骨髓基质干细胞治疗骨不连的新进展

背景:骨髓基质干细胞体外培养增殖力强、易于向成骨细胞及软骨细胞方向分化且成骨性能稳定等特点,成为骨组织工程中合适的种子细胞.目的:总结分析采用骨髓基质干细胞作为种子细胞,分析其直接移植于骨不连部位或复合支架或转基因治疗骨不连所具有的优劣势.方法:检索1992/2011西文生物医学期刊文献数据及CNKI 数据库有关骨不连研究,骨髓基质干细胞分离、培养,在骨不连方面的应用,骨组织工程细胞支架方面的文献,英文检索词为"bone marrow stromal stem cells,nonunions,repairing,tissue engineering",中文检索词为"骨髓基质干细胞,骨修复,骨不连,组织工程".排除重复性研究,保留23篇进一步归纳总结.结果与结论:利用骨髓基质干细胞作为种子细胞,直接植入骨不连部位,或与适当的支架材料结合,或用骨髓基质干细胞作为靶细胞,导入外源目的基因诱导成骨的基因治疗来修复骨缺损的方法,给骨缺损的治疗带来光明的前景.但同时也存在骨髓基质干细胞增殖、分化合适条件难以准确确定,经皮移植自体骨髓基质干细胞植入体内后容易流失,不能在植入部位形成有效的细胞浓度,支架材料尚不能完全符合临床要求,以及如何将骨组织工程与基因治疗的方法结合起来等问题,需要进一步的研究.     

骨髓基质干细胞治疗骨不连的新进展

背景：骨髓基质干细胞体外培养增殖力强、易于向成骨细胞及软骨细胞方向分化且成骨性能稳定等特点,成为骨组织工程中合适的种子细胞.目的：总结分析采用骨髓基质干细胞作为种子细胞,分析其直接移植于骨不连部位或复合支架或转基因治疗骨不连所具有的优劣势.方法：检索1992/2011西文生物医学期刊文献数据及CNKI 数据库有关骨不连研究,骨髓基质干细胞分离、培养,在骨不连方面的应用,骨组织工程细胞支架方面的文献,英文检索词为＂bone marrow stromal stem cells,nonunions,repairing,tissue engineering＂,中文检索词为＂骨髓基质干细胞,骨修复,骨不连,组织工程＂.排除重复性研究,保留23篇进一步归纳总结.结果与结论：利用骨髓基质干细胞作为种子细胞,直接植入骨不连部位,或与适当的支架材料结合,或用骨髓基质干细胞作为靶细胞,导入外源目的基因诱导成骨的基因治疗来修复骨缺损的方法,给骨缺损的治疗带来光明的前景.但同时也存在骨髓基质干细胞增殖、分化合适条件难以准确确定,经皮移植自体骨髓基质干细胞植入体内后容易流失,不能在植入部位形成有效的细胞浓度,支架材料尚不能完全符合临床要求,以及如何将骨组织工程与基因治疗的方法结合起来等问题,需要进一步的研究.     

骨髓基质细胞移植治疗外伤性脑损伤的效应

目的:综述骨髓基质细胞移植治疗外伤性脑损伤的效应。资料来源:应用计算机检索PUBMED 1996-10/2006-10期间的相关文章,检索词为“bone marrow stromal cells(BMSCs),traumatic brain in jury(TBI),cell transplantation”,并限定文章语言种类为English。资料选择:对资料进行初审,并查看每篇文献后的引文。纳入标准:文章所述内容应与骨髓基质细胞移植治疗外伤性脑损伤研究相关。排除标准:重复研究或Meta分析类文章。资料提炼:共收集到138篇相关文献,31篇文献符合纳入标准,排除的107篇文献为内容陈旧或重复。符合纳入标准的31篇文献中,分别涉及骨髓基质细胞的生物学特性、体外诱导分化、移植途径、对外伤性脑损伤的修复以及修复中枢神经系统损伤的机制。资料综合:骨髓间充质干细胞是一群存在于骨髓中的非造血干细胞,具有较强的自我更新能力和多向分化潜能,在体外不同的诱导条件下可分化为骨细胞、软骨细胞、肌腱细胞、脂肪细胞、神经细胞、平滑肌细胞等多种细胞。通过脑实质内直接注射、脑脊液内注射或血管内注射移植后,骨髓基质细胞可透过血脑屏障集中于损伤区并在中枢神经系统内长时间存活。通过替代受损细胞和激活内源性修复机制,骨髓基质细胞可改善损伤的神经功能、促进外伤后神经重塑。随着对骨髓基质细胞研究的深入,以下几个方面还需进一步研究:骨髓基质细胞特异性标志的确定及快速分离纯化、归巢及体内分化的相关机制、促进损伤后神经组织重塑的分子机制。结论:骨髓基质细胞于脑实质内直接注射、脑脊液内注射或血管内注射移植后均能促进外伤性脑损伤修复,作用机制主要为替代受损细胞和激活内源性修复。目前尚存在一些未解决的问题,但骨髓基质细胞治疗外伤性脑损伤仍是一种很有前途的治疗方法。     

膝关节半月板损伤与骨髓基质干细胞

背景：骨髓基质干细胞移植是解决软骨组织缺损最有前景的手段之一,骨髓基质干细胞也成为软骨组织修复应用较广泛的重要种子细胞。目的：分析骨髓基质干细胞在半月板损伤和软骨损伤方面的应用情况,为膝关节半月板损伤的修复和治疗提供理论参考。方法：由第一、二作者共同检索1997/2011PubMed数据库及清华同方数据库,中文关键词为＂骨髓间充质干细胞,膝关节半月板损伤＂,英文关键词为＂bone marrow stromal cells;knee joint meniscus injury＂。纳入骨髓基质干细胞修复膝关节半月板损伤的相关文献26篇进行分析。结果与结论：骨髓基质干细胞在治疗和修复膝关节半月板损伤方面具有独到的优势,包括其损伤面较小,方便易行,没有后遗症,并克服了切除半月板引起的形态和生物功能的改变,避免了因异体材料移植引起的膝载荷传导紊乱和晚期骨性关节炎发病的可能,与保留半月板修复其功能的临床治疗理念较为接近,并成为当前和今后治疗与修复膝关节半月板损伤的新思路。在其修复治疗的过程中,其关键问题在于一方面如何获得大量纯化的骨髓基质干细胞并在体外定向诱导为半月板软骨表型的细胞,另一方面是诱导分化的确切机制研究。     

骨髓基质干细胞与骨缺损的修复

背景：骨髓基质干细胞来源广泛,分离方便,扩增迅速,具有多向分化潜能,适合自体移植的特点,同时是骨组织工程中合适的种子细胞。目的：总结分析采用骨髓基质干细胞作为种子细胞,并利用转基因或其复合支架修复骨缺损所具有的优势。方法：检索1990/2009西文生物医学期刊文献数据及CNKI数据库有关骨髓基质干细胞分离、培养,在骨缺损方面的应用,骨组织工程方面的文献,英文检索词为＂Bone marrow stem cells,Repairing,Bone defect＂,中文检索词为＂骨髓基质干细胞,修复,骨缺损＂。排除重复性研究,保留28篇进一步归纳总结。结果与结论：文章从骨髓基质干细胞作为种子细胞的优势,骨髓基质干细胞的分离和培养,骨髓基质干细胞的成骨诱导及骨髓基质干细胞修复骨缺损等方面进行了总结。利用骨髓基质干细胞作为种子细胞,与适当的支架材料结合,或用骨髓基质干细胞作为靶细胞,导入外源目的基因诱导成骨的基因治疗来修复骨缺损的方法,给骨缺损的治疗带来光明的前景。但同时也存在骨髓基质干细胞存化,骨髓基质干细胞的增殖、分化合适的条件,哪些因子能有效促进新骨形成,载体材料及体内植入方式,以及如何将骨组织工程与基因治疗的方法结合起来等问题需要进一步的研究。     

骨髓基质干细胞与骨缺损的修复

背景:骨髓基质干细胞来源广泛,分离方便,扩增迅速,具有多向分化潜能,适合自体移植的特点,同时是骨组织工程中合适的种子细胞.目的:总结分析采用骨髓基质干细胞作为种子细胞,并利用转基因或其复合支架修复骨缺损所具有的优势.方法:检索1990/2009 西文生物医学期刊文献数据及CNKI数据库有关骨髓基质干细胞分离、培养,在骨缺损方面的应用,骨组织工程方面的文献,英文检索词为"Bone marrow stem cells,Repairing,Bone defect",中文检索词为"骨髓基质干细胞,修复,骨缺损".排除重复性研究,保留28篇进一步归纳总结.结果与结论:文章从骨髓基质干细胞作为种子细胞的优势,骨髓基质干细胞的分离和培养,骨髓基质干细胞的成骨诱导及骨髓基质干细胞修复骨缺损等方面进行了总结.利用骨髓基质干细胞作为种子细胞,与适当的支架材料结合,或用骨髓基质干细胞作为靶细胞,导入外源目的基因诱导成骨的基因治疗来修复骨缺损的方法,给骨缺损的治疗带来光明的前景.但同时也存在骨髓基质干细胞存化,骨髓基质干细胞的增殖、分化合适的条件,哪些因子能有效促进新骨形成,载体材料及体内植入方式,以及如何将骨组织工程与基因治疗的方法结合起来等问题需要进一步的研究.     

Bone marrow extracellular matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors.

Direct contact between hematopoietic cells and viral packaging cell lines or other sources of stroma has been shown to increase the efficiency of retroviral-mediated gene transfer into these target cells compared with infection with viral supernatant. We have investigated the role of defined bone marrow extracellular matrix molecules (ECM) in this phenomenon. Here we report that infection of cells adhering to the carboxy-terminal 30/35-kD fragment of the fibronectin molecule (30/35 FN), which contains the alternatively spliced CS-1 cell adhesion domain, significantly increases gene transfer into hematopoietic cells. Two retroviral vectors differing in recombinant viral titer were used. Gene transfer into committed progenitor cells and long-term culture-initiating cells, an in vitro assay for human stem cells, was significantly increased when the cells were infected while adherent to 30/35 FN-coated plates compared with cells infected on BSA-coated control plates or plates coated with other bone marrow ECM molecules. Although gene transfer into committed progenitor cells and to a lesser degree into long-term culture-initiating cells was increased on intact fibronectin as well, increased gene transfer efficiency into hematopoietic cells on 30/35 FN was dependent on CS-1 sequence since infection on a similar FN fragment lacking CS-1 (42 FN) was suboptimal. 30/35 FN has previously been shown by our laboratory and other investigators to mediate adhesion of primitive murine and human hematopoietic stem cells to the hematopoietic microenvironment. Additional studies showed that neither soluble 30/35 FN nor nonspecific binding of hematopoietic cells to poly-L-lysine-coated plates had any appreciable effect on the infection efficiency of these cells. Our findings indicate that hematopoietic stem cell adhesion to specific ECM molecules alters retroviral infection efficiency. These findings should aid in the design of gene transfer protocols using hematopoietic progenitor and stem cells for somatic gene therapy.     

Bone marrow stromal cells prepared using AB serum and bFGF for hematopoietic stem cells expansion

BACKGROUND: An ex vivo culture system was previously established for stem cell expansion using human marrow stromal cells and serum-free medium. However, the stromal cells were prepared using long-term culture medium containing horse serum and FCS, which may transmit infectious diseases of xenogeneic origin. In this study, therefore, a method was established to prepare stromal cells using an AB serum-based medium. In the case that serum from a transplant recipient or PBPC donor is available, additional infectious diseases would not be transmitted. STUDY DESIGN AND METHODS: Cord blood CD34+ cells were cultured with thrombopoietin, stem cell factor, and flt3/flk2 ligand on a monolayer of human marrow primary stromal cells prepared using long-term culture medium or AB serum-based medium. After 2 weeks, clonogenic progenitor activity and SCID mouse-reconstituting cell activity were assayed. mRNA expression of cytokines and Notch ligand by stromal cells was also examined. RESULTS: There were no remarkable differences in expansion-supporting activity and mRNA expression between stromal cells established by the two methods. CONCLUSION: An ex vivo expansion system completely based on AB serum has been established.     

Bone marrow stromal cells mediate androgenic suppression of B lymphocyte development

Castration of normal male mice induces expansion of the bone marrow B cell population, an effect that can be reversed by androgen replacement. We employed in vitro cultures and two in vivo models to investigate whether androgens exert these effects directly on marrow lymphoid precursors or whether actions on marrow stromal elements are required. Immature B cells from normal mouse bone marrow were not responsive to the suppressive effect of androgens unless they were cocultured with marrow stromal cells or with supernatants from androgen-treated stromal cells, suggesting that the androgen effects are exerted through marrow stromal elements by production of a diffusible mediator. Further experiments revealed that bone marrow stromal cells produced TGF-beta in response to dihydrotestosterone (DHT), and neutralization of TGF-beta in the DHT-treated stromal cells reversed the suppressive effects. The stromal cell requirement for androgen-mediated effects was confirmed in vivo by experiments using chimeric animals created by bone marrow transplantation in which androgen receptor expression was restricted to either the stromal or lymphoid cells of the bone marrow. Androgens only affected B cell development in chimeric mice with androgen-sensitive stromal cells. These experiments suggest that effects of androgens on developing B cells are mediated through androgen receptors in bone marrow stromal cells. TGF-beta is a candidate mediator for these hormonal effects.     

Cryopreserved primate bone marrow cells can be used for retroviral-mediated gene transfer.

Retroviral-mediated gene transfer into Rhesus monkey bone marrow cells, which were cryopreserved, stored, and then transduced at the time of thawing, was studied for potential application in gene therapy protocols. Albumin density gradient fractionation was used to define subpopulations of cryopreserved cells transduced by a murine retroviral vector. The transfer of the bacterial neomycin phosphotransferase gene into Rhesus monkey bone marrow that was cryopreserved and thawed was found to be preferential in a light-density population enriched for granulocyte-macrophage colony-forming units (CFU-GM). These results are similar to results obtained with freshly harvested bone marrow in which this population is enriched for CD34 antigen-positive cells, as well as for cells that were undergoing cell division. This method may be useful in enriching for transduced precursors in future gene transfer experiments in primates.     

改良Dexter法与IMDM培养基培养骨髓基质细胞的比较

背景:体外分离培养骨髓基质细胞,并于骨髓移植后将其注入严重联合免疫缺陷鼠体内,对于促进造血及免疫重建尤为必要.目的:比较骨髓基质细胞在不同培养条件下的生长状况,寻找一种体外分离培养扩增骨髓基质细胞简单而实用的方法.设计、时间及地点:体外对比观察实验,于2006-07/2007-01在太原市中心医院皮肤科实验室完成.材料:人骨髓取自太原市中心医院血液科经筛选的正常骨髓(取材均经患者同意).方法:分离人骨髓单个核细胞,对照组采用改良Dexter法培养:将骨髓单个核细胞用5×10-7mol/L氢化可的松的RPMI1640培养基调整细胞浓度为1×109L-1,以每孔1 mL接种于24孔培养板,培养2 d后全量换液,去除非贴壁细胞,之后每3 d半量换液1次.细胞生长至半融合状态时传代,传两三代后用胰酶消化,收集细胞,液氮冷冻保存.实验组用IMDM培养基培养:用不含氢化町的松的IMDM培养基培养,其余成分及培养条件均与对照组相同.2 d后全量换液,去除非贴壁细胞,之后每4天半量换液1次.传代及收集方法同对照组.主要观察指标:通过倒置显微镜观察细胞贴壁情况、形态变化及增殖状态,MTT比色法检测细胞增殖活性.结果:倒置显微镜下,两组培养细胞在接种24 h后细胞大量贴壁并伸展开,随着培养时间延长,贴壁细胞数量增多,14 d左右细胞铺满板底达融合状态.传代细胞经消化后变形,24 h后恢复原来形态,并贴壁增殖,形态和原代细胞相似,4.0～5.0d后呈融合状态.两种方法培养的骨髓基质细胞增殖活性差异无显著性意义(P>0.05).结论:改良Dexter法及IMDM培养基培养均可使骨髓基质细胞在短期内贴壁及增殖.IMDM培养基由于不需加氢化可的松,故比改良Dexter法更易于操作.     

Stable marker gene transfer into human bone marrow stromal cells and their progenitors using novel herpesvirus saimiri-based vectors

We have evaluated the ability of new herpesvirus saimiri (HVS)-based vectors to deliver a marker gene green fluorescent protein (GFP) into human bone marrow (BM) stromal cells and their progenitors. Stromal cells expanded from adherent layers of long-term BM cultures (LTC) were susceptible to HVS-based infection in a dose-dependent manner, and the efficiency of 94.8 +/- 2.0% was achieved using single exposure with HVS/EGFP vector at multiplicity of infection (moi) of approximately 50. Colony-forming unit-fibroblast (CFU-F) assay established the ability of HVS-based vectors to infect progenitors for bone marrow stroma fibroblasts and transfer the marker gene over multiple cell divisions in the absence of selective pressure. HVS was not toxic for stromal cells and progenitors and no viral replication was detected upon growth of modified stroma. On the basis these data, we believe that HVS-based constructs can offer a new opportunity for selective gene delivery into bone marrow stromal cells and progenitors. The ability of HVS to infect nondividing cells can be considered advantageous in the development of both ex vivo and in vivo strategies.     

Epidermal stem cells as targets for gene transfer

One of the characteristics of the epidermis that makes it an attractive tissue for gene therapy is that it is renewed through proliferation of stem cells. If the stem cells can be transduced with the gene of interest then expression of that gene should continue throughout adult life. This article discusses current research on epidermal stem cells, highlighting progress in their identification and in discovering the mechanisms that regulate exit from the stem cell compartment.     

Bone marrow graft rejection as a function of antibody-directed natural killer cells

There is conclusive evidence that acute bone marrow transplant rejection in lethally irradiated mice is caused by natural killer (NK) cells. The rejection of marrow allografts is exquisitely specific and is controlled by antigenic determinants encoded in or near the H-2 gene complex. The specificity of in vivo marrow graft rejection contrasts with the in vitro specificity pattern of NK cells in cytotoxicity assays. We therefore examined how NK cells cause H-2-specific marrow graft rejection in vivo. Several experimental approaches are presented that suggest that natural antibody, present in responder strains of mice, specifically directs NK cells in an antibody-dependent cytolytic and/or cytostatic reaction, resulting in marrow graft rejection. The following evidence for this mechanism is documented. The ability to reject a marrow graft can be passively transferred by serum from responder to allogeneic nonresponder mice and the specificity of rejection can be mapped within the H-2 region. Serum-induced marrow graft rejection is abrogated following depletion of immunoglobulin, and the serum of responder mice is able to induce a specific antibody-dependent cytotoxic reaction in vitro.     

骨髓基质干细胞移植治疗帕金森病的研究与进展

目的:探讨骨髓基质干细胞分化为神经元样细胞的潜能和在治疗帕金森病方面的应用及其作用机制。资料来源:应用计算机检索PUBMED 1997-01/2006-03期间与骨髓基质干细胞和帕金森病有关的文章,检索词为“bone marrow stromal cells”,以及“neuron,Parkinson’s disease,transplantation,in brain”组合检索,并限定文章语言种类为“English”。同时计算机检索中国期刊全文数据库2003-01/2006-03相关的文章,检索词为“骨髓基质干细胞,神经元,帕金森病,移植,脑内”,并限定文章语言种类为中文。资料选择:对资料进行初审,并查看每篇文献后的引文。纳入标准:文章所述内容应与骨髓基质干细胞分化为神经元样细胞及其应用于治疗帕金森病等中枢神经系统疾病的研究相关;以近5年且发表在较权威杂志者优先。排除标准:重复研究或Meta分析类文章。资料提炼:共收集到330篇相关文献,排除277篇内容陈旧或重复的文献,53篇符合纳入标准,有33篇检索到全文,选用其中的30篇作为本文参考文献:16篇涉及骨髓基质干细胞多向分化潜能;8篇涉及骨髓基质干细胞移植治疗中枢神经系统疾病;6篇涉及骨髓基质干细胞应用于治疗帕金森病的基础研究。资料综合:骨髓基质干细胞具有多向分化潜能,在特定条件下可分化为具有神经元形态并表达神经元标记物的细胞,在脑内可与损伤的细胞融合,能分泌营养因子或者刺激损伤部位产生内源性因子,促进损伤组织的修复并减少细胞凋亡,已成为细胞移植治疗各种疾病的理想种子细胞。结论:在适宜的条件下,骨髓基质干细胞经过诱导在体内、外均可分化为神经元样细胞,将其注入帕金森病模型大鼠纹状体内能形成表达神经标志性蛋白的神经元样细胞或星形胶质细胞,产生的细胞可在受损部位周围存活甚至移行至全脑,并明显恢复运动功能。     

骨髓基质细胞作为人骨形态发生蛋白-2基因受体细胞的可行性研究

目的:通过检测hBMP-2基因在受体细胞中瞬时表达水平,探讨骨髓基质细胞作为hBMP-2基因受体细胞的可行性。方法:应用脂质体介导的方法将携带hBMP-2基因的重组载体PcDNA3-hBMP-2导入体外培养的骨髓基质细胞中,分别应用原位技术和酶联免疫吸附方法检测目的基因在受体细胞内mRNA和蛋白质的存在与表达状况。结果:在mRNA水平,可检测到hBMP-2基因在阳性细胞进行了转录;在蛋白质水平,从细胞的培养基中可检测到hBMP-2蛋白质。结论:骨髓基质细胞可以作为hBMP-2基因的受体细胞,基因表达可分泌hBMP-2蛋白质。     

mdx小鼠与C57小鼠骨髓基质细胞体外分化能力的比较

背景:肌营养不良症是一种渐进性致死性X连锁隐性遗传性肌肉疾病,目前无特效治疗.肌营养不良症模型鼠(mdx小鼠)的骨髓基质细胞增殖及定向分化能力是否正常,自身骨髓移植是否合适还有待研究.目的:观察mdx小鼠骨髓基质细胞体外培养时的增殖及多向分化能力.方法:取mdx小鼠与C57小鼠胫股骨骨髓基质细胞体外培养,经吉姆萨染色后观察其形成成纤维细胞集落形成单位的能力:通过不同诱导液使骨髓基质细胞定向分化为成骨、成脂、成肌细胞,观察其形态学特性;并分别用Von kossa染色、油红O染色、免疫荧光检测desmin阳性细胞对已分化细胞进行鉴定和分化率比较;培养1周时,提取分化细胞总RNA,反转录后,用real-time PCR检测各分化细胞相关基因表达.结果与结论:mdx小鼠骨髓基质细胞形成的成纤维细胞集落形成单位数目和体积均小于C57小鼠.其成骨、成肌分化的效率均明显低于C57小鼠(P<0.01),两种小鼠的骨髓基质细胞成脂分化效率差异无显著性(P>0.05).real-time PCR检测结果显示,与C57小鼠相比,mdx小鼠的骨髓基质细胞成骨、成肌基因表达均有不同程度下降,而成脂基因表达无明显差异.结果提示,mdx小鼠的骨髓基质细胞体外培养时的增殖及定向分化能力较C57小鼠下降,与Dystrophin基因缺失有关,mdx小鼠自体骨髓移植将会受限.     

Liquid storage of marrow stromal cells

BACKGROUND: Marrow stromal cells (MSCs) for clinical trials are inevitably stored before administration, but little is known about the effects of storage on MSCs. The effects of short-term liquid storage on the in vitro function of MSCs intended for a clinical trial were studied.
STUDY DESIGN AND METHODS: Early-passage human MSCs were suspended in 0.9% saline or culture medium and stored at 4°C or room temperature for up to 72 hours followed by assessment of cell loss, viability, and growth in culture.
RESULTS: When stored in saline at 4°C, MSC counts decreased by 5% to 20%, MSC viability decreased 17% to 37%, and MSC growth decreased 65% to 100% after 24-hour storage. Similar results were obtained by MSC storage at room temperature or in Dulbecco's modified Eagle's medium or by addition of 1% human serum albumin (HSA) from two manufacturers. Storage of MSCs in saline with HSA from a third manufacturer maintained MSC viability at prestorage levels and improved poststorage MSC growth versus saline (32 ± 9% vs. 9 ± 9%; p < 0.05) or saline with two other HSA preparations (4 ± 4 and 8 ± 11%; p < 0.05).
CONCLUSION: MSCs stored at 4°C or room temperature, in saline or culture medium, rapidly become nonviable. HSA preparations vary significantly in their ability to maintain poststorage MSC viability and growth. These results provide insight regarding the effects of storage on MSCs and indicate the need to screen HSA preparations before their use as additives to preserve MSCs during short-term liquid storage.