which indicated their haematopoietic derivation. On the contrary, MSC-like cells shared a similar phenotype to bone marrow MSCs. They were CD34- CD45- CD44+ CD71+ CD90+ CD105+. They could be induced to differentiate into osteogenic, adipogenic and neural lineage cells. Single cell clones also showed similar phenotype and differentiation ability. Our results suggest that early fetal blood is rich in MSCs but term UCB is not.     

还原型谷胱甘肽对脐带间质干细胞生物学特性的影响

目的 探讨还原型谷胱甘肽(GSH)对脐带间质干细胞生物学特性的影响.方法 用MTT法检测脐带间质干细胞活力、细胞计数法绘制细胞生长曲线、流式细胞仪检测细胞周期和表面标志,成骨诱导后组织化学检测碱性磷酸酶(ALP)表达.结果 GSH浓度低于0.15 mg/ml时可促进脐带间质干细胞的增殖,高于此浓度时对细胞的抑制作用随浓度加大而逐步增强.GSH可缩短脐带间质干细胞到达生长平台期的时间并延长细胞传代的代数,细胞周期检测显示S期显著增加,同时GSH对脐带间质干细胞表面标志无明显影响,成骨诱导分化后ALP表达阳性率无明显改变.结论 低浓度GSH可促进脐带间质干细胞的增殖,0.15 mg/ml浓度可以达到最佳促进增殖效果,同时对细胞表面标志与向成骨细胞分化的能力无明显影响,可以用于体外对脐带间质干细胞的扩增.     

Safety and effect of umbilical cord blood –derived mesenchymal stem cells on apoptosis of human cardiomyocytes

Objective To study the safety and effect of the umbilical cord blood （UCB）-derived mesenchymal stem cells （MSCs） on apoptosis of human cardiomyocytes （HCM）. Methods UCB was collected at the time of delivery with informed consent obtained from 10 donors. The UCB-derived MSCs were treated with 5-azaserube （5-AZA） and were further induced to differentiate into cardiomyocytes. Telomerase activity, G-banding patterns of chromosomal karyotypes, tumor formation in nude mice, RT-PCR, and the effect of inhibiting apoptosis of HCM were investigated. Results MSCs derived from UCB were differentiated into cardiomyocytes in vitro, which possessed telomerase activity after 5-AZA induction, and no abnormal chromosomal karyotypes were observed. Expression of p53, cyclin A, cdk2, ~3 -actin, C-fos, h-TERT and c-myc were similar in MSCs before and after 5-AZA treatment. There was no tumor formation in nude mice after injection of UCB-derived MSCs. UCB-derived MSCs significantly inhibited apoptosis of HCM. Conclusion UCB-derived MSCs are a valuable, safe and effective source of cell-transplantation treatment .     

脐血间充质干细胞的体外扩增及向类肝细胞分化的实验研究

目的探讨人脐带血间充质干细胞（UBC-MSCs）向类肝细胞定向分化的可能性。方法无菌条件下采集新生儿脐带血75份,采用密度梯度离心法与直接贴壁法相结合分离UBC－MSCs,体外培养传代,获得第4代集落生长细胞作流式细胞仪表面抗原测定,并应用a－FGF、b－FGF、HGF、OSM等多种成分,分阶段向肝细胞定向诱导分化。倒置相差显微镜观察细胞形态变化,取诱导后4周和6周的细胞分别采用RT-PCR法检测ALB、AFP和CK－19基因mRNA水平,分析类肝细胞诱导表达情况。结果按10。／ml接种,用含15％FBS的DMEM／F12培养基培养可成功获取UBC—MSCs。细胞呈长梭形,细胞表面抗原测定显示强烈表达CD29、CD44,而不表达造血细胞的标志物CD34、CD45。分阶段诱导6周后,细胞形态呈多角形,并检测到肝细胞表面标志物白蛋白、AFP和CK-19。结论新生儿脐血中可分离出MSCs,并可在体外进行培养扩增,分阶段诱导可分化为类肝细胞。     

人脐血间充质干细胞抑制心肌细胞凋亡的作用与安全性

目的探讨人脐带血来源的间充质干细胞(mesenchymal stem cells,MSCs)诱导后的安全性及其与人心肌细胞共培养下的抗凋亡作用。方法在知情同意的原则下,取得10位供者分娩时的脐带血。脐带血来源的MSCs经5-氮杂胞苷处理后向心肌细胞诱导分化。观察诱导分化的MSCs端粒酶活性,G带显带技术下的染色体组型,裸鼠体内诱导肿瘤形成,RT-PCR以及共培养下对心肌细胞凋亡的抑制作用。结果脐带血来源的MSCs可在体外经5-氮杂胞苷诱导分化为心肌细胞,诱导后的细胞具有端粒酶活性,未观察到异常的染色体组型。p53、cyclinA、Cdk2、β-肌动蛋白、c-fos、h-TERT和c-myc在5-氮杂胞苷处理前后的MSCs中表达相似。注射到裸鼠体内未见肿瘤形成。脐带血来源的MSCs对人心肌细胞凋亡有明显的抑制作用。结论脐带血来源的MSCs是一种安全有效的移植治疗的细胞来源,共培养下能抑制人心肌细胞的凋亡。     

人脐带间充质干细胞体外培养过程中的表观遗传学改变

目的分离、培养人脐带间充质干细胞(hUC-MSCs),并探讨体外培养过程中表观遗传学的改变。方法随机选取足月顺产健康胎儿脐带,采取复合酶消化法从脐带胶质中获得hUC-MSCs。流式细胞术检测细胞表面标志物。分化培养基诱导hUC-MSCs分化。β-半乳糖苷酶染色检测细胞衰老。甲基化特异性PCR(MSP)检测hUC-MSCs在不同培养时期特定基因的甲基化状态。蛋白免疫印记(Western blot)检测hUC-MSCs在不同培养时期MGMT基因的蛋白表达。结果按整根脐带30 cm计算,可收获(3～6)×106hUC-MSCs。培养的hUC-MSCs高表达CD29、CD73、CD105、CD90和CD44,不表达CD45、CD34和HLA-DR。hUC-MSCs可定向分化为成脂、成骨和成软骨细胞。随着细胞的传代,衰老细胞占细胞总数的比例增加。培养后期的hUC-MSCs出现MGMT基因甲基化。结论复合酶消化法可从脐带胶质中获得具有间充质干细胞(MSC)特性的细胞。随着传代次数的增多,DNA修复基因MGMT出现甲基化,可能参与hUC-MSCs衰老调控。     

Maternal plasma or human serum albumin in wash buffer enhances enrichment and ex vivo expansion of human umbilical cord blood CD34+ cells

Umbilical cord blood is a valuable source of haemopoietic stem/progenitor cells (HSC) for transplantation. This study explored the effect of maternal plasma/human serum albumin (HSA) in the purification and culture conditions of CD34+ cells derived from human umbilical cord blood. During CD34+ cell enrichment, including maternal plasma or HSA instead of fetal bovine serum (FBS) in the wash buffer, significantly increased the purity and the fold expansion of CD34+ cells. The increase in fold expansion of CD34+ cells was independent of CD34+ cell purity before expansion. With FBS, the mean fold expansion of CD34+ cells and total nucleated cells on day 7 was 9.7 +/- 5.5 and 39.7 +/- 13.7 respectively. The use of maternal plasma increased the mean fold expansion of CD34+ cells and total nucleated cells on day 7 to 28.2 +/- 6.7 and 71.5 +/- 15.4 respectively. When HSA was added to wash buffer, the mean fold expansion of CD34+ cells and total nucleated cells were 30.4 +/- 10.5 and 83.5 +/- 24.8 respectively. No statistical significance was found between using HSA and maternal plasma on total cell and CD34+ cell expansion. We propose that HSA in maternal plasma was responsible for the positive effect on CD34+ cell enrichment and expansion.     

Experimental treatment of radiation pneumonitis with human umbilical cord mesenchymal stem cells

Objective:To evaluate of the curative effect of human umbilical cord mesenchymal stem cells(hUC-MSCs)on rat acute radiation pneumonitis.Methods:Fourty rats were randomly divided into control group,radiation group,stem cell prevention group,stem cell treatment group and prednisone treatment group.All rats except those in the control group were radiated with X ray to establish the acute radiation pneumonitis damage model.The hUC-MSCs cultured in vitro was administrated to the rats of the prevention group via tail vein(1×10~6 cells/kg BW)24 h before the radiation,while the same administration was performed in the rats of the treatment group 24 h after the radiation.After 24 h post the radiation,the rats in tbe radiation group were given 0.4 mL physiological saline,and those in the prednisone group were given 1 mg/kg prednisone.All rats were,observed and executed 72 h after the radiation to defect lung histological changes.Results:After the administration of hUC-MSCs,the survival status of the rats in the prevention group and treatment group was obviously better than that in the control group.As shown by the histological staining,the morphology,proliferation activity aad bronchial state of lung tissues were better in the prevention group and treatment group than in the control group.Conclusion:The hUC-MSCs have definite therapeutic effects on acute radiation pneumonitis in rats.     

脐带间充质干细胞 nestin mRNA表达的研究

目的：从基因角度求证脐带间充质干细胞（MSCs）神经巢蛋白nestin mRNA表达。方法取新鲜脐带组织行MSCs分离、培养及鉴定。采用胰酶消化法计数MSCs,流式细胞仪检测细胞活性,提取原代、一代、二代细胞的mR-NA,采用实时荧光定量PCR法检测nestin mRNA表达。结果脐带中分离出的细胞贴壁生长状况较好,其中大部分细胞呈梭形生长,传代后细胞与原代细胞生长形态相似,生长加快;个别细胞呈神经样生长。 MSCs呈nestin mRNA阳性表达,其表达量在传代后有明显减少;与nestin蛋白在神经前体细胞向神经细胞分化时的变化一致。结论脐带MSCs存在nestin mRNA表达;传代后nestin mRNA表达量虽减小,但仍可作为备选种子细胞进行移植。     

肝衰竭患者血清诱导脐血间充质干细胞表达白蛋白和甲胎蛋白的研究

目的：建立分离、纯化脐血间充质干细胞（HUMSCs）的方法,探讨肝衰竭患者血清对HUMSCs的诱导分化作用。方法：采用密度梯度离心和贴壁培养法相结合分离培养HUMSCs并鉴定,以舍重型肝病患者血清的低糖DMEM培养基诱导培养,不同时间点采用免疫组织化学方法（sP）检测甲胎蛋白（AFP）和白蛋白（Alb）的表达。结果：分离获得高纯度的HUMSCs,HUMSCs高表达CD44和CD29,不表达CD34,可以分化为脂肪细胞;SP结果表明,对照组HUMSCs不表达AFP和Alb,肝衰竭患者血清在培养7天时,大部分细胞呈AFP阳性表达（75．2±7．8）％,与对照组比较差异有显著性意义（P〈0．05）;诱导培养14天、21天和28天时AFP表达阳性率分别为（40．1±8．2）％、（10．4±3．5）％和（6．8±2．3）％;肝衰竭患者血清培养7天时仅少数细胞内可见Alb表达,表达阳性率为（9．8±2．7）％;培养14天、21天和28天,Alb表达阳性率分别为（25．4±5．2）％、（67．2±7．8）％和（83．4±7．9）％,与对照组比较差异有显著性意义（P〈0．05）。结论：密度梯度离心和贴壁培养法相结合是一种较好的分离纯化HUMSCs的方法;肝衰竭患者血清可以诱导HUMSCs表达AFP和Alb。     

脐血造血干细胞向巨核细胞诱导分化的研究

目的:建立脐血CD34+造血干细胞向巨核细胞诱导分化的体系,探讨最佳的扩增方法。方法:免疫磁珠法分离获得CD34+细胞培养在无血清无基质培养液中,采用TPO加SCF加IL-3加IL-6、TPO加SCF加IL-3、TPO加SCF3种不同因子组合对其诱导分化及扩增。收集3、7、10、14d的扩增产物,运用荧光显微镜检测巨核细胞的表面标志;流式细胞术(FCM)检测巨核细胞的凋亡;并对巨核细胞形成单位(CFU-MK)及DNA含量进行检测。结果:分离获得的CD34+细胞在体外可以有效扩增,随培养时间的延长CD34+/CD41+细胞数第7天达最高值,之后逐渐下降;而CD41+、CD42b+、CD61+细胞随培养时间的延长表达量逐渐增高。加入IL-3和IL-6后,Annexin Ⅴ阳性细胞由(8.26±2.49)%降至(3.51±1.24)%。CFU-MK的数量在第10天时最高,且8倍体及8倍体以上的巨核细胞所占的的百分比增加,即成熟产板型巨核细胞增加。结论:脐血CD34+造血干细胞在体外可向巨核细胞诱导分化及有效扩增。3种因子组合中TPO加SCF加IL-3加IL-6组扩增效率最高。     

脐血间充质干细胞培养条件的优化

目的探讨脐血间充质干细胞(mesenchymal stem cell,MSC)的培养扩增条件。方法取2004-10~2005-02广州华侨医院产科胎儿脐带血,比较不同的分离方法、接种密度、首次换液时间、不同的培养基对脐血中的间充质干细胞原代培养过程的影响,以流式细胞仪对优化条件下培养的间充质干细胞进行细胞周期分析,细胞表面标志检测,多种成分联合诱导其向脂肪、成骨方向分化,细胞化学染色检测诱导后的细胞变化。结果在其他条件不变的情况下,羟乙基淀粉沉淀法优于Ficoll分离法,5×106/cm2是脐血MSC培养的适宜接种密度,首次换液时间为7d。优化条件下培养的间充质干细胞不表达造血细胞系的标志(CD_(34)/CD_(45)/CD_(14))及内皮细胞的标志CD106,强表达CD_(29)、CD_(44)、CD_(13),在适当的诱导条件下可向脂肪及成骨方向分化。结论脐血中含有MSC,可以作为组织工程的种子细胞。     

人脐带血间充质干细胞对人胃癌MGC80-3细胞增殖的影响

目的探讨人脐带血间充质干细胞(h UCB-MSCs)对人胃癌MGC80-3细胞体外增殖的影响。方法原代培养h UCBMSCs,通过流式细胞仪进行细胞表型鉴定;采用MTT法测定不同浓度h UCB-MSCs条件培养基作用于胃癌MGC80-3细胞后,胃癌细胞24 h、48 h、72 h的抑制率;倒置相差显微镜观察胃癌MGC80-3细胞形态的变化;流式细胞技术测定胃癌MGC80-3细胞凋亡率。结果采用流式细胞仪表型分析获得的第4代h UCB-MSCs均质性好;MTT法显示,不同浓度h UCB-MSCs条件培养基处理MGC80-3细胞24 h、48 h、72 h后,各实验组抑制率均有不同程度升高;倒置相差显微镜观察显示细胞形态发生变化;流式细胞术分析结果显示,MSC-CM能诱导MGC80-3细胞发生凋亡,细胞凋亡率随着MSC-CM的升高而升高,各实验组与空白对照组比较及各实验组之间比较,差异均有统计学意义(P0.05)。结论 h UCB-MSCs能明显抑制人胃癌细胞株MGC80-3细胞的体外增殖与转移,且作用效果具有剂量和时间依赖性。     

c-Met基因过表达脐带间充质干细胞株的构建*

目的 建立c-Met慢病毒载体转染人脐带间充质干细胞（hUMSC）,为治疗肝衰竭作为种子细胞。方法 培养hUMSC,经流式细胞技术检测细胞表面表型,转染c-Met慢病毒载体,在荧光显微镜下观察转染效率,确定最佳多重感染复数（MOI）。采用嘌呤霉素抗性筛选稳定表达c-Met的hUMSC细胞系,采用Western-blot法检测细胞c-met蛋白表达量。结果 P5代细胞高表达CD44、CD90和CD105抗原,不表达CD31、CD45和CD34相关造血细胞抗原,符合人脐带间充质干细胞的特性;构建成功的c-Met慢病毒载体转染人脐带间充质干细胞最佳MOI=80,经嘌呤霉素筛药,在荧光显微镜下观察发现荧光阳性率为100%,且经Western-blot法检测证实该细胞过表达c-Met蛋白。结论 成功构建过表达c-Met基因的脐带间充质干细胞,为进一步实验打下了基础。     

5-Azacytidine-treated human mesenchymal stem/progenitor cells derived from umbilical cord, cord blood and bone marrow do not generate cardiomyocytes in vitro at high frequencies

Background and Objectives   Mesenchymal stem/progenitor cells (MSCs) are multipotent progenitors that differentiate into such lineages as bone, fat, cartilage and stromal cells that support haemopoiesis. Bone marrow MSCs can also contribute to cardiac repair, although the mechanism for this is unclear. Here, we examine the potential of MSCs from different sources to generate cardiomyocytes in vitro , as a means for predicting their therapeutic potential after myocardial infarction.
Materials and Methods   Mesenchymal stem/progenitor cells were isolated from the perivascular tissue and Wharton's jelly of the umbilical cord and from cord blood. Their immunophenotype and differentiation potential to generate osteoblasts, chondrocytes, adipocytes and cardiomyoxcytes in vitro was compared with those of bone marrow MSCs.
Results   Mesenchymal stem/progenitor cells isolated from umbilical cord and cord blood were phenotypically similar to bone marrow MSCs, the exception being in the expression of CD106, which was absent on umbilical cord MSCs, and CD146 that was highly expressed in cord blood MSCs. They have variable abilities to give rise to osteoblasts, chondrocytes and adipocytes, with bone marrow MSCs being the most robust. While a small proportion (~0·07%) of bone marrow MSCs could generate cardiomyocyte-like cells in vitro, those from umbilical cord and cord blood did not express cardiac markers either spontaneously or after treatment with 5-azacytidine.
Conclusion   Although MSCs may be useful for such clinical applications as bone or cartilage repair, the results presented here indicate that such cells do not generate cardiomyocytes frequently enough for cardiac repair. Their efficacy in heart repair is likely to be due to paracrine mechanisms.     

Cytokine mediated expansion of human umbilical cord blood CD34+ cells: comparison of the use of partially purified and pure CD34+ target cells

To determine the optimal cell population for cytokine mediated expansion, we compared the use of Magnetic Cell Sorting (MACS) system enriched CD34+ human umbilical cord blood (HUCB) cells with that of MACS enriched, flow purified CD34+ HUCB cells. Both MACS enriched CD34+ cells and MACS enriched, flow purified CD34+ cells (mean starting purity of CD34+ SC 51.27 ± 7.6% and 96.36 ± 1.34% respectively n = 6) were incubated for seven days with Interleukin-1 (IL-1)+IL-3+Stem Cell Factor (SCF) and showed a fold increase in the number of nucleated cells (10.02 ± 2.6 and 18.23 ± 4.73 respectively) and a reduction in the percentage of CD34+ cells (5.55 ± 1.23% and 12.21 ± 3.29% respectively). An increase in the absolute numbers of CD34+ cells (4.8 × 10⁴ ± 2.3 × 10⁴) was observed with MACS enriched CD34+ cells as compared to no change (1.3 × 10⁵ ± 8.8 × 10⁴) with MACS enriched, flow purified CD34+ cells. An increase in IL-3+GM-CSF+SCF responsive colony forming unit (CFU) (1.7 × 10⁴ ± 9.4 × 10³ and 1.6 × 10⁵ ± 7.7 × 10⁴ respectively) was also observed as compared with input values (1.5 × 10⁴ ± 1 × 10⁴ and 2.3 × 10⁴ ± 8.9 × 10³ respectively). We conclude that MACS enriched, flow sorted CD34+ HUCB cells have greater cytokine mediated expansion potential as measured by progenitor expansion, than MACS enriched CD34+ HUCB cells.     

Xenogeneic immunosuppression of human umbilical cord mesenchymal stem cells in a major histocompatibility complex-mismatched allogeneic acute graft-versus-host disease murine model

Mesenchymal stem cells (MSCs) hold great promise for treating immune disorders owing to their immunoregulatory capacity, but the mechanism remains controversial. As we show here, the mechanism of human umbilical cord mesenchymal stem cell (HUCMSC)‐mediated immunosuppression involves TGF‐β and indoleamine 2,3‐dioxygenase (IDO). In this study, we investigated the influence of xenogeneic HUCMSCs on acute graft‐versus‐host disease (aGVHD) in murine allogeneic bone marrow transplantation (BMT). In the HUCMSC‐treated group, lethally irradiated DBA/2(H‐2Kd) mice were adoptively transferred with expanded HUCMSCs, bone marrow (BM), and splenocytes (SCs) from C57BL/6 (H‐2Kb) mice. Recipients in the control group were transferred only BM and SCs. The two groups were compared in survival, weight, histopathologic specimens, and aGVHD scoring. In the HUCMSC‐treated group, 60% of the mice survived past day 30 after BMT, but in the control group, all mice died within 18 d. The mice treated with HUCMSCs exhibited light symptoms of aGVHD after day 30. The results suggest that xenogeneic HUCMSCs could alleviate aGVHD symptoms and prolong survival after allogeneic BMT. Our study suggests that in vitro expanded HUCMSCs might be used to inhibit severe aGVHD effectively in allogeneic hematopoietic cell transplantation clinically.     

An experimental study of preventing and treating acute radioactive enteritis with human umbilical cord mesenchymal stem cells

ObjectiveTo test the curative effect of human umbilical cord-derived mesenchymal stem cells on rat acute radioactive enteritis and thus to provide clinical therapeutic basis for radiation sickness.MethodsHuman umbilical cord-derived mesenchymal stem cells were cultivated in vitro and the model of acute radioactive enteritis of rats was established. Then, the umbilical cord mesenchymal stem cells were injected into the rats via tail vein. Visual and histopathological changes of the experimental rats were observed.ResultsAfter the injection, the rats in the prevention group and treatment group had remarkably better survival status than those in the control group. The histological observations revealed that the former also had better intestinal mucosa structure, more regenerative cells and stronger proliferation activity than the latter.ConclusionsHuman umbilical cord-derived mesenchymal stem cells have a definite therapeutic effect on acute radioactive enteritis in rats.