Shockwave stimulates oxygen radical-mediated osteogenesis of the mesenchymal cells from human umbilical cord blood.

Human umbilical cord blood (HUCB) mesenchymal progenitor cells expressed stro-1 or CD44 or CD29, and subsequently, differentiated toward osteogenic lineage. Physical shockwave treatment increased osteogenic activity of HUCB mesenchymal progenitor cells through superoxide-mediated TGF-beta1 induction. Transplantation of shockwave-treated HUCB mesenchymal progenitor cells enhanced healing of segmental femoral defect in severe combined immunodeficiency disease (SCID) mice. INTRODUCTION: Mesenchymal progenitor cells (MPCs) in the bone marrow are precursors to bone development. It remains uncertain whether MPCs are present in human umbilical cord blood (HUCB) and are capable of differentiating into osteogenic cell lineage. Extending from a model of shockwave (SW) promotion of bone marrow stromal cell differentiation toward osteoprogenitors in rats, we further investigated how physical SW mediated biological responses in regulating osteogenic differentiation of HUCB MPCs. MATERIALS AND METHODS: HUCB was subjected to SW treatment at different energy flux densities and impulses. Colony-forming units-stroma (CFU-Stroma), osteogenic activities (Cbfa1/Runx2 expression, bone alkaline phosphatase activity, and bone nodule formation), and bone formation by heterologous transplantation into SCID mice were assessed. RESULTS: Few CD34+ stem cells (1.3%) and stro-1+ cells (1.0%) were present in the freshly prepared mononuclear cells (MNCs) from HUCB. The number of stro-1+ cells, but not CD34+, increased to 72.4% in the adherent cell culture over 6 days. Stro-1+ cells co-expressed CD44 and CD29 markers and grew into CFU-Stroma that matured into bone nodules. We found that the SW treatment (0.16 mJ/mm2 energy flux density, 200 impulses) elicited superoxide production and promoted formation of CFU-Stroma, but not of hematopoietic CFU-Mix. SW also enhanced the production of transforming growth factor (TGF)-beta1, but not of interleukin (IL)-3 or granulocyte monocyte-colony stimulating factor (GM-CSF). Neutralization of TGF-beta1 significantly reduced SW-promoted CFU-Stroma formation. Superoxide scavenging by superoxide dismutase blocked SW enhancement of TGF-beta1 production and formation of CFU-Stroma. Administration of SW-treated HUCB MPCs to SCID mice with femoral segmental defects facilitated dense, bridging callus and gap closure. CONCLUSION: HUCB MPCs subjected to SW treatment is a potential source for stem cells useful in the treatment of orthopedic disorders. An optimal physical SW treatment enhanced osteogenesis through superoxide-mediated TGF-beta1 production. Physical stimulation is an alternative method for extending mesenchymal stem cells of HUCB.     

人脐带间充质干细胞向血管内皮祖细胞诱导分化的实验研究

目的探讨人脐带间充质干细胞定向诱导分化为血管内皮祖细胞的可行性,为构建组织工程血管瓣膜内皮化提供新的细胞来源。方法无菌条件下取剖宫产新生儿脐带,复合胶原酶消化法获取脐带间充质干细胞进行培养,以流式细胞仪检测脐带间充质细胞的表面标志。取扩增3～6代的脐带间充质干细胞用VEGF和b-FGF诱导分化。用免疫荧光法鉴定内皮祖细胞的标志。结果脐带间充质干细胞可以诱导分化为血管内皮样细胞,表达CD34、CD133和vWF,且Dil-acLDL实验阳性。结论脐带间充质干细胞可以诱导分化为血管内皮祖细胞,可为构建组织工程血管瓣膜提供新的种子细胞来源。     

CD34+细胞在干细胞旁分泌下向内皮细胞分化的研究

目的 探讨人脐血CD34+细胞在脐带间充质干细胞(UC-MSCs)旁分泌作用下向内皮细胞诱导分化的可行性.方法 收集20份脐血,体积(103.80±19.77)ml.免疫磁珠(MACS)分选CD34+细胞;取脐带用消化贴壁法获得UC-MSC.流式鉴定干细胞表型.实验分单纯培养组、诱导组、共培养组.结果 流式鉴定CD34+细胞纯度(95.02±3.81)%.培养14 d流式检测共培养组表达CD31、CD144、VWF分别为(65.43±5.61)%、(54.40±4.13)%、(47.53±3.96)%(与单纯培养组比较P＜0.05),部分表达CD34,阴性表达CD45,这与诱导组及成熟脐静脉内皮细胞表达率一致.结论 UC-MSCs旁分泌作用与外源性细胞因子都具有促分化作用,均能使脐血CD34+细胞向内皮细胞分化.

Abstract：

Objective To study whether the paracrine action of umbilical cord-derived mesenchymal stem cells (UC-MSC) can induce differentiation of human umbilical cord blood-derived CD34+ cells into endothelial cells in vitro. Methods The 20 fresh umbilical cord blood samples were collected with volume of (103. 80 ± 19. 77) ml. CD34+ cells were isolated from the mononuclear cells by magnetic activated cell sorting system (MACS) , and mesenchymal stem cells (MSCs) were isolated from umbilical cord by collagenase and trypsin digestion. Three groups were set up: CD34+ cells pure culture group, cytokineinduced group and two stem cells co-culture group with noncontact. Results The average purity of enriched CD34+ cells as assessed by FACS was (95. 02 ± 3. 81) %. Freshly isolated CD34+ cells were small and round which suspended in culture medium. Attached cord-like structure cells of CD34+ cells appeared after 7 days coculture with noncontacted MSC, and when the CD34+ cells grew into large number, they formed colonies. These cells expressed endothelial specific markers, including CD144 (54. 40 ±4. 13)% ,vWF (47. 53 ± 3. 96) % , CD31 (65. 43 ± 5. 61) % ( P ＜ 0. 05, as compared with CD34+ cells pure culture group) , partially expressed CD34 and, the leukocyte common antigen CD45 was negatively expressed.Conclusion Human umbilical cord blood-derived CD34+ cells could be induced into endothelial cells under the paracrine action of umbilical cord-derive mesenchymal stem cells which has the same effect of cytokines.     

人脐血中血管内皮祖细胞体外扩增和鉴定的实验研究

目的：探讨从人脐血中分离、培养、体外扩增血管内皮祖细胞(EPC)的方法及体外进行EPC的鉴定。方法：采用密度梯度离心法从脐血中分离EPC，体外分别培养在包被有FN和不包被FN的培养皿中，采用免疫组织化学技术(SABC法)鉴定培养贴壁细胞表面标志CD31、CD34及KDR的表达；流式细胞仪检测细胞表面标志CD133及表达CD133细胞比例。结果：包被FN的培养皿中细胞贴壁及增殖均比未包被的多，免疫组化染色CD31、CD34及KDR均呈阳性，培养第4天流式细胞仪检测CD133^ 占21．3％。结论：密度梯度离心法可用于体外分离脐血中EPC进行实验研究，FN对于体外培养EPC有非常重要的作用。     

Living patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells.

OBJECTIVE: A major shortcoming in contemporary congenital heart surgery is the lack of viable replacement materials with the capacity of growth and regeneration. Here we focused on living autologous patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells (EPCs) as a ready-to-use cell source for paediatric cardiovascular tissue engineering. METHODS: EPCs were isolated from 20 ml fresh umbilical cord blood by density gradient centrifugation and myofibroblasts were harvested from umbilical cord tissue. Cells were differentiated and expanded in vitro using nutrient media containing growth factors. Before seeding, cell-phenotypes were assessed by immuno-histochemistry. Biodegradable patches fabricated from synthetic polymers (PGA/P4HB) were seeded with myofibroblasts followed by endothelialization with EPCs. All patches were cultured in a perfusion bioreactor. A subgroup of patches was additionally stimulated by cyclic strain. Analysis of the neo-tissues comprised histology, immuno-histochemistry, extracellular matrix (ECM) analysis and biomechanical testing. RESULTS: Endothelial phenotypes of EPCs before seeding were confirmed by Ac-Dil-LDL, CD 31, von-Willebrand-Factor and eNOS staining. Histology of the seeded patches demonstrated layered viable tissue formation in all samples. The cells in the newly formed tissues expressed myofibroblast markers, such as desmin and alpha-SMA. The EPCs derived neo-endothelia showed constant endothelial phenotypes (CD 31, vWF). major constituents of ECM such as collagen and proteoglycans were biochemically detected. Stress-strain properties of the patches showed features of native-analogous tissues. CONCLUSIONS: Living tissue engineered patches can be successfully generated from human umbilical cord derived myofibroblasts and EPCs. This new cell source may enable the tissue engineering of versatile, living, autologous replacement materials for congenital cardiac interventions.     

脐血间充质干细胞的分离扩增及向成骨及脂肪细胞的分化

目的探讨新生儿脐血间充质干细胞（mesenchymal stem cells，MSCs）体外分离、纯化、扩增，以及向成骨及脂肪细胞定向诱导分化的方法与条件。方法无菌条件下收集新生儿脐血60～120ml，枸橼酸钠抗凝，以Ficoll—Hypaque淋巴细胞分离液密度梯度法、沉降红细胞后密度梯度法及CD34^＋免疫磁珠负选法分离单个核细胞（mononuclear cells，MNCs）。分离获得的MNCs采用L—DMEM培养基或Mesencult^TM培养基／10％胎牛血清进行MSCs培养传代，获得第3代集落生长细胞作流式细胞仪表面抗原测定，并向成骨及脂肪细胞定向诱导分化，成骨细胞钙沉积经茜素红染色鉴定，脂肪细胞胞浆油滴经油红染色鉴定。结果经沉降红细胞后分离的MNCs，使用Mesencult^TM培养基／10％胎牛血清培养成功率高，第3代可出现明显的集落生长，而另两种方法分离培养的细胞则难以形成集落；集落细胞表面抗原测定表达CD29、CD59、CD71而不表达CD34、CD45及HLA—DR等分子。成骨定向诱导分化的集落细胞经茜素红染色胞浆中出现有大量的钙沉积；成脂肪定向诱导分化的集落细胞油红染色示胞浆充满油滴空泡。结论新生儿脐血中可分离出MSCs，并可在体外进行培养扩增。以甲基纤维素沉降红细胞后密度梯度离心分离的MNCs培养较为有效，集落细胞表达基质细胞表面抗原，能够向成骨细胞及成脂肪细胞定向诱导分化。     

An in vitro study demonstrating that haematomas found at the site of human fractures contain progenitor cells with multilineage capacity

We isolated multilineage mesenchymal progenitor cells from haematomas collected from fracture sites. After the haematoma was manually removed from the fracture site it was cut into strips and cultured. Homogenous fibroblastic adherent cells were obtained. Flow cytometry revealed that the adherent cells were consistently positive for mesenchymal stem-cell-related markers CD29, CD44, CD105 and CD166, and were negative for the haemopoietic markers CD14, CD34, CD45 and CD133 similar to bone-marrow-derived mesenchymal stem cells. In the presence of lineage-specific induction factors the adherent cells could differentiate in vitro into osteogenic, chondrogenic and adipogenic cells. Our results indicate that haematomas found at a fracture site contain multilineage mesenchymal progenitor cells and play an important role in bone healing. Our findings imply that to enhance healing the haematoma should not be removed from the fracture site during osteosynthesis.     

血小板裂解液对人脐带间充质干细胞体外成软骨分化的影响

目的探讨血小板裂解液(platelet lysate,PL)在体外定向诱导人脐带间充质干细胞(human umbilical cord derived mesenchymal stem cells,hUCMSCs)分化成软骨细胞中的作用。方法取健康产妇自愿捐赠脐带,采用胶原酶消化法分离hUCMSCs,体外培养扩增,流式细胞仪进行细胞表型鉴定。根据加入诱导培养基成分不同将实验分为以下3组:A组为H-DMEM培养基、10%FBS及10%PL,B组为H-DMEM培养基、10%FBS、10 ng/mL TGF-β1、1×10-7 mol/L地塞米松、50μg/mL维生素C及1%胰岛素铁硒传递蛋白(insulin-transferrin-selenium,ITS),C组为H-DMEM培养基、10%FBS、10 ng/mL TGF-β1、1×10-7 mol/L地塞米松、50μg/mL维生素C、1%ITS及10%PL。诱导培养2周,甲苯胺蓝染色检测各组软骨细胞基质的分泌,免疫荧光检测软骨特异性Ⅱ型胶原表达,半定量RT-PCR检测蛋白聚糖(Aggrecan)和Ⅱ型胶原表达。结果分离得到的hUCMSCs不表达造血细胞的表面标记CD45、CD34和HLA-DR,而表达黏附分子和MSCs表面标记CD44、CD105和CD146。甲苯胺蓝染色和Ⅱ型胶原免疫荧光染色示C组呈阳性,B组呈弱阳性,而A组均呈阴性。半定量RT-PCR检测示Aggrecan和Ⅱ型胶原在B、C组中均有表达,A组中未见表达;C组Aggrecan mRNA和Ⅱ型胶原mRNA表达明显高于B组,差异均有统计学意义(P<0.05)。结论单纯10%PL不能诱导hUCMSCs成软骨分化,但它可当作成软骨诱导培养基的辅助添加剂,对hUCMSCs成软骨分化有明显促进作用,为构建组织工程软骨提供了新的可利用条件。     

重组人骨形态发生蛋白-2质粒转染诱导人脐血间充质干细胞软骨分化研究

目的探究从人脐血中提取间充质干细胞，重组质粒pIRES2-EGFP-hBMP-2转染干细胞并诱导其成软骨化的可能性。 方法采用密度梯度离心方法获取脐带血中细胞，依据贴壁时间不同获得间充质干细胞，流式细胞仪检测表面抗原表达鉴定细胞；然后把重组有pIRES2-EGFP-hBMP-2的质粒导入间充质干细胞，观察EGFP的表达；ELISA方法检测在不同时间收集的培养基上清中hBMP-2蛋白含量，采用免疫荧光和RT-PCR方法检测目的蛋白和基因表达。转染成功后继续培养细胞2 w，免疫组化检测细胞Ⅱ型胶原的表达和RT-PCR检测软骨特异性标志物软骨连接蛋白（CRLT1）的表达。 结果两种方法可以获取人脐血间充质干细胞，流式细胞术鉴定发现CD90、CD105、CD146高表达，CD34、CD45、Anti-HLA-DR不表达。非脂质载体包裹重组质粒pIRES2-EGFP-hBMP-2可成功导入脐血间充质干细胞，转染率为（27.7±7.6）%。ELISA检测实验组和对照组hBMP-2的表达结果有统计学差异（t=3.355，P<0.01）。RT-PCR结果表明hBMP-2基因稳定转录，免疫荧光标记hBMP-2蛋白呈红色荧光。Ⅱ型胶原免疫组化染色示部分细胞被染成棕黄色，RT-PCR结果表明加入转染后的干细胞组CRLT1的表达量比未转染的干细胞组高（t=59.700，P<0.05）。 结论重组hBMP-2基因可以成功转染人脐血间充质干细胞并在胞内稳定表达，且有hBMP-2分泌，并可促进其表达Ⅱ型胶原蛋白及软骨连接蛋白，可向软骨细胞化诱导。     

hBMP-2基因改良修饰骨髓间充质干细胞和人脐血间充质干细胞研究

目的研究hBMP-2基因改良修饰骨髓和人脐血间充质干细胞的方法,并比较修饰结果。 方法联合应用密度梯度离心法和贴壁培养法分离、培养骨髓和人脐血间充质干细胞,通过高效非脂质体试剂X-treme GENE介导重组hBMP-2质粒转染骨髓和人脐血间充质干细胞,倒置荧光显微镜检测荧光强度并计算转染效率,qPCR检测hBMP-2及软骨连接蛋白在两种细胞中的表达,免疫组化检测细胞中Ⅱ型胶原的变化。 结果成功分离、培养出骨髓和人脐血间充质干细胞,两种细胞具有不同的生长特性。高效非脂质体试剂介导的重组hBMP-2质粒成功转染骨髓和人脐血间充质干细胞,转染骨髓间充质干细胞效率[（18.44±5.94）%]低于人脐血间充质干细胞[（27.74±7.59）%],且差异有统计学意义（t=3.027,P<0.05）。转染后的两种细胞均检测到hBMP-2、软骨连接蛋白及Ⅱ型胶原的表达。 结论hBMP-2基因可以有效改良修饰骨髓和人脐血间充质干细胞,且能促进其向软骨细胞方向分化。     

Human umbilical cord blood cells can be induced to express markers for neurons and glia

Rare cells are present in human umbilical cord blood that do not express the hematopoietic marker CD45 and in culture do not produce cells of hematopoietic lineage. These umbilical cord multipotent stem cells (UC-MC) behave as multilineage progenitor cells (stem cells) and can be expanded in tissue culture. Exposure to basic fibroblast growth factor (bFGF) and human epidermal growth factor (hEGF) for a minimum of 7 days in culture induces expression of neural and glial markers. Western immunoblots demonstrate expression of both beta-tubulin III and glial fibrillary acidic protein (GFAP). Immunocytochemistry of the cells showed intense labeling to both compounds on the intracellular cytoskeleton. The oligodendrocyte cell surface marker galactocerebroside (Gal-C) was present on most cells. Many cells show dual labeling, expressing both neuronal and glial markers.     

人脐血CD133+血管内皮祖细胞的培养、鉴定与分化研究

目的探讨从人脐血中分离、体外培养CD133^＋血管内皮祖细胞（EPCs）的方法及其生长特性和鉴定。方法采用密度梯度离心法结合MACS分选法提取人脐血中CD133^＋细胞,进行流式细胞仪检测纯度,予EBM-2培养液接种培养,观察其生长特性;利用Dil-LDL及FITC-Lectin摄取实验、细胞免疫组织化学等进行鉴定。结果经流式细胞仪检测CD133^＋细胞平均占单核细胞的（1．13±0．10）％,磁珠分选所得CD133^＋细胞平均纯度为（91．45±1．04）％;CD133^＋细胞贴壁生长,可分化为梭形血管内皮细胞及形成集落;CD133^＋细胞培养过程中Dil-LDL及FITC-Lectin摄取阳性,双染阳性率为（95．83±1．72）％;CD133^＋细胞培养1周后经免疫组织化学检测CD34、Ⅷ因子阳性率分别为（95．83±2．23）％和（95．92±1．43）％,与人脐静脉内皮细胞比较差异无统计学意义;CD133^＋细胞体外培养4d、1周可形成小血管结构。结论免疫磁珠分选法可获取较高纯度CD133^＋血管内皮祖细胞,在生长因子作用下诱导其分化为成熟血管内皮细胞。     

Quantification and Immunophenotypic Characterization of Bone Marrow and Umbilical Cord Blood Mesenchymal Stem Cells by Multicolor Flow Cytometry

In recent years, mesenchymal stem cells (MSC) have been attracting the greatest interest in the regeneration of injured tissues, autoimmune diseases, and transplantation of hematopoietic progenitor cells. Bone marrow (BM) represents the major source of MSC; however, umbilical cord blood (UCB) MSC has some advantages over BM, such as the higher differentiation capability and noninvasive collection methods. We sought to establish a 7-color, single-tube flow cytometric assay to quantify MSC in fresh tissues, namely BM and UCB, based on phenotypic markers of these cells. Moreover, we evaluated the differential expression of these markers in BM and UCB MSC. We used 5 UCB samples and 5 BM samples obtained from individuals without hematologic disease. To characterize MSC we used the following combination of monoclonal antibodies: CD71-FITC; CD105-PE; CD184-PE-Cy5; CD34-PE-Cy7; CD133-APC; CD45-APC-H7; CD44-Pacific blue, acquiring at least 1 million nucleated cells. We observed a greater number of BM MSC when compared with UCB MSC as well as some differences in the expression of some MSC antigens, particularly CD105 and CD44. Based on our preliminary results, phenotypic identification of MSC by flow cytometry is possible using a 7-color, single-tube assay. However, culture assays after sorting of cells characterized in this study are required to prove that they correspond to MSC.     

人脐血来源内皮祖细胞的纯化鉴定及定向分化的研究

目的 检测CD133^＋血管内皮祖细胞（EPC）的细胞表面标志物，鉴定其生物学特性。方法 采用免疫磁珠法分离人脐血EPC，用EGM-2MV培养基[含表皮生长因子（EGF）、血管内皮生长因子（VEGF）、成纤维细胞生长因子（FGF）2等具有诱导分化作用的细胞因子]进行体外培养。应用流式细胞术、免疫组织化学等方法检测CD133^＋细胞的比例、原代EPC的生长曲线及生长特性。透射电镜查找Weibel-Palade小体。同时设计EPC裸鼠成瘤实验，观察瘤体生长及血管增生情况，以鉴定EPC的生物学特性。结果 CD133^＋细胞在免疫磁珠分离前后的比例各为0．91％及85．52％。EPC原代培养时细胞形态正常、密度均匀，前3d为相对抑制期，此后快速增殖，10d后达80％～90％融合。EPC生长曲线显示，接种后5d内细胞数量无明显变化，从第7天开始明显增加，第17天达高峰。光学显微镜下可见，原代EPC贴壁后呈梭形，接种后7d数量明显增加，呈克隆状生长。透射电镜观察到，细胞膜伸出许多伪足丝，基底膜呈多层；细胞质内可见一种短棒状小体，含平行管状的内部结构，即Weibel-Palade小体，确定其为EPC。裸鼠成瘤实验可见EPC组肿瘤瘕体及血管数量大于或多于对照组；抗人血管性假血友病因子（vWF）免疫荧光染色显示，大量EPC参与肿瘤血管生成。结论本实验分离培养的CD133^＋细胞具有分化为成熟血管内皮细胞的能力，即为EPC。裸鼠成瘤实验初步证明EPC参与血管重建，具有促进血管新生、加速缺血组织血管化的作用。     

人脐带间充质干细胞获得和纯化及向软骨细胞分化能力的研究

目的 研究脐带间充质干细胞获得纯化的高效方法及向软骨细胞的分化能力.方法 取人的正常分娩或剖腹产胎儿的脐带,用胶原酶和胰酶初步消化后,将流式细胞仪筛选得到的CD45(-)、CD90(+)细胞作为原代细胞进行培养,并用复合胶原酶差速消化法逐级传代、纯化细胞,每次传代后取部分细胞用作流式细胞仪分析细胞的CD45、CD90阳性比率;对P3代细胞向软骨细胞进行诱导.结果 流式细胞仪检测提示,采用流式细胞仪进行初次筛选并用复合胶原酶差速消化法逐级传代获得的P1、P2、P3代细胞中CD90的阳性率为(80.86±7.85)%、(95.86±3.28)%、(97.15±1.43)%,而CD45的阳性率为(2.53±0.28)%、(0.97±0.48)%、(0.05±0.01)%;向软骨细胞诱导结果提示P3代细胞有向终末软骨细胞分化能力,具有干细胞的特性.结论 采用流式细胞仪进行初次筛选并用复合胶原酶差速消化法能快速获得高纯度的具有向终末软骨细胞分化能力的人脐带间充质干细胞.     

Phenotype and chondrogenic differentiation of mesenchymal cells from adipose tissue of different species

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into several mesoderm lineages. They have been isolated from different tissues, such as bone marrow, adult peripheral blood, umbilical cord blood, and adipose tissue. The aim of this study was to analyze the differences in proliferation and phenotype of adipose tissue‐derived MSCs from three different species, and to evaluate their capacity to differentiate into chondrocytes in vitro. A comparative study of cultured human, rabbit, and sheep mesenchymal cells from adipose tissue was carried out, and the main morphological parameters, proliferative activity, and expression of surface markers were characterized. Proliferation and flow cytometry data showed species‐related differences between animal and human MSCs. Histological staining suggested that rabbit and sheep mesenchymal cells were able to differentiate into chondrocytic lineages. Human mesenchymal cells, though they could also differentiate, accomplished it with more difficulty than animal MSCs. These results could help to explain the differences in the chondrogenic capacity of sheep and rabbit MSCs when they are used as animal models compared to human mesenchymal cells in a clinical assay. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1499–1507, 2009     

脐带间充质干细胞向汗腺样细胞分化后的表型变化

目的：比较人脐带间充质干细胞（hUC-MSCs）向汗腺样细胞诱导前后表型特征的变化.方法：对hUC-MSCs进行分离、培养、鉴定,通过显微镜观察、免疫细胞化学、流式细胞术等方法比较hUC-MSCs经汗腺分化诱导培养液培养前后细胞形态变化及癌胚抗原（CEA）、角蛋白7（CK7）、CK8、CK14、CK18、CK19表达的差异.结果：hUC-MSCs向汗腺样细胞诱导前呈梭形,呈成纤维细胞样;流式细胞仪检测发现CD29、CD90表达阳性;而CD34、CEA、CK14、CK19表达阴性.经汗腺诱导培养基诱导后hUC-MSCs分化为外形肥大、不规则、类似铺路石样的细胞,聚集性增殖;免疫细胞化学检测显示CK7、CK8、CK18抗原表达阳性;流式细胞仪检测结果显示CEA、CK14、CK19的阳性表达率分别为77.98%,48.47%,20.85%.结论：hUC-MSCs经过汗腺分化诱导培养基培养后能够分化为表达汗腺细胞标记物抗原的汗腺样细胞.     

Isolation of endothelial cells and their progenitor cells from human peripheral blood

Purpose: We have developed techniques to isolate endothelial cell (EC) progenitors from human peripheral and umbilical cord blood. Methods: Human adult peripheral and umbilical cord blood monocytes were isolated by centrifugation, and progenitor cells were separated with the use of magnetic polystyrene beads that were coated with a monoclonal antibody specific for the CD34 cell–membrane antigen. Cells were propagated in selective media, and developing cultures were immunostained for CD31, CD34, factor VIII, and vascular endothelial growth factor cell receptors. ECs that developed were transfected with a gene for prourokinase and used to line ePTFE grafts, which were evaluated in vitro in a pulsatile flow system. Results: Umbilical cord monocyte cultures demonstrated colonies that resembled ECs at approximately 2 weeks, with growth being best supported by EC growth media plus 20% calf serum with iron. Immunostaining of colonies was positive for CD31 and factor VIII. After 18 days in culture, CD34⁺ cells from adult peripheral blood were noted, which had the typical cobblestone appearance of ECs and immunostained positively for CD31 and factor VIII–related antigens. Cultures of umbilical cord–derived cells and adult peripheral blood–derived cells developed complex line formations within 1 week in culture that stained positively for vascular endothelial growth factor receptor-2. Urokinase-transfected ECs were shown to overexpress urokinase. Prosthetic grafts lined with transfected cells showed 87.33% ± 4.97% cell adherence after 2 hours in a pulsatile flow system at clinically relevant shear stress. Conclusion: We conclude that endothelial progenitor cells can be isolated from human adult peripheral and umbilical cord blood and developed into EC cultures as a source of cells for vascular graft seeding and gene therapy. (J Vasc Surg 2000;31:181-9.)     

黄芪诱导人脐带间充质干细胞分化为神经样细胞的实验研究

目的 研究原代人脐带间充质干细胞(UCMSCs)的生物学特性及其体外向神经细胞诱导分化的条件,为组织化人工神经的制备提供干细胞资源.方法 无菌条件下从人脐带华尔通胶(Wharton jelly)分离并贴壁培养人UCMSCs,免疫组化技术检测UCMSCs表面特异标志物表达;四组细胞分别加入单纯培养液空白对照组(A组)、单纯生长因子诱导组(B组)、单纯黄芪诱导组(C组)和黄芪联合生长因子诱导液(D组),倒置显微镜下观察其形态并检测NSE、NF和GFAP的阳性表达,结果进行统计学分析.结果 人UCMSCs表面标记CD29、CD44、CD105强阳性,CD106弱阳性,CD34、CD45阴性,符合人UCMSCs的特征;人UCMSCs经黄芪诱导后细胞表面GFAP、NSE均为强阳性表达.结论 人UCMSCs可以在体外采用组织块贴壁培养法培养成功,黄芪可将人UCMSCs成功的诱导为神经样细胞,为组织化人工神经的制备和周围神经缺损的治疗提供了新的方法.

Abstract：

Objective To investigate the biological characteristics of human umbilical cord derived mesenchymal stem cells(UCMSCs) and inducing them to differentiate into neurons in vitro,in order to provide stem cell resource for the tissue engineering artificial nerve. Methods UCMSCs were cultured from Wharton jelly of human umbilical cord in the condition of sterilitas,surface antigens of UCMSCs were detected by immunohistochemistry.The frist group of cells were added by virgin nutrient,the second group of cells were induced by merely growth factors,the third of cells were induced by merely astragalus mongholicus and the fourth by the astragalus mongholicus with growth factor,observed the morphology of the cells of the two groups under inverted microscope,and determine the positive expression rate of nerve cells' markers,such as NSE,NF and GFAP.The results were statistically analyzed.Results UCMSCs were strongly positive for CD29,CD44,CD105,weakly positive for CD105 and negative for CD34,CD45.After induced by Astragalus mongholicus,UCMSCs were strongly positive for GFAP,NSE. Conclusion UCMSCs can be successfully cultured from the adherent tissue pieces,Astragalus mongholicus can successfully induce them to differentiate into neurons in vitro,to provide a new method of making the tissue engineering artificial nerve and treat theperipheraly nervus defect.     

黄芪诱导人脐带间充质干细胞分化为神经样细胞的实验研究

目的 研究原代人脐带间充质干细胞(UCMSCs)的生物学特性及其体外向神经细胞诱导分化的条件,为组织化人工神经的制备提供干细胞资源.方法 无菌条件下从人脐带华尔通胶(Wharton jelly)分离并贴壁培养人UCMSCs,免疫组化技术检测UCMSCs表面特异标志物表达;四组细胞分别加入单纯培养液空白对照组(A组)、单纯生长因子诱导组(B组)、单纯黄芪诱导组(C组)和黄芪联合生长因子诱导液(D组),倒置显微镜下观察其形态并检测NSE、NF和GFAP的阳性表达,结果进行统计学分析.结果 人UCMSCs表面标记CD29、CD44、CD105强阳性,CD106弱阳性,CD34、CD45阴性,符合人UCMSCs的特征;人UCMSCs经黄芪诱导后细胞表面GFAP、NSE均为强阳性表达.结论 人UCMSCs可以在体外采用组织块贴壁培养法培养成功,黄芪可将人UCMSCs成功的诱导为神经样细胞,为组织化人工神经的制备和周围神经缺损的治疗提供了新的方法.