不同来源骨髓间质干细胞分离培养及成骨分化比较

目的：间质干细胞（MSCs）是多能成体干细胞,近年关于其在肿瘤相关领域的研究得到了国内外广泛关注。我们分离、鉴定并纯化不同来源的骨髓间质干细胞,比较其在成骨分化的差异,为下一步试验选择良好的细胞来源。方法：采用差速贴壁法分离SD大鼠及Balb/c小鼠骨髓间质干细胞,通过传代纯化,并观察MSCs在此过程中的纯化程度及增殖状况。流式细胞术鉴别两种MDSCs细胞表面抗原的表达。比较不同来源间质干细胞在成骨分化的差异。结果：在SD大鼠MDSCs分离和培养过程中,其增殖能力显著强于Balb/c小鼠MDSCs,在第三代时即可见典型的漩涡状贴壁生长特性。经流式细胞术检测两种来源的细胞均表达间质干细胞表面抗原。在成骨方面SD大鼠MDSCs表现出了较强的多能特性,且SD大鼠出现程度大于Balb/c小鼠。结论：SD大鼠来源MDSCs较易获得纯度较高且增殖旺盛的细胞源,在成骨方面的能力强于Balb/c小鼠,在一定程度上其可成为良好的细胞来源。     

脐带来源的华通氏胶间充质干细胞的分离、特性及应用前景

间充质干细胞(MSCs)的来源非常广泛。虽然各种组织来源的MSCs生物学特性不尽相同，但在标准培养条件下该类细胞均可以贴壁生长；细胞群体中有 95%以上的细胞表达典型的间充质标记分子，如 CD73、CD90 和 CD105，但缺乏造血标记分子CD14、CD19、CD34、CD45和CD79a的表达；特别是均具有分化为成脂细胞、成骨细胞和成软骨细胞的能力。这些性质是国际细胞治疗协会给出的定义该类细胞的最低标准。脐带间充质干细胞是指来源于新生儿脐带组织中的多能干细胞。目前来看，从脐带不同解剖学部位(区室)获得的MSCs的生物学性质也存在差异，其中从脐带华通氏胶中分离获得的华通氏胶间充质干细胞，在增殖能力、分化潜能、免疫调节能力等方面均明显优于其他区室来源的间充质干细胞，是用于组织器官损伤修复及免疫调节的一种理想的“种子”细胞，被认为具有更好的临床应用前景。本文围绕近年来华通氏胶间充质干细胞的分离建系、增殖特性、分化潜能以及临床应用方面的研究进展进行了综述，其中重点介绍了化学诱导分化方案的建立和优化，认为与通过遗传改变诱导重编程方法相比，化学诱导分化方法具有更好的临床应用潜力。     

七氟烷预处理对大鼠骨髓间充质干细胞在缺氧复氧环境下的作用和机制初探

目的:研究七氟烷预处理对大鼠骨髓间充质干细胞（MSCs）在缺氧复氧环境下的作用和机制初探。方法:分离培养SD大鼠骨髓间充质干细胞，通过流式细胞仪和诱导分化培养鉴定其免疫表型和分化潜能。细胞随机分为3组:组1空白对照组；组2缺氧复氧组（缺氧24 h，复氧24 h）；组3七氟烷预处理组（2%七氟烷预处理2 h后，缺氧24 h，复氧24 h）。处理后收集各组细胞，用流式细胞仪测定其凋亡率，线粒体膜电位变化及细胞周期。Western blot测定细胞外调节蛋白激酶（ERK）的蛋白表达。结果:与组2相比，组3 MSCs凋亡率明显下降，处于S期细胞增多，细胞增殖能力增强。Western blot结果显示:与组2相比，组3磷酸化ERK蛋白表达上升。结论:七氟烷预处理能提高缺氧复氧环境下骨髓间充质干细胞存活率，降低MSCs凋亡数量，并能增强MSCs的增殖能力，这一机制与七氟烷促进MSCs细胞p-ERK蛋白表达相关。     

妇科肿瘤相关间充质干细胞研究进展

目的 总结目前妇科肿瘤相关间充质干细胞(mesenchymal stem cells,MSCs)的研究进展.方法 应用PubMed和CNKI期刊全文数据库检索系统,以“宫颈肿瘤、卵巢肿瘤、子宫体肿瘤、外阴肿瘤、阴道肿瘤、输卵管肿瘤和间充质干细胞”为关键词,检索2000-01-01-2014-10-31的相关文献,纳入标准:1)MSCs相关文献;2)正常组织来源的MSCs与肿瘤;3)肿瘤组织中的MSCs;4)妇科肿瘤组织中的MSCs.根据纳入标准符合分析的文献29篇.结果 1)MSCs是一种机体内部间质内广泛存在的非造血系成体干细胞,可分化为骨骼、软骨和脂肪细胞等.2)正常组织来源的MSCs能调控肿瘤生长,起抑制还是促进作用与宿主免疫功能状态、肿瘤细胞类型和所处微环境等因素有关.肿瘤相关的炎症环境能改变正常MSCs的功能,使之获得高效招募炎性单核及巨噬细胞的能力,从而达到促进肿瘤生长的目的.MSCs能分化为成纤维细胞,为肿瘤进展提供结构和功能支持,形成血管细胞,促进微血管形成,形成间质网络改善肿瘤微环境,从而促进肿瘤的增殖.3)肿瘤组织中也存在MSCs,在肿瘤增殖调控中起重要作用,在肺癌、胃癌和骨肉瘤等实体瘤中得到分离和验证.4)妇科常见恶性肿瘤中的宫颈癌、卵巢癌和子宫内膜癌组织中均能分离出具有多向分化潜能的MSCs.宫颈癌组织标本的MSCs亚群,形态似成纤维细胞,呈克隆样增殖,免疫表型为CD13、CD29、CD44、CD105和HLA-I阳性,细胞保持正常的核型,在适当的诱导条件下,这些细胞能够分化成骨细胞等;宫颈癌相关MSCs能促进肿瘤细胞增殖,机制与血管生成和上皮间质转化相关.卵巢癌腹水和肿瘤组织中均可分离出MSCs,高表达CD44、MMP9和Oct4,具有正常的形态和核型,能分化成脂肪、软骨和骨,本身无致瘤性,能通过增加肿瘤干细胞的数目以促进肿瘤的生长.子宫内膜基质中的MSCs可以长期增殖,有分化和克隆形成能力.异位子宫内膜的MSCs侵袭和浸润能力更强.子宫内膜癌中分离出的侧群细胞,能分化成MSCs,可以在裸鼠体内形成大的浸润性肿瘤,具有丰富的细胞外基质,有肿瘤细胞和间质细胞样细胞.但目前鲜见内膜癌相关的MSCs调控内膜癌细胞增殖的研究报道.结论 通过对妇科肿瘤组织中MSCs的研究,有望深入了解肿瘤侵袭、转移发生的机制,从而为妇科肿瘤的治疗提供新的理论依据和临床靶点.     

自发恶性转化的大鼠骨髓间充质干细胞基因表达谱的芯片分析

目的:解析骨髓间充质干细胞(MSCs)作为组织工程种子细胞的自发恶性转化的分子遗传学基础,探讨其临床可用性和安全性.方法:密度梯度离心法和贴壁筛选法联合应用,分离大鼠MSCs,流式细胞仪分析细胞同源性,体外培养6个月后获得自发恶性转化的MSCs.Trizol总RNA抽提法获取足量的RNA,用于MSCs基因表达谱的分析.实时定量RT-PCR法对在自发恶性转化的MSCs中呈差异表达的基因进行扩增和检测,以验证基因芯片分析结果.结果:流式细胞仪检测表明了所分离MSCs的高度同源性.MSCs发生自发恶性转化后,有44条差异表达基因,其中21条基因表达上调,23条基因表达下调.经实时定量RT-PCR检测差异表达基因结果与基因芯片结果一致.结论: Wnt、SHH、Notch、TGFβ/BMPs等信号转导通路上的若干基因在MSCs自发恶性转化中起到重要作用.     

骨髓间充质干细胞表达外源性IL-12对胶质瘤C6细胞增殖的影响

目的: 探讨以骨髓间充质干细胞(mesenchymal stem cells,MSCs) 为基因治疗载体表达外源性IL-12对胶质瘤C6细胞增殖的影响.方法:分离培养大鼠MSCs, 腺病毒介导IL-12基因转染大鼠MSCs(AdIL-12-MSCs),RT-PCR 及Western Blotting检测AdIL-12-MSCs中IL-12基因mRNA及蛋白表达.MTT法检测AdIL-12-MSCs分泌的外源性IL-12对C6胶质瘤细胞增殖活性的影响,光镜下观察外源性IL-12对C6细胞形态的影响.结果:腺病毒介导IL-12基因成功转染MSCs形成AdIL-12-MSC,其IL-12基因在mRNA及蛋白水平均有明显表达.AdIL-12-MSC分泌的外源性IL-12显著抑制胶质瘤C6细胞的增殖(P<0.05).结论:转染IL-12的MSCs(AdIL-12-MSC)能够在mRNA及蛋白水平表达外源性IL-12基因,显著抑制胶质瘤C6细胞的增殖.     

静脉注射骨髓基质干细胞对放射性脑损伤模型大鼠认知障碍的影响

目的观察静脉注射骨髓基质干细胞(MSCs)对放射性脑损伤模型大鼠认知功能的影响。方法离体分离、培养、增殖成年大鼠MSCs。采用6 MV X线对8～10周龄雌性SD大鼠做全脑照射,照射剂量20 Gy。照后1周采用尾静脉注射Hoechst33342荧光标记的MSCs(4×10~6),注射后4、8周观察大鼠认知功能变化,应用免疫荧光技术检测大鼠脑内Hoechst33342标记细胞及分化的功能细胞。结果与正常对照组相比,模型组大鼠的学习记忆能力明显受损(P<0.01),静脉注射后4、8周,照射组大鼠认知功能明显改善(P<0.05)。结论静脉注射MSCs可显著改善全脑照射大鼠认知功能,MSCs可在放射性脑损伤大鼠脑组织中存活和分化。     

羊膜间充质干细胞对异体外周血淋巴细胞转化的影响

[目的]研究羊膜间充质干细胞（MSCs）对成人外周血淋巴细胞的免疫调节作用。[方法]采用组织块培养法从羊膜分离、培养MSCs,通过细胞形态的均一性和流式细胞术检测细胞表面标志以鉴定其纯度。将分离培养的羊膜MSCs按不同浓度接种于96孔板中,72h待其贴壁后,用丝裂霉素处理。将外周血淋巴细胞接种于羊膜MSCs上,加入植物凝集素（PHA）以刺激淋巴细胞转化。以单纯淋巴细胞（不含MSCs）经PHA刺激转化者为对照组,设单纯淋巴细胞（不加PHA）作空白对照。各组细胞培养72h后,收集各孔悬浮细胞,用CCK-8法测定细胞增殖率,观察羊膜MSCs对异体外周血淋巴细胞转化的影响。[结果]羊膜MSCs的形态和骨髓MSCs类似,具有和骨髓MSCs相似的细胞表型,可以显著抑制异体外周血淋巴细胞转化。羊膜MSCs浓度为2×106/ml、1×106/ml、5×105/ml、2.5×105/ml、1.25×105/ml时的平均转化抑制率分别为88.92%、83.38%、62.10%、32.22%、17.35%。[结论]从羊膜中可以成功分离、培养出MSCs,为MSCs提供了新的来源。羊膜MSCs可以抑制PHA诱导的异体外周血淋巴细胞转化,对同种异体免疫反应具有负调节作用,且该作用具有剂量依赖性。     

间充质干细胞的历史沿革、生物学特性与应用前景

上世纪70年代,首次有报道骨髓中少部分塑性贴附细胞能够分化形成类似骨或软骨的集落,这些细胞最终可以分化为间质系统细胞,被定义为"间质干细胞(mesenchymal sten cells,MSCs)".MSC能表达多种表面抗原,但不具有特定的特征.MSCs具有自身更新能力和多向分化潜能,向骨、脂肪、软骨、骨骼肌细胞、甚至肝细胞、神经细胞分化,近年还发现MSC易于外源基因的转染和表达,被认为是很好的基因载体.MSC支持造血的作用已得到公认.目前的MSC的移植实验多为先在体外诱导分化为相应组织细胞后再植入体内,诱导分化操作易改变细胞特性,运用到临床治疗上,会发生种种无法预测的可能.     

三氧化二砷负向调控MDSC的肿瘤免疫抑制功能

目的:研究三氧化二砷(arsenic trioxide,As2O3)是否通过抑制髓系来源抑制性细胞(myeloid-derived suppressor cells,MDSCs)负向调控MDSCs诱导的肿瘤免疫耐受作用.方法:C57BL/6小鼠皮下注射黑素瘤B16细胞和肝癌H22细胞构建移植瘤模型,As2O3处理,观察移植瘤生长情况,流式细胞术检测荷瘤小鼠脾脏内MDSCs和其他免疫细胞的免疫表型,流式细胞术检测2 μmol/L As2O3对B16模型小鼠来源的MDSCs分化的影响.取B16模型小鼠随机分为As2O3处理及对照组,混合淋巴细胞反应检测MDSCs对T细胞免疫抑制活性的改变,ELISA检测B16模型小鼠血清及MDSCs培养上清中TNF-α、IL-10的水平.结果:As2O3抑制B16和H22模型鼠的肿瘤生长,延长B16模型小鼠的生存期,并可显著下调小鼠脾脏内MDSCs的比例.体外经2 μmol/L As2O3处理5d后,B16模型小鼠来源的MDSCs中成熟DCs(CD11c+ CD40+)的比例较对照组显著升高[(27.38±4.57)％ vs (17.44±4.51)％,P=0.0078];As2O3组来源的MDSCs对T细胞的免疫抑制活性明显低于对照组(P =0.016);As2O3组小鼠血清及MDSCs培养上清中TNF-α(F=5.78,P=0.014)和IL-10(F=17.83,P=0.045)的含量均较对照组显著降低.结论:As2O3可通过诱导MDSCs向成熟表型分化、下调其免疫抑制活性等,负调控MDSCs的肿瘤免疫抑制功能.     

骨髓间充质干细胞多向分化潜能和微环境关系的研究

目的:深入研究骨髓间充质干细胞(mesenchymal stem cells,MSCs)多向分化的潜能与微环境的关系,从而为促进MSCs向目标组织细胞诱导分化创建新的实验方法。方法:大鼠MSCs进行分离,体外培养、扩增、鉴定。将MSCs向脂肪细胞和胰岛细胞方向诱导分化,并深入研究在不同的微环境下,其分化能力的差别,对照组诱导剂为含有角朊细胞生长因子(KGF)、胰岛素-转铁蛋白-硒(ITS)、尼克酰胺的无血清DMEM/F12培养基,实验组在对照组基础上添加胰腺条件培养液;对诱导的胰岛细胞进行观察、双硫腙染色,并进行葡萄糖刺激实验,测定细胞分泌胰岛素及C-肽功能。结果:培养的MSCs表现为非造血干细胞特性。其可向脂肪细胞,胰岛细胞等不同组织细胞分化。对照组和实验组均可分化为胰岛细胞,但实验组分化而成的胰岛细胞在数量和功能上均高于对照组。结论:MSCs具有多向分化潜能,其分化能力在特定的微环境下更强。     

A Descriptive Study of the Physical Direct Interaction between Adipose Tissue-Mesenchymal Stem Cells and Colo 205 Cells: Impact on Cancer Cells Stemness,and Intracellular Reactive Oxygen Species Levels

Background: Mesenchymal stem cells (MSCs) are widely used in clinical research to treat a wild spectrum of diseases due to their homing ability to damaged tissues, self-renewal capacity, and differentiation ability into various types of cells. In this research, we are describing the physical direct interaction between AT-MSCs and colon cancer cells, its impact on the stemness of colon cancer cells, along with the levels of intracellular Reactive Oxygen Species (ROS) levels in both types of cells. Methods: Adipose-tissue mesenchymal stem cells (AT-MSCs) were characterized by the means of MSCs classical markers expression using flow cytometry, and multilineage differentiation through osteogenic and adipogenic differentiation. MSCs and colo205 cells were cocultured in monolayer and 3D techniques in a ratio of 1:3 for 72 hours without media exchange and compared to monocultured cells. The physical direct interaction of cells in adhered culture and spheroids formation in ULA plates was observed using a light-inverted microscope. MSCs classical markers and cancer stem cells (CSCs) associated surface proteins were quantified in MSCs and colo205 cells. Intracellular ROS level was measured in both cell types. Surface protein and intracellular ROS quantification were carried out using flow cytometry. Results: CRC cells (colo205 cells) utilized MSCs as a feeder layer to grow and generate spheroids. The interaction increased the percentage of CSCs in colo205 population which was attributed to the increased expression of CD133, and reduced the levels of intracellular ROS in MSCs. Results indicated that MSCs support the growth, spheriod formation, and the stemness of colon cancer cells, while reducing the levels of intracellular ROS in MSCs.     

骨髓间充质干细胞多向分化潜能和微环境关系的研究

目的：深入研究骨髓间充质干细胞（mesenchymal stem cells,MSCs）多向分化的潜能与微环境的关系,从而为促进MSCs向目标组织细胞诱导分化创建新的实验方法。方法：大鼠MSCs进行分离,体外培养、扩增、鉴定。将MSCs向脂肪细胞和胰岛细胞方向诱导分化,并深入研究在不同的微环境下,其分化能力的差别,对照组诱导剂为含有角朊细胞生长因子（KGF）、胰岛素-转铁蛋白-硒（ITS）、尼克酰胺的无血清DMEM／F12培养基,实验组在对照组基础上添加胰腺条件培养液;对诱导的胰岛细胞进行观察、双硫腙染色,并进行葡萄糖刺激实验,测定细胞分泌胰岛素及C-肽功能。结果：培养的MSCs表现为非造血干细胞特性。其可向脂肪细胞,胰岛细胞等不同组织细胞分化。对照组和实验组均可分化为胰岛细胞,但实验组分化而成的胰岛细胞在数量和功能上均高于对照组。结论：MSCs具有多向分化潜能,其分化能力在特定的微环境下更强。     

Impaired osteogenic differentiation of mesenchymal stem cells derived from bone marrow of patients with lower-risk myelodysplastic syndromes

The pathogenesis of myelodysplastic syndromes (MDS) has not been completely understood, and insufficiency of the hematopoietic microenvironment can be an important factor. Mesenchymal stem cells (MSCs) and osteoblasts are key components of the hematopoietic microenvironment. Here, we measured the expression of multiple osteogenic genes in 58 MSCs from MDS patients with different disease stages and subtypes by real-time PCR and compared the osteogenic differentiation of MSCs from 20 MDS patients with those of MSCs from eight normal controls quantitatively and dynamically. The mRNA level of Osterix and RUNX2, two key factors involved in the early differentiation process toward osteoblasts, was significantly reduced in undifferentiated MSCs from lower-risk MDS. After osteogenic induction, lower-risk MDS showed lower alkaline phosphatase activity, less intense alizarin red S staining, and lower gene expression of osteogenic differentiation markers; however, higher-risk MDS was normal. Finally, in bone marrow biopsy, the number of osteoblasts was significantly decreased in lower-risk MDS. These results indicate that MSCs from lower-risk MDS have impaired osteogenic differentiation functions, suggesting their insufficient stromal support in MDS.     

Bone marrow-derived mesenchymal stem cells

Human mesenchymal stem cells (MSCs) contribute to the regeneration of mesenchymal tissues, and are essential in providing support for the growth and differentiation of primitive hemopoietic cells within the bone marrow microenvironment. Techniques are now available to isolate human MSCs and manipulate their expansion in vitro under defined culture conditions without change of phenotype or loss of function. Mesenchymal stem cells have generated a great deal of interest in many clinical settings, including that of regenerative medicine, immune modulation and tissue engineering. Studies have already demonstrated the feasibility of transplanted MSCs providing crucial new cellular therapy. In this review, many aspects of the MSC will be discussed, with the main focus being on clinical studies that describe the potential of MSCs to treat patients with hematological malignancies who are undergoing chemotherapy and/or radiotherapy.     

骨髓增生异常综合征患者骨髓来源间充质干细胞的成骨分化特点及其临床意义

目的：探讨骨髓增生异常综合征（myelodysplastic syndrome,MDS)患者骨髓来源间充质干细胞( mesenchymal stemcell, MSC)成骨分化特点及其临床意义。方法: 骨髓标本取自河北大学附属医院血液内科30 例未经治疗的新诊断的MDS患者。分离培养MDS患者及正常人的MSC,体外培养并观察MSC的形态学特点。在适当条件下诱导MSC向成骨细胞和成脂细胞分化,茜素红染色观察成骨诱导分化第14 天钙结节形成情况,实时荧光定量PCR法检测未分化MSC中成骨分化转录因子Ostefix、RUNX2 以及参与造血细胞向白血病细胞转化的Jagged-1 的mRNA表达水平。结果：MDS患者的骨髓来源的MSC表现为细胞体积增大、分化潜能下降。与对照组相比,成骨分化转录因子Osterix 和RUNX2 表达水平明显下降(均P<0.05);茜素红染色结果显示,MDS组钙结节含量也明显少于正常对照组,而Jagged-1 的表达水平明显升高(均P<0.05)。结论：MDS骨髓来源的MSC细胞体积增大、成骨分化能力明显减弱以及Jagged-1 的表达水平升高,可能在MDS造血功能衰竭和向急性髓系白血病转化的过程中发挥了重要作用。     

Production and characterization of monoclonal antibodies to mesenchymal stem cells derived from human bone marrow

The bone marrow (BM) serves as a reservoir for different classes of stem cells. In addition to haematopoietic stem cells, bone marrow contains a population of mesenchymal stem cells (MSCs). These cells have a multilineage differentiation capacity and are able to generate progenitors with restricted developmental potential, which include fibroblasts, osteoblasts, adipocytes, and chondrocytes. Characteristic markers have been reported for expanded MSCs, but none of these markers are specific for MSCs. Thus, the objective of this study was to produce monoclonal antibodies against MSCs. MSCs derived from human bone marrow were cultured, expanded, and immunized into mice, and spleen cells subsequently harvested were used to generate hybridoma cell lines secreting antibodies against MSCs. Hybridoma culture supernatants were screened for antibodies against MSCs by enzyme-linked immunosorbent assay (ELISA), and 33 positive clones were then screened against cell suspensions of MSCs by immunofluorescence staining and flow cytometry. Ten clones were positive in immunofluorescence staining. Among these, three hybridoma cell lines, namely, YS08, YS14, and YS18 were found to be reactive with MSC by flow cytometry, but non-reactive with human tumor cell lines and hematopoietic stem cells. YS08 and YS14 showed specific bands in Western blotting. In conclusion, we developed three monoclonal antibodies, YS08, YS14, and YS18, that recognize human MSC cell surface antigen.