Respiratory syncytial virus infection in human bone marrow stromal cells

Respiratory syncytial virus (RSV) is the most common respiratory pathogen in infants and young children. The pathophysiology of this infection in the respiratory system has been studied extensively, but little is known about its consequences in other systems. We studied whether RSV infects human bone marrow stromal cells (BMSCs) in vitro and in vivo, and investigated whether and how this infection affects BMSC structure and hematopoietic support function. Primary human BMSCs were infected in vitro with recombinant RSV expressing green fluorescent protein. In addition, RNA from naive BMSCs was amplified by PCR, and the products were sequenced to confirm homology with the RSV genome. The BMSC cytoskeleton was visualized by immunostaining for actin. Finally, we analyzed infected BMSCs for the expression of multiple cytokines and chemokines, evaluated their hematopoietic support capacity, and measured their chemotactic activity for both lymphoid and myeloid cells. We found that BMSCs support RSV replication in vitro with efficiency that varies among cell lines derived from different donors; furthermore, RNA sequences homologous to the RSV genome were found in naive primary human BMSCs. RSV infection disrupted cytoskeletal actin microfilaments, altered cytokine/chemokine expression patterns, decreased the ability of BMSCs to support B cell maturation, and modulated local chemotaxis. Our data indicate that RSV infects human BMSCs in vitro, and this infection has important structural and functional consequences that might affect hematopoietic and immune functions. Furthermore, we have amplified viral RNA from naive primary BMSCs, suggesting that in vivo these cells provide RSV with an extrapulmonary target.     

Simian immunodeficiency virus infection of macaque bone marrow macrophages correlates with disease progression in vivo.

The pathogenesis of hematopoietic abnormalities associated with infection of susceptible hosts with either simian immunodeficiency virus (SIV) or human immunodeficiency virus (HIV) is not fully understood. To determine if bone marrow cells are infected with SIV and if the pattern of viral infection is correlated with the severity of disease and abnormalities in hematopoiesis, 23 SIV-infected rhesus monkeys were examined by immunohistochemistry and in situ hybridization. By immunohistochemistry, only four monkeys were positive for SIV core protein p27, while in situ hybridization revealed viral RNA in the bone marrow of 15 monkeys. Simian immunodeficiency virus RNA was consistently expressed in the bone marrow from monkeys with severe lymphoid depletion (11 of 11), but less so in monkeys with follicular hyperplasia (0 of 2) or mild lymphoid depletion (4 of 10). In animals with mild lymphoid depletion, bone marrow cells infected with SIV were mainly mononuclear cells that appeared to be of myelomonocytic lineage. In contrast, monkeys with severe lymphoid depletion had SIV RNA localized to larger mononuclear cells with abundant cytoplasm often located in small lucent areas of the stroma. These SIV RNA-positive mononuclear cells were positive for the macrophage determinant CD68 as demonstrated by immunohistochemistry. Furthermore the stage of simian acquired immune deficiency syndrome, as indicated by lymphoid morphology, and SIV localization in the bone marrow were correlated with the incidence of anemia, bone marrow hyperplasia, and abnormal distribution of macrophages in the bone marrow. These results indicate that, in common with other animal lentiviral infections, the macrophage is a major target of SIV infections in the bone marrow.     

脂肪基质干细胞和骨髓基质干细胞的体外分化能力

背景：脂肪基质干细胞和骨髓基质干细胞具有很多相似的生物学特性。 目的：比较脂肪基质干细胞和骨髓基质干细胞与受损PC12细胞分别共培养后定向分化能力的差异。 方法：分别分离培养脂肪组织来源和骨髓组织来源的基质干细胞,取第5代细胞进行实验,2种细胞分别与正常或受损PC12细胞培养上清液共培养,或仅单独培养。 结果与结论：脂肪基质干细胞和骨髓基质干细胞均表达较高水平的CD44和CD29,而后者表达的CD45、CD56在前者几乎未检测到。单独培养的2种细胞均表达较高水平的Nanog、Oct4、Sox2,不表达神经元特异性烯醇酶。其中经受损PC12细胞干预的2种细胞Nanog、Oct4、Sox2表达水平显著降低,而脂肪基质干细胞中神经元特异性烯醇酶阳性细胞数更多,提示受损PC12细胞对于脂肪基质干细胞可能具有更强的诱导分化作用。     

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。     

Control of hematopoietic stem cells by the bone marrow stromal niche: the role of reticular cells

In the bone marrow, hematopoietic stem cells (HSCs) are maintained by special microenvironments, termed niches. The nature and function of these niches, however, remains unclear. HSCs are thought be in contact with bone-lining osteoblasts, but recent studies have suggested that only a small subpopulation of HSCs reside in this endosteal niche. By contrast, many HSCs are associated with the sinusoidal endothelium, which is referred to as the vascular niche. Recent data have suggested that primitive mesenchymal cells, including CXC chemokine ligand 12-abundant reticular cells and nestin-expressing cells act as HSC niches. Here, we review HSC niches, with an emphasis on the emerging role of reticular niches for maintaining HSCs in a proliferative and undifferentiated state.     

Kinetics of hematopoietic stem cells and supportive activities of stromal cells in a three-dimensional bone marrow culture system

Abstract

In the bone marrow, hematopoietic cells proliferate and differentiate in close association with a three-dimensional (3D) hematopoietic microenvironment. Previously, we established a 3D bone marrow culture system. In this study, we analyzed the kinetics of hematopoietic cells, and more than 50% of hematopoietic progenitor cells, including CFU-Mix, CFU-GM and BFU-E in 3D culture were in a resting (non-S) phase. Furthermore, we examined the hematopoietic supportive ability of stromal cells by measuring the expression of various mRNAs relevant to hematopoietic regulation. Over the 4 weeks of culture, the stromal cells in the 3D culture are not needlessly activated and “quietly” regulate hematopoietic cell proliferation and differentiation during the culture, resulting in the presence of resting hematopoietic stem cells in the 3D culture for a long time. Thus, the 3D culture system may be a new tool for investigating hematopoietic stem cell–stromal cell interactions in vitro.     

Long-term hematopoietic stem cells require stromal cell-derived factor-1 for colonizing bone marrow during ontogeny

The physiological role of SDF-1 on hematopoietic stem cells (HSCs) remains elusive. We show that colonization of bone marrow by HSCs in addition to myeloid cells is severely impaired in SDF-1(-/-) embryos by a long-term repopulation assay. Colonization of spleen by HSCs was also affected, but to a lesser extent. Enforced expression of SDF-1 under the control of vascular-specific Tie-2 regulatory sequences could completely rescue the reduction of HSCs but not myeloid cells in SDF-1(-/-) bone marrow. SDF-1 was detected in the vicinity of the vascular endothelial cells in fetal bone marrow. SDF-1 plays a critical role in colonization of bone marrow by HSCs and myeloid cells during ontogeny, and the mechanisms by which SDF-1 functions are distinct between HSCs and myeloid cells.     

骨髓基质细胞衰老对造血细胞衰老的影响

目的探讨骨髓基质细胞(BMSCs)衰老对骨髓造血细胞衰老的影响及其可能的机制。方法贴壁培养大鼠骨髓基质细胞,传代BMSCs至P3代时分组。对照组:常规培养;衰老组:常规培养基础上加入D-半乳糖(D-Gal)诱导BMSCs衰老,两组细胞分别培养48h。收集培养上清液,ELISA法检测细胞因子:粒细胞巨噬细胞集落刺激因子(GM-CSF)、干细胞因子(SCF)、白细胞介素(IL)-6、IL-1β,IL-2和肿瘤坏死因子-α(TNF-α)含量,CCK-8检测细胞增殖能力,衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测BMSCs衰老。分离提取正常骨髓单个核细胞(BMMNCs),在衰老组与对照组BMMSCs上种植正常BMMNCs共培养24h,收集上层悬浮BMMNCs。CCK-8法测定细胞增殖能力;多向造血祖细胞集落(CFU-Mix)半固体培养法检测细胞增殖及分化能力;SA-β-Gal染色观察衰老细胞百分率;流式细胞术分析细胞周期与细胞凋亡;DCFH-DA荧光染色检测细胞活性氧簇(ROS)水平;酶学法检测细胞内丙二醛(MDA)含量和总超氧化物歧化酶(SOD)活性。结果衰老组BMSCs增殖能力显著下降,SA-β-Gal染色阳性细胞百分率增加,细胞分泌GM-CSF、SCF、IL-6、IL-1β水平明显降低,IL-2、TNF-α水平显著升高。与衰老BMSCs共培养后,BMMNCs的增殖能力显著下降,SA-β-Gal染色阳性细胞百分率显著上升,BMMNCs形成CFUMix集落数量明显降低,细胞周期呈现阻滞且细胞凋亡率上升,细胞内ROS、MDA含量上升,SOD含量下降。结论本实验中D-Gal成功复制了BMSCs衰老,衰老的BMSCs可诱导骨髓造血细胞衰老,其机制可能与BMSCs导致造血细胞氧化损伤有关。     

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。     

骨髓基质细胞衰老对造血细胞氧化应激的影响

目的探讨骨髓基质细胞衰老对骨髓造血细胞氧化应激的影响。方法取6~8周雄性健康C57小鼠,全骨髓贴壁法培养骨髓基质细胞(BMSCs),建立衰老骨髓基质细胞体外模型。实验分为常规对照组和衰老组。对照组:常规培养基培养48h;衰老组:常规培养基内加入30g/L D-半乳糖,作用48h。衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测衰老骨髓基质细胞百分率;流式细胞术分析骨髓基细胞周期。骨髓单个核细胞(BMNCs)与骨髓基细胞共培养,锥虫蓝计数BMNCs活细胞数;流式细胞术检测骨髓基细胞周期;髓系多向性造血祖细胞(CFU-Mix)半固体培养集落计数;DCFH-DA荧光染色流式检测BMNCs和骨髓基细胞的活性氧簇(ROS)水平;激光扫描共焦显微术半定量检测骨髓基细胞连接蛋白43(Cx43)表达。结果 D-半乳糖体外诱导骨髓基细胞衰老,骨髓基细胞SA-β-Gal染色阳性率显著上升,细胞周期G1期阻滞。BMNCs与衰老组骨髓基细胞共培养48h,锥虫蓝计数BMNCs活细胞数量下降;细胞周期阻滞;CFU-Mix集落形成数量较对照组显著下降。衰老组骨髓基细胞胞内ROS含量增加,Cx43表达明显下调;与衰老骨髓基细胞共培养的BMNCs胞内ROS含量较与常规对照骨髓基细胞共培养的BMNCs胞内ROS含量上升。结论衰老骨髓基质细胞抑制骨髓造血细胞增殖分化,其机制可能与衰老骨髓基质细胞氧化应激增强,缓解造血细胞氧化应激能力下降有关。     

Effect of serotonin on hematopoietic stem cells in bone marrow

Laboratory for Control of Biosynthesis of Animal Tissues, Institute of Biophysics, Siberian Branch, Academy of Sciences of the USSR, Krasnoyarsk. (Presented by Academician of the Academy of Medical Sciences of the USSR Yu. A. Vladimirov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 11, pp. 488–489, November, 1991.     

骨髓间质细胞中组织定向干细胞的鉴定

目的:验证骨髓中存在组织定向干细胞(TCSCs),为心血管组织工程提供理想的种子细胞来源。方法:采用SDF-1α梯度趋化从骨髓中分离CXCR4阳性细胞,免疫细胞化学法检测CXCR4与PDGFR-β表达,流式细胞仪检测PDGFR-β阳性细胞,从而确定平滑肌祖细胞(SMPCs)的比例;从骨髓分离培养内皮祖细胞(EPCs),免疫细胞化学和流式细胞仪检测FLK-1、CD34、CXCR4与vWF的表达情况。结果:SDF-1α趋化分离出的细胞几乎都为CXCR4阳性,其中4·67%的细胞呈PDGFR-β阳性;EPCs形态圆形或近三角形贴壁生长,原代细胞CXCR4、VEGFR-2、CD34明显阳性,传代细胞vWF阳性。结论:在骨髓基质细胞中存在SMPCs和EPCs等TCSCs。     

骨髓基质干细胞与癫痫细胞凋亡的相关性

BACKGROUND:There is a close relationship between epilepsy and apoptosis. The appearance of epilepsy can lead to the loss of neurons in the hippocampus, triggering a series of programmed cell death. OBJECTIVE:To investigate the effect of bone marrow stromal stem cell transplantation on apoptosis in epilepsy. METHODS:After modeled to be of epilepsy 45, Sprague-Dawley model rats were randomly divided into three groups, followed by given no intervention (moldel group), normal saline (normal saline group) or bone marrow stromal stem cell transplantation (transplantation group). At 1, 2 and 4 weeks after modeling, the number of Bax-positive cells, Bcl-2-positive cells and Bax/Bcl-2 were detected by immunohistochemistry. RESULTS AND CONCLUSION:The number of Bax-positive cells, Bcl-2-positive cells and Bax/Bcl-2 presented no obvious changes in the normal saline group at different time points. However, the number of Bax-positive cells and Bax/Bcl-2 in the transplantation group was significantly decreased, while the number of Bcl-2-positive cells significantly increased compared with the other two groups at 1, 2 and 4 weeks after modeling (P < 0.05). Moreover, the above indicators varied significantly in the transplantation group at different time points after modeling (P < 0.05). These results show that bone marrow stromal stem cell transplantation can affect the apoptosis and effectively reduce the apoptosis in rats with epilepsy by up-regulating the number of Bax-positive cells and down-regulating the number of Bcl-2-positive cells.     

转FL基因基质细胞对脐血造血干细胞的作用

造血干细胞不足，限制了造血干细胞移植在临床的应用。我们曾探讨了持续稳定表达人FL(FLT3配体)和GMCSF(粒-巨噬细胞集落刺激因子)的转基因基质细胞系对CD34^ 细胞具有扩增作用，而在CD34^ 细胞中只有CD34^ 、CD38^-细胞才可使造血功能重建。本实验拟通过已建立的持续稳定表达人FL转基因基质细胞系，进一步探讨FL基因修饰的骨髓基质细胞对人脐血造血干细胞的体外扩增作用。     

脱基质小牛骨粉复合骨髓基质干细胞的成骨作用

背景：利用骨组织工程技术在上颌窦提升中的成骨研究是目前口腔种植学到研究热点。 目的：探讨骨组织工程在上颌窦底提升同期牙种植中的成骨效果。 方法：体外分离培养犬骨髓基质干细胞,将细胞与脱基质小牛骨粉复合培养并向成骨细胞定向诱导分化。健康成年犬12只行双侧上颌窦底提升同期牙种植,一侧植入复合物,另一侧植入脱基质小牛骨粉做对照。 结果与结论：种植体稳固无松动,上颌窦黏膜完整。实验侧可见新生骨形成较早、骨量较多;对照侧新生骨形成较慢。X射线显示实验侧新生骨质致密,与种植体结合紧密。随时间的增加,牵出力增大,12,24周时两侧差异有显著性意义(P < 0.05)。组织形态学检测显示新生骨面积逐渐增加,12,24周时差异有显著性意义(P < 0.05)。提示组织工程化骨在上颌窦提升同期牙种植中可获得良好的成骨效果。     

胎肝条件培养液体外诱导人骨髓间充质干细胞定向分化为造血细胞的研究

目的：探讨人骨髓间充质干细胞（hMSCs）体外造血分化潜能。方法： 选用孕12.5-14.5 d（12.5-14.5 dpc）的昆明小鼠，分别制备小鼠胎肝基质细胞条件培养液（FLSC-CM）及胚胎成纤维细胞饲养层（FD），将体外扩增的CD34-CD45-hMSCs分别接种于含FLSC-CM、FD和IL-6及SCF组合的培养体系中，培养7 d后，通过形态学、表型、粒-单/巨噬细胞系集落培养（CFU-GM）对分化细胞进行鉴定。结果： hMSCs与FLSC-CM共培养组产生的非贴壁细胞明显增多，形态类似于单核或小淋巴细胞，部分细胞可表达人造血细胞特异性表面分子（CD34和CD45），在含人粒-单集落刺激因子（GM-CSF）的甲基纤维素培养体系中能够形成CFU-GM，而FD和IL-6+SCF诱导组无上述作用。结论： FLSC-CM可诱导CD34-CD45-hMSCs分化为造血细胞，提示hMSCs具有体外造血分化潜能。     

人骨髓基质干细胞的单细胞克隆培养与鉴定***☆

背景：骨髓基质干细胞缺乏特异的表面识别分子,其鉴定一直是研究中的难题。 目的：探索人骨髓基质干细胞培养条件,获得体外克隆化培养的成人骨髓基质干细胞,并对其进行表型分析及分化潜能鉴定。 方法：外科手术取髂骨术中抽取骨髓,采用密度梯度离心法初步分离骨髓基质干细胞,用极限稀释法进行克隆化培养;流式细胞仪对克隆化培养的骨髓基质干细胞进行细胞表面标志检测,并进行体外成软骨、成骨及心肌细胞诱导,免疫组织化学及RT-PCR检测成软骨、成骨及心肌细胞表达,确定其表型及分化潜能。 结果与结论：单个细胞来源骨髓基质干细胞在体外1∶3传代一般可以传28代左右,24代以前生长状态良好;经流式细胞仪检测,骨髓基质干细胞表达CD29,CD44,CD106,不表达CD14,CD34,CD45,HLA-DR;骨髓基质干细胞体外可向成骨、成软骨及心肌细胞分化,提示体外克隆化培养的骨髓基质干细胞能维持良好的成体干细胞生物学特性。 关键词：单克隆培养;骨髓基质干细胞;多能成体干细胞;鉴定;分化 doi:10.3969/j.issn.1673-8225.2012.10.008     

从骨髓和脐血造血干细胞中诱导成骨样细胞

骨髓有2个重要的功能:造血和成骨。骨髓中存在造血干细胞(hem atopoietic stem cells,HSCs)(CD34 细胞)和间充质干细胞(m esenchym al stem cells,MSCs)。这2种干细胞共同存在于骨髓腔中,MSC对HSC不仅有空间位置的机械支持作用,还分泌多种造血因子支持其造血功能,有助于HSC未分