大鼠骨髓间充质干细胞增殖及定向分化能力的增龄性变化

背景：自体骨髓间充质干细胞移植治疗心血管疾病已经成为当前研究的热点之一,但是患者自体骨髓间充质干细胞的增殖能力及向心肌样细胞方向分化的能力是否随年龄的改变而有所变化尚不清楚。 目的：探讨大鼠骨髓间充质干细胞生物学特性的增龄性差异。 方法：纯化培养不同年龄组SD大鼠骨髓间充质干细胞,流式细胞仪分析其细胞周期,同时骨髓间充质干细胞与乳鼠心肌细胞共培养,细胞免疫荧光技术分析骨髓间充质干细胞心肌肌钙蛋白T的表达。 结果与结论：流式细胞仪分析显示处于G₀/G₁期骨髓间充质干细胞的百分率随年龄的增加而增加;骨髓间充质干细胞的肌钙蛋白T表达率亦随年龄的增加而减少。结果表明大鼠骨髓间充质干细胞的增殖与定向分化能力随着年龄的增长而下降。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Roles of activated astrocytes in bone marrow stromal cell proliferation and differentiation

Local microenvironment plays an important role in determining the fate choice of stem cells in the central nervous system (CNS). Astrocytes, a major component of local microenvironment in the CNS, have been demonstrated to influence the proliferation and neural differentiation of stem cells including neural stem/progenitor cells, embryonic stem cells and bone marrow stromal cells (BMSCs). However, it has remained to be ascertained if inflammation-activated astrocytes can affect the behavior of BMSCs. To this end, astrocyte-conditioned medium (ACM) was prepared in this study for treatment of BMSCs. The ACM derived from Wistar rat astrocytes stimulated by lipopolysaccharide for 12, 36 or 72 h, respectively, served as inflammatory ACM (12 h ACM, 36 h ACM and 72 h ACM), while that from unstimulated astrocytes was used as normal control astrocyte-conditioned medium (N-ACM). The results showed that the proliferation and neural differentiation of BMSCs grown in inflammatory ACM were significantly increased compared with those grown in N-ACM. The efficiency of BMSCs exposed to 36 h ACM was significantly greater than that of those exposed to 12 or 72 h ACM. Following neutralization of interleukin-6 (IL-6) of the ACM, both the proliferation and astrocytic differentiation of BMSCs were decreased; on the other hand, the neuronal differentiation was significantly increased. The present findings suggest that inflammation-activated astrocytes can facilitate the proliferation and neural differentiation of BMSCs and activated astrocytes at different phase after CNS injuries might have distinct effects on BMSCs. Moreover, astrocyte-derived IL-6 participates in the proliferation and neural differentiation of BMSCs.     

Thy-1 modulation and cell proliferation at early steps of intrathymic bone marrow cell differentiation.

Intrathymic (IT) transfer of bone marrow (BM) precursor cells in sublethally irradiated hosts has been widely used to study T cell differentiation and maturation. In this report we have used double congenic mice Ly 5.1 Thy 1.1 (host) and Ly 5.2 Thy 1.2 (donor) and detected cycling Ly 5.2+ BM cells by in vivo bromodeoxyuridine incorporation, before induction of the Thy 1.2 antigen. Until Day 9 post-transfer, some donor type cells express a high level of Thy 1.2 together with macrophage and granulocyte markers. A few days later, a Thy 1.2low population transiently B220+ was detected. Thereafter, donor type cells expressed an intermediate Thy 1.2 brightness; this population then persisted and surpassed the other subsets. Our findings permitted to establish a relationship between cell cycle and Thy 1 fluorescence intensity according to the sequence: Thy 1low resting, Thy 1low cycling, Thy 1high cycling, Thy 1high resting. Moreover, we have shown that cells from the myelo?d and B lineages can, in vivo, transiently express the Thy 1 antigen, develop and differentiate within the thymus microenvironment.     

不同材料因素影响骨髓间充质干细胞的增殖及成骨分化

文题释义：表面微观形貌：材料表面所具有的所有微观几何形状及相关物理量统称为表面微观形貌,包括孔径大小、孔径率、表面凹凸程度、底物刚度、弹性模量、表面能等方面,同一种材料不同的表面微观形貌将会对材料表面的物质产生不同的影响。 表面能/润湿性：表面能是指恒温、恒压、恒组成情况下可逆地增加物系表面积须对物质所做的非体积功,润湿性是指一种液体在一种固体表面铺展的能力或倾向性。 背景：在利用组织工程技术治疗骨缺损或骨损伤的过程中,生物材料对移植的骨髓间充质干细存活率与增殖分化潜能有影响。 目的：对生物材料如何影响骨髓间充质干细胞的增殖及成骨分化的研究进展进行综述,指导材料合理应用。 方法：应用计算机检索中国知网、PubMed、Web of science及万方数据库,检索词为“骨髓间充质干细胞、成骨分化、生物材料、微观形貌、Bone marrow mesenchymal stem cells、Osteogenic differentiation、Biological materials、The microstructure”,根据标准纳入不同生物材料因素影响骨髓间充质干细胞增殖及成骨分化的文献。结果与结论：①金属材料具有良好的生物相容性、骨传导性、机械性能,非金属材料具有良好的生物相容性、骨传导性、再吸收性及三维塑形性;②材料的表面微观形貌因素众多,即使是同一种材料,表面能/润湿性越高越利于骨髓间充质干细胞的增殖与成骨分化,表面粗糙程度相对越高越利于细胞的增殖、黏附及分化,孔径直径越大、孔径率越小越利于细胞的成骨分化,底物刚度相对高及弹性模量相对大有利于骨髓间充质干细胞的成骨分化,以上材料的各种因素等都能够影响骨髓间充质干细胞的增殖及成骨作用,这些新的研究进展进一步提高了骨髓间充质干细胞种植于各类生物材料用于临床治疗的应用。ORCID: 0000-0001-8325-9867(王旸昊) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

Klotho对大鼠骨髓间充质干细胞增殖和分化的影响

目的 探讨延缓衰老基因Klotho对骨髓间充质干细胞（BMSCs）增殖和分化的影响。 方法 体外条件下培养大鼠骨髓间充质干细胞,构建分泌型Klotho(sKL)过表达的BMSCs。Western blotting检测细胞及培养基中sKL的表达;MTT法检测细胞增殖能力;Western blotting检测衰老标志物P53、P21蛋白的表达。利用成骨诱导液定向诱导BMSCs向成骨细胞分化,应用碱性磷酸酶(ALP)染色鉴定成骨效果;利用成脂诱导液定向诱导BMSCs向脂肪细胞分化,应用油红O染色鉴定成脂效果。 结果 Western blotting结果显示,sKL组细胞和培养基中sKL蛋白表达显著上调（P<0.05）,P53、P21表达显著下调（P<0.05）;MTT结果显示,各组细胞吸光度值（A值）差别无显著性（P>0.05）,sKL组成骨及成脂能力明显强于对照组（P<0.05）。 结论 sKL增强了大鼠BMSCs的延缓衰老能力,对BMSCs的成骨分化和成脂分化产生一定的促进作用,而对BMSCs的增殖无显著影响。     

Effect of the chemical composition of Ricinus communis polyurethane on rat bone marrow cell attachment,proliferation, and differentiation

Alterations in the chemical composition of a polymer may be undertaken to improve its biological properties. The aim of this study was to investigate the in vitro biocompatibility of Ricinus communis polyurethane (RCP) with three different chemical compositions: RCPp (pure RCP), RCP+CaCO(3), and RCP+Ca(3)(PO(4))(2). Rat bone marrow cells were cultivated under conditions that allowed osteoblastic differentiation and were evaluated for cell attachment, cell proliferation, cell morphology, total protein content, alkaline phosphatase (ALP) activity, and bonelike nodule formation. For the evaluation of attachment, cells were cultured for 4 h. After 3 days, cell morphology was evaluated. Cell proliferation was evaluated after 7 and 14 days. Total protein content and ALP activity were evaluated after 14 days. For bonelike nodule formation, cells were cultured for 21 days. Data were compared with an analysis of variance and Duncan's multiple range test when appropriate. Cell attachment and ALP activity were not affected by RCP chemical composition. Proliferation, total protein content, and bonelike nodule formation were all affected by RCP chemical composition. These results suggest that initial cell events are not affected by RCP chemical composition, whereas RCPs blended with calcium carbonate or, better yet, calcium phosphate, by favoring events that promote matrix mineralization, are more biocompatible materials.     

Regulation of constitutive bone marrow cell proliferation by bone marrow suppressor cells

Bone marrow (BM) cells have previously been shown to suppress specific immune responses of cells from peripheral lymphoid organs. The present report describes a suppressor cell present in normal rabbit BM, which regulated the constitutive proliferation of other BM cells. The suppressor cells were Fc gamma-receptor-positive (Fc gamma R+) complement-receptor-negative, and nonadherent or weakly adherent. Similar suppressive activity was not detected among rabbit spleen cells. Removal of Fc gamma R+ suppressor cells allowed greater than 10-fold increases in the proliferation of Fc gamma R- BM cells. Addition of Fc gamma R+ BM cells to Fc gamma R- cells efficiently blocked proliferation. The suppressor cells acted by inhibiting the elaboration of a soluble growth-promoting factor by cells in the Fc gamma R- population. The growth-promoting factor enhanced proliferation of unseparated rabbit BM cells and peripheral blood mononuclear cells.     

骨髓基质干细胞抑制大鼠肝纤维化细胞生长的实验研究

目的 探讨骨髓基质干细胞(BMSCs)对大鼠肝纤维化细胞(CFSCs)生长的抑制作用.方法 采用不同培养时间BMSCs的培养上清培养大鼠正常肝细胞系(BRL)以及CFSCs,MTT法检测CFSCs的增殖情况;Transwell建立BMSCs与CFSCs的双层共培养体系,经TUNEL法检测与BMSCs按不同比例进行培养后的CFSCs凋亡情况.结果 BMSCs培养上清作用于CFSCs后,其增殖能力明显低于对照组,有统计学意义(P＜0.05),并表现出剂量依赖性,而对BRL没有作用.CFSCs与BMSCs按不同比例共培养后,随着BMSCs比例的增加,CFSCs调亡率上升.结论 BMSCs能够不可逆的抑制CFSCs生长而不影响正常的肝细胞,这种生长抑制是通过诱导CFSCs的凋亡发生的.     

Effects on proliferation and differentiation of multipotent bone marrow stromal cells engineered to express growth factors for combined cell and gene therapy

A key mechanism for mesenchymal stem cells/bone marrow stromal cells (MSCs) to promote tissue repair is by secretion of soluble growth factors (GFs). Therefore, clinical application could be optimized by a combination of cell and gene therapies, where MSCs are genetically modified to express higher levels of a specific factor. However, it remains unknown how this overexpression may alter the fate of the MSCs. Here, we show effects of overexpressing the growth factors, such as basic fibroblast growth factor (bFGF), platelet derived growth factor B (PDGF-BB), transforming growth factor β(1) (TGF-β(1) ), and vascular endothelial growth factor (VEGF), in human bone marrow-derived MSCs. Ectopic expression of bFGF or PDGF-B lead to highly proliferating MSCs and lead to a robust increase in osteogenesis. In contrast, adipogenesis was strongly inhibited in MSCs overexpressing PDGF-B and only mildly affected in MSCs overexpressing bFGF. Overexpression of TGF-β(1) blocked both osteogenic and adipogenic differentiation while inducing the formation of stress fibers and increasing the expression of the smooth muscle marker calponin-1 and the chondrogenic marker collagen type II. In contrast, MSCs overexpressing VEGF did not vary from control MSCs in any parameters, likely due to the lack of VEGF receptor expression on MSCs. MSCs engineered to overexpress VEGF strongly induced the migration of endothelial cells and enhanced blood flow restoration in a xenograft model of hind limb ischemia. These data support the rationale for genetically modifying MSCs to enhance their therapeutically relevant trophic signals, when safety and efficacy can be demonstrated, and when it can be shown that there are no unwanted effects on their proliferation and differentiation.     

骨形态发生蛋白2及碱性成纤维生长因子2对大鼠骨髓间充质干细胞膜片增殖和成骨分化的影响

文题释义：细胞膜片技术：是在体外接种培养高密度的细胞,使其相互融合生长至100%而形成的透明致密膜状物。该技术不需要胰酶消化即可收集细胞,因此保留了大量的胞外基质、细胞间连接以及细胞-基质连接等结构。目前细胞膜片技术已成为组织工程领域的研究热点,已被推广应用于牙周膜、角膜、心脏、软骨、食管等多种组织器官修复。成骨细胞：主要由内外骨膜和间充质始祖细胞分化而来,在复杂的骨形成过程中发挥着主要的功能,承担着骨基质的合成、分泌和矿化。骨髓间充质干细胞具有多向分化潜能,能定向分化为成骨细胞,其成骨分化过程可受多种因素的影响,如细胞因子的调控、遗传因素和激素水平等。背景：现阶段骨形态发生蛋白2和碱性成纤维生长因子2对骨髓间充质干细胞膜片增殖、成骨分化的影响和作用机制还尚未可知,如何将生长因子与组织工程细胞膜片技术相整合,最终将其用于骨缺损修复具有重要意义。目的：探讨单独及联合应用骨形态发生蛋白2和碱性成纤维生长因子2对骨髓间充质干细胞膜片增殖和成骨分化的影响。方法：体外分离培养鉴定SD大鼠骨髓间充质干细胞并构建细胞膜片,选用不同质量浓度的骨形态发生蛋白2和碱性成纤维生长因子2单独及联合诱导骨髓间充质干细胞膜片,CCK-8法结合碱性磷酸酶活性检测确定2种因子促进膜片增殖和成骨分化的最佳有效质量浓度;然后对骨髓间充质干细胞膜片进行成骨诱导,通过大体及显微镜观察、Vonkossa染色、茜素红染色、RT-PCR检测相关成骨标志物来评估诱导效果。结果与结论：单独应用骨形态发生蛋白2可增强骨髓间充质干细胞膜片的碱性磷酸酶活性,最佳质量浓度为100 μg/L(P < 0.001),单独应用碱性成纤维生长因子2能加速骨髓间充质干细胞膜片的增殖,最佳质量浓度为20 μg/L(P < 0.001),而联合应用既可以促进膜片增殖又能提高其碱性磷酸酶活性(P < 0.001);经成骨诱导后,4组膜片在形态学上无明显差异,均能诱导骨髓间充质干细胞膜片的成骨分化,其中联合组钙结节最明显(P < 0.001),可显著促进膜片晚期成骨分化并抑制其早期成骨分化,具有明显的协同促进作用(P < 0.001)。结果表明,骨形态发生蛋白2和碱性成纤维生长因子2联合应用时具有协同作用,既可以促进骨髓间充质干细胞膜片增殖,又能显著增强其成骨诱导能力。ORCID: 0000-0003-1918-579X(何惠宇)中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

碱性成纤维细胞生长因子和5-氮杂胞苷对大鼠骨髓间充质干细胞分化潜能和细胞增殖的影响

目的:探讨碱性成纤维细胞生长因子(bFGF)和5-氮杂胞苷(5-aza)对体外骨髓间充质干细胞(MSCs)细胞增殖与分化潜能的影响。方法:大鼠骨髓中获得MSCs,培养传代,在倒置相差显微镜和透射电镜下观察细胞形态变化;流式细胞仪检测细胞周期改变;MTT法检测bFGF和5-aga对MSCs生长的影响;免疫细胞化学方法检测actin、desmin、myoglobin、NSE、GFAP的表达。结果:有心肌样细胞和神经元样细胞的形态变化;MSCs经5-aza以及5-aza加bFGF联合诱导actin、desmin、myoglobin、NSE和GFAP的表达均较对照组显著增高;而经bFGF诱导后,前4种蛋白表达无明显变化,仅GFAP蛋白表达显著增高。结论:5-aza对MSCs细胞生长有抑制作用,而bFGF有明显促增殖作用。MSCs被5-aza诱导分化成心肌样细胞和神经元样细胞,bFGF可使MSCs分化为GFAP阳性的神经胶质样细胞。     

Human recombinant elastin-like protein coatings for muscle cell proliferation and differentiation

Recombinant proteins represent a new and promising class of polymeric materials in the field of biomaterials research. An important model for biomaterial design is elastin, the protein accounting for the elasticity of several tissues. Human elastin-like polypeptides (HELPs) have been developed as recombinant versions of elastin with the purpose of enhancing some peculiar characteristics of the native protein, like self-assembling. In this paper, we report on a comparative study of rat myoblasts response to coatings based on two different HELP macromolecules, with respect to control cultures on bare cell culture polystyrene and on a standard collagen coating. Cell behavior was analyzed in terms of adhesion, proliferation and differentiation. The collected data strongly suggest the use of HELPs as excellent biomaterials for tissue engineering and regenerative medicine applications.     

Human bone marrow heparan sulfate induces leukemia cell differentiation

The proliferation and development of hematopoietic cells occurs in close association with bone marrow stroma. Heparan sulfate is a major component of the stroma. We have isolated a form of heparan sulfate proteoglycan from a human stromal cell line grown in vitro in the presence of [35S]sulfate. This proteoglycan contains a phosphatidylinositol component which likely anchors it to the stromal cell membrane. The glycosaminoglycan chains of this proteoglycan could induce maturation of the HL-60 myeloid leukemia cell line. A less hydrophobic heparan sulfate proteoglycan was also present in the stroma, but could not induce HL-60 maturation. The two heparan sulfates had glycosaminoglycan chains that were similar in size (36 Kd) and charge density. Structural studies suggested only minor but perhaps significant differences in the carbohydrate sequences of the two heparan sulfates. The relationship of these subtle structural differences to the difference observed in differentiation-inducing activity remains to be elucidated.     

衰老模型骨髓基质细胞抑制造血细胞增殖分化

目的研究衰老骨髓基质细胞对骨髓造血细胞增殖分化能力的影响,为阐述机体造血微环境衰老对造血干/祖细胞增殖的影响提供实验依据。方法全骨髓贴壁法体外培养大鼠骨髓基质细胞,分为对照组和衰老组。衰老组:常规培养基内加入30 mg/m L D-半乳糖作用48 h。CCK-8法测定BMSCs增殖;流式细胞术分析细胞周期;β-半乳糖苷酶(SA-β-Gal)染色观察衰老BMSCs百分率;Western blot检测P16、P21和P53蛋白表达。骨髓造血细胞与BMSCs共培养,集落计数检测髓系多向性造血祖细胞(CFU-Mix)增殖分化。ELISA检测BMSCs培养上清液中IL-1β、GM-CSF和SCF含量;DCFH-DA流式荧光检测BMSC活性氧簇(ROS)水平;酶学法检测BMSCs内过氧化物丙二醛(MDA)含量和总超氧化物歧化酶(SOD)活性。结果与对照组相比,D-半乳糖诱导BMSCs衰老,细胞阻滞于G0/G1期(P0.01),增殖能力显著下降,SA-β-Gal染色阳性率升高(P0.01);衰老相关蛋白P16、P21和P53表达明显上调(P0.01)。与衰老BMSCs共培养的骨髓造血细胞增殖分化能力减弱。衰老BMSCs内ROS、MDA氧化损伤指标上升,SOD抗氧化指标下降(P0.01);BMSCs培养上清液IL-1β、GM-CSF和SCF含量明显下降(P0.01)。结论衰老骨髓基质细胞抑制造血细胞增殖、分化能力,其机制可能与骨髓基质细胞氧化损伤,分泌活性因子改变有关。     

纤维蛋白胶复合重组人骨形态发生蛋白2微球对犬骨髓基质细胞增殖与分化的影响

背景：前期实验证实聚乳酸-聚乙醇酸微球/纤维蛋白胶能作为重组人骨形态发生蛋白2的良好可注射性缓释载体。 目的：观察可注射性骨形态发生蛋白缓释体系对犬骨髓基质细胞增殖与分化的影响。 方法：采用复乳-溶剂挥发法制备重组人骨形态发生蛋白2/聚乳酸-聚乙醇酸共聚物载药微球,然后将微球与纤维蛋白胶复合制备出重组人骨形态发生蛋白2/聚乳酸-聚乙醇酸共聚物/纤维蛋白胶复合材料,采用细胞培养及组织化学等方法观察微球对犬骨髓基质细胞的增殖与分化的影响。 结果与结论：重组人骨形态发生蛋白2/聚乳酸-聚乙醇酸共聚物/纤维蛋白胶微球对骨髓基质细胞的增殖无明显影响,但对细胞的分化功能有明显的促进作用。说明纤维蛋白胶复合重组人骨形态发生蛋白2微球能够提高骨髓基质细胞的体外成骨能力,可作为骨形态发生蛋白的良好载体。     

骨髓间充质干细胞增殖与成骨分化中电磁场激活ERK通路的作用

背景：研究表明电磁场可调节骨髓间充质干细胞的增殖和分化,但其具体机制尚不清楚。 目的：从ERK信号途径探讨电磁场诱导大鼠骨髓间充质干细胞增殖与分化成骨的作用。 方法：取第3代生长良好的大鼠骨髓间充质干细胞,暴磁组给予15 Hz、1 mT的正弦波电磁场刺激,PD98059+暴磁组在电磁场刺激前给予20 μmol/L ERK阻断剂PD98059,PD98059组仅给PD98059不进行电磁场刺激,对照组正常培养。电磁场刺激后,收集各组细胞,Western blot法检测ERK通路的活性,MTT法检测细胞增殖活性,碱性磷酸酶试剂盒检测细胞碱性磷酸酶活性。 结果与结论：电磁场刺激后,细胞ERK1/2磷酸化水平、细胞的增殖活性、及碱性磷酸酶活性均明显升高(P < 0.01);PD98059可明显抑制ERK1/2磷酸化水平及细胞增殖活性的升高(P < 0.01),而在一定程度上提高细胞的碱性磷酸酶活性(P < 0.01)。说明电磁场刺激可通过激活骨髓间充质干细胞ERK信号通路,并且主要通过该途径促进骨髓间充质干细胞的增殖;而在脉冲电磁场促进骨髓间充质干细胞分化成骨的过程中,激活ERK信号通路所起的作用较小。     

Effect of culture substrates and fibroblast growth factor addition on the proliferation and differentiation of rat bone marrow stromal cells

This study is an investigation of the proliferation and differentiation of bone marrow stromal cells (BMSCs) on film substrates with different surface properties. Films of noncharged polymers with several water wettabilities; cell culture plates coated with collagen type I or IV, gelatin, or basic fibroblast growth factor (bFGF); and glass were used. BMSCs isolated from rat bone marrow were cultured on the various substrates in medium with dexamethasone [Dex(+)] or without dexamethasone [Dex(-)] to assess cell proliferation and differentiation. The number of proliferated BMSCs depended on the water wettability of substrates, although the cell number was greater in Dex(-) medium than in Dex(+) medium. Protein-coated substrates exhibited a high proliferation rate compared with noncoated substrates. Alkaline phosphatase (ALP) activity increased with increasing cell number, whereas ALP activity per cell correlated well with cell number. When cultured in Dex(+) medium containing bFGF or on culture plates coated with bFGF, BMSC proliferation tended toward enhancement with an increase in the amount of bFGF added in solution form, whereas it did not depend on the amount of bFGF in coated form. On the other hand, ALP activity and calcium content of BMSCs became maximal with bFGF coated at about 1 x 10(3) to 2 x 10(3) ng, in contrast to bFGF in solution form. Irrespective of the amount of bFGF, ALP activity and calcium content levels for bFGF in coated form were higher than for bFGF in solution form. It is concluded that the type of culture substrate and the manner of addition of bFGF affect the proliferation and differentiation of BMSCs.