小鼠骨髓基质细胞的生物学特性及血管新生能力

目的 探讨体外培养的骨髓基质细胞的一些生物学特性及体内移植后在缺血区新血管生成中的作用。方法分离5—6周龄的小鼠胫骨、股骨，用预冷的DMEM／F12培养基冲洗出骨髓，经密度梯度离心分离出骨髓单个核细胞，接种后12～16d形成单层贴壁的成纤维样细胞。体外诱导分化鉴定分离的细胞，用传代的细胞进行生长曲线测定，观察其接种贴壁率、分裂指数，检测细胞周期和超微结构，并建立下肢缺血模型。荧光标记的体外扩增的骨髓单个核细胞被移植入缺血组织。移植后2周，荧光显微镜及内皮细胞碱性磷酸酶染色，检查荧光阳性细胞与染色阳性细胞的时空关系。结果体外传代培养的单个核细胞倍增时间约为42h。传代10h贴壁率达90％以上。分裂指数曲线与生长曲线相似。细胞周期显示约83％的细胞处于G1期。结论 体外培养的骨髓基质细胞生长稳定，传代后的细胞适应性强，增殖较快，表现出较早期细胞特点，在体外及移植入体内缺血区能分化为血管内皮细胞，有望用于改善组织缺血。     

缺氧预处理提高骨髓基质细胞促进血管新生能力

目的 研究缺氧对骨髓基质细胞(bone marrow stromal cells，BMSCs)表达血管内皮细胞生长因子(vascular endothelial growth factor，VEGF)及促进血管新生能力的影响。方法 将BMSCs置于缺氧环境下培养24h后，研究其VEGF表达的变化。将急性心肌梗死大鼠随机分为3组。未处理组在梗死后4w移植未经缺氧处理的BMSCs；缺氧组将经缺氧预处理的BMSCs注射到心肌梗死区；对照组仅注射无血清的培养液。在梗死后6w取各组标本观察梗死区及周围VEGF的表达和血管新生状况。结果 体外培养的BMSCs经缺氧预处理后VEGF的表达明显增加。缺氧组和未处理组梗死区及周围VEGF的表达和毛细血管密度较对照组明显增加。缺氧组VEGF的表达和毛细血管密度又较未处理组明显增加。结论 体外缺氧可诱导BMSCs高表达VEGF。BMSCs移植后促进梗死区及周围VEGF的表达，对血管新生有积极作用。移植前缺氧预处理使BMSCs促进缺血心肌组织表达更多的VEGF从而提高其促进血管新生作用。     

小鼠骨髓基质细胞成内皮细胞分化及其缺血区存活能力研究

目的 探讨体外培养的骨髓基质细胞的部分生物学特性、成内皮细胞分化能力及在缺血区存活能力？方法 2005年1月至11月，第三军医大学新桥医院全军心血管内科中心分离5～6周龄的小鼠胫骨、股骨，预冷的DMEM／F12培养基冲洗出骨髓，密度梯度离心分离出骨髓单核细胞，接种后12～16d形成单层贴壁的成纤维样细胞：体外诱导分化鉴定分离的细胞：建立下肢缺血模型，荧光标记的体外扩增的骨髓基质细胞被移植入缺血组织，移植后1周，荧光显微镜及HE染色，检查荧光强弱分布与HE染色密度的时空关系。结果 体外传代培养的基质细胞诱导后分泌NO，移植1周在缺血区检测到大量荧光阳性细胞存活。结论在本实验条件下，体外培养的骨髓基质细胞生长稳定，传代后的细胞增殖较快，表现出较早期细胞特点，在体外能分化为血管内皮细胞，可望用作缺血性心脏病的细胞治疗的供体细胞。     

小鼠骨髓基质细胞成内皮细胞分化及其缺血区存活能力研究

目的探讨体外培养的骨髓基质细胞的部分生物学特性、成内皮细胞分化能力及在缺血区存活能力。方法2005年1月至11月,第三军医大学新桥医院全军心血管内科中心分离5~6周龄的小鼠胫骨、股骨,预冷的DMEM/F12培养基冲洗出骨髓,密度梯度离心分离出骨髓单核细胞,接种后12~16d形成单层贴壁的成纤维样细胞。体外诱导分化鉴定分离的细胞。建立下肢缺血模型,荧光标记的体外扩增的骨髓基质细胞被移植入缺血组织,移植后1周,荧光显微镜及HE染色,检查荧光强弱分布与HE染色密度的时空关系。结果体外传代培养的基质细胞诱导后分泌NO,移植1周在缺血区检测到大量荧光阳性细胞存活。结论在本实验条件下,体外培养的骨髓基质细胞生长稳定,传代后的细胞增殖较快,表现出较早期细胞特点,在体外能分化为血管内皮细胞,可望用作缺血性心脏病的细胞治疗的供体细胞。     

骨髓动员在糖尿病小鼠缺血肢体新生血管形成中的作用

目的探讨骨髓动员在糖尿病小鼠缺血肢体新生血管形成中的作用。方法采用链脲佐菌素建立糖尿病鼠模型,切除一侧肢体股动脉,注射粒细胞集落刺激因子(G-CSF)进行骨髓动员,以观察缺血肢体新生血管的形成。结果糖尿病小鼠模型建立8周后,血浆一氧化氮(NO)水平开始下降,骨髓动员后8 d,外周血CD~+_(34)细胞明显增加[从(0．15±0．05)%到(2．30±0．41)%],半定量RT-PCR检测缺血肢体肌肉组织的血管内皮细胞生长因子mRNA表达与正常对照组比较明显增强；动员后2周,激光多普勒血流检测骨髓动员组的缺血肢体血流明显高于未动员组[(76．37±6．10)%vs(18．07±3．40)%,P＜0．05],vWF免疫组化微血管密度检测动员组也明显高于未动员组[(26．6±4．8)个/每视野vs(11．5±2．6)个/每视野,P＜0．05]。结论糖尿病能降低小鼠血管内皮细胞的功能,但骨髓动员能促进糖尿病小鼠缺血肢体的新生血管形成。     

骨髓基质细胞的血管新生作用及对心功能的影响

目的　观察骨髓基质细胞在缺血心肌内诱导血管新生的作用及对心功能的影响。方法　复制兔心肌梗死动物模型 ,3d后将体外分离扩增的自体骨髓基质细胞用荧光标记物DAPI标记后移植到梗死周围缺血区心肌 (细胞移植组 ,n =8) ,对照组注射等量培养基 (n =6 )。 4W后 ,先进行心脏超声检查分别测定对照组和细胞移植组左室射血分数 (EF)和短轴缩短率 (FS) ,然后采集移植区心肌标本 ,用荧光示踪方法观察移植细胞在缺血坏死区心肌存活的情况 ,用CD31单克隆抗体免疫组化染色法测定毛细血管密度。结果　细胞移植组心肌组织和小血管内壁均可见蓝色荧光的DAPI标记的移植细胞 ;细胞移植组心肌毛细血管密度显著高于对照组 [(16 3 0 0± 2 5 85 )vs (96 0 0± 16 6 1) ,P <0 0 5 ];细胞移植组EF较对照组增高 [(0 5 0± 0 0 3)vs(0 4 6± 0 0 3) ,P <0 0 5 ],短轴缩短率FS也高于对照组[(2 7 0 2± 1 2 7)vs (2 3 85± 1 6 9) ,P <0 0 5 ]。结论　体外扩增的骨髓基质细胞可在缺心肌内存活 ,并能促进缺血组织血管新生、改善心功能。     

人骨髓基质细胞的培养及鉴定

目的:探讨骨髓基质细胞的分离、体外培养方法,为基因治疗提供载体细胞.方法:采用密度梯度离心法结合贴壁筛选法分离人骨髓进行体外培养扩增,用倒置显微镜观察细胞形态学特征并用流式细胞仪鉴定其CD90表达.结果:分离培养的骨髓基质细胞似成纤维状,原代细胞呈集落生长,并可表达CD90,传代后细胞形态较一致,且随着传代次数的增加CD90表达越高,第3代可达94.31%.结论:骨髓基质细胞可通过体外培养纯化获得,随着传代次数增加,纯度越高,该方法是一种较理想的骨髓基质细胞培养方法.     

白血病干细胞和骨髓基质细胞共培养的方法学探讨

目的 探讨白血病干细胞和骨髓基质细胞共培养的可行方法及扩增白血病干细胞的特点.方法 体外用两种方法共培养急性髓系白血病（AML）患者的骨髓单个核细胞（MNCs）：一种方法是将MNCs中的贴壁基质细胞单独培养,第2代或第3代基质细胞用丝裂霉素处理后做饲养层,再同悬浮造血细胞共培养;另一种方法是将MNCs持续培养即原代共培养.观察MNCs形态学特征、长期存活情况及增殖特性,用流式细胞仪检测分析细胞表面标志.结果 两种方法共培养的细胞部分可以长期存活,在共培养中有卵石样区域细胞及悬浮细胞团的形成.原代共培养的细胞所形成的卵石样区域数目多,排列规律.结论 两种方法都可以起到扩增白血病干细胞的作用.同经过丝裂霉素处理之后的饲养层相比,没有经过处理的骨髓基质细胞在共培养时能够更有效地扩增白血病干细胞.     

Thymocyte rosettes: multicellular complexes of lymphocytes and bone marrow-derived stromal cells in the mouse thymus.

We describe the isolation and purification of multicellular complexes composed of lymphocytes and bone marrow-derived stromal cells ("thymocyte rosettes") from the mouse thymus. These rosettes are the structural in vitro correlate of in vivo associations between lymphoblasts and I-A/E negative macrophages or medullary I-A/E positive dendritic-like cells. Both types of rosettes are preformed in vivo. The rosette-associated thymocytes display a surface antigen phenotype typical of immature thymocytes. In radiation chimeras, replacement of host thymocytes by injected bone marrow cells follows a regular pattern: donor type T cells appear first at day 11 as clusters around I-A negative macrophages and approximately 2 days later as similar clusters associated with either I-A positive cortical epithelial cells or I-A positive medullary dendritic cells. These data suggest (a) a defined sequence of lymphostromal interactions during intrathymic maturation and (b) a rapid proliferation of thymocytes after interaction with stromal cells.     

Human B-lymphopoiesis is supported by bone marrow-derived stromal cells

We have recently reported an in vitro culture system that allows the clonal growth and differentiation of normal human bone marrow B-lineage cells. In the report presented here, we have used this B-cell colony assay to study the influence of cellular components of the human bone marrow microenvironment on B-lymphopoiesis. It is demonstrated that bone marrow stromal cells were able to provide all the necessary requirements for the growth and differentiation of B-lineage cells under the conditions of the B-cell colony assay. These stromal cells were obtained from long-term bone marrow cultures (LTBMC) that had been established from the spicules in human bone marrow. When these stromal cells were plated as an adherent underlayer in the double-agar B-cell colony assay, both immature and mature B-lineage cells were induced to differentiate into colonies containing cells that secreted immunoglobulin. The stromal cells from these spicule-derived LTBMCs maintained the capacity to support B-cell colony formation for up to 9 months.     

CD34 expression on murine marrow-derived mesenchymal stromal cells: impact on neovascularization

OBJECTIVE: CD34 is a sialomucin often expressed by cells with hemangiopoietic potential and widely serves as a surrogate marker of stem cell potential. Mesenchymal stromal cells (MSCs) also express CD34, although the functional significance of its expression remains undefined. In this study, we determined whether CD34(pos) MSCs are functionally distinct from CD34(null) MSCs. MATERIALS AND METHODS: MSCs derived from C57Bl/6 mice were transduced to express the green fluorescent protein (GFP) from which pure CD34(pos) MSC and CD34(null) MSC clones were selected. In vitro, clones were examined by microarray analysis, while in vivo subcutaneous implantation of matrix-embedded MSCs was used to assess cell survival, differentiation, and neovascularization. RESULTS: The flow cytometric phenotype of CD34(pos) and CD34(null) MSCs were similar, as was gene expression of vascular endothelial growth factors (VEGFs) A and B. However, CD34(pos) MSCs upregulated a number of supplementary angiogenesis-associated genes and showed a greater expression of gene associated with vascular differentiation. At 15 days postimplantation, cell survival between CD34(pos) and CD34(null) MSCs was similar, however, CD34(pos) MSCs evoked a significantly greater host-derived response (4.2 +/- 0.7 vs 1.9 +/- 0.5 x 10(6) cells; p < 0.05). GFP-expressing CD34(pos) MSC implants acquired significantly more CD31 expression compared to CD34(null) MSC cells (10.7% +/- 8.4% vs 3.1% +/- 0.6%; p < 0.05), as well as a significantly greater host-derived endothelial cell influx (CD31(+)/CD45(-)). CONCLUSION: CD34 expression by MSCs correlates with enhanced vasculogenic and angiogenic potential in vivo.     

Role of bone marrow-derived lymphatic endothelial progenitor cells for lymphatic neovascularization

The lymphatic vasculature plays a pivotal role in maintaining tissue fluid homeostasis, immune surveillance, and lipid uptake in the gastrointestinal organs. Therefore, impaired function of the lymphatic vessels caused by genetic defects, infection, trauma, or surgery leads to the abnormal accrual of lymph fluid in the tissue and culminates in the swelling of affected tissues, known as lymphedema. Lymphedema causes impaired wound healing, compromised immune defense, and, in rare case, lymphangiosarcoma. Although millions of people suffer from lymphedema worldwide, no effective therapy is currently available. In addition, recent advances in cancer biology have disclosed an indispensable function of the lymphatic vessel in tumor growth and metastasis. Therefore, understanding the detailed mechanisms governing lymphatic vessel formation and function in pathophysiologic conditions is essential to prevent or treat these diseases. We review the developmental processes of the lymphatic vessels and postnatal lymphatic neovascularization, focusing on the role of recently identified bone marrow-derived podoplanin-expressing (podoplanin(+)) cells as lymphatic endothelial progenitor cells.     

Emerging roles for multipotent, bone marrow-derived stromal cells in host defense

Multipotent, bone marrow-derived stromal cells (BMSCs, also known as mesenchymal stem cells [MSCs]), are culture-expanded, nonhematopoietic cells with immunomodulatory effects currently being investigated as novel cellular therapy to prevent and to treat clinical disease associated with aberrant immune response. Emerging preclinical studies suggest that BMSCs may protect against infectious challenge either by direct effects on the pathogen or through indirect effects on the host. BMSCs may reduce pathogen burden by inhibiting growth through soluble factors or by enhancing immune cell antimicrobial function. In the host, BMSCs may attenuate pro-inflammatory cytokine and chemokine induction, reduce pro-inflammatory cell migration into sites of injury and infection, and induce immunoregulatory soluble and cellular factors to preserve organ function. These preclinical studies provide provocative hints into the direction MSC therapeutics may take in the future. Notably, BMSCs appear to function as a critical fulcrum, providing balance by promoting pathogen clearance during the initial inflammatory response while suppressing inflammation to preserve host integrity and facilitate tissue repair. Such exquisite balance in BMSC function appears intrinsically linked to Toll-like receptor signaling and immune crosstalk.     

Immunophenotypic heterogeneity of bone marrow-derived mesenchymal stromal cells from patients with hematologic disorders: correlation with bone marrow microenvironment

The immunophenotypic analysis of ex vivo-expanded mesenchymal stromal cells (MSC) has so far been confined to single or dual staining analysis in normal subjects. In this study, using a four-color cytofluorimetric protocol, we demonstrated that cultured MSC derived from the bone marrow of patients with hematologic malignancies showed alterations in the expression of CD105, CD90, CD184, and HLA-DR molecules. The decrease in the percentage of CD105+ and CD90+ MSC correlated with an increased bone marrow angiogenesis. This paper provides evidence that multiparametric flow cytometry is essential for the establishment of a standardized protocol to identify various MSCs subsets and aberrant phenotypes.     

Stimulation of interleukin-6 production by endothelin in rat bone marrow-derived stromal cells

Endothelin (ET) produced by endothelial cells has recently been found to be a potent vasoconstricting hormone. In this report, ET is shown to be a potent stimulator of interleukin-6 (IL-6) production by rat bone marrow (BM)-derived stromal cells. It was also shown that ET increased the level of mRNA for IL-6 in these cells. The two types of ET receptor (R), ETAR and ETBR, were shown to be expressed on both BM-derived stromal cells in culture and ex vivo in BM tissue, suggesting that ET works as a physiologic stimulator of IL-6 production in the BM. It was shown that ETAR is coupled to phospholipase C activation, leading to the production of inositol 1,4,5-trisphosphate (IP3) and 1,2- diacylglycerol (DAG) as second messengers in BM-derived stromal cells. This was corroborated by data showing that IL-6 production in these cells was induced by combined stimulation with ionomycin and phorbol myristate acetate, thereby bypassing the effects of IP3 and DAG, respectively. This is the first report on the hormonal regulation of IL- 6 production by BM stromal cells, indicating that hematopoiesis is subject to endocrinologic regulation under physiologic conditions. ET has recently been reported to be produced by macrophages in response to bacterial lipopolysaccharide and human immunodeficiency virus-1 glycoprotein 120. These facts, taken together with our findings, raise the possibility that ET shares the same role of IL-1 as a local cytokine, mediating an intercellular signal between macrophages and BM stromal cells in response to bacterial or viral stimulation.