慢性粒细胞白血病患者的骨髓间充质干细胞对树突状细胞功能的调控

目的 探讨慢性粒细胞白血病(CML)患者骨髓间充质干细胞(MSC)对单个核细胞来源的树突状细胞(DC)的功能的调控及其机制.方法 采用细胞贴壁法获取CML患者骨髓MSC,在低血清培养液中培养和扩增.获取正常成人志愿者外周血单个核细胞,直接在体外诱导生成DC(正常DC组),另加入正常志愿者MSC(正常诱导组)或CML患者骨髓MSC(CML诱导组)以诱导生成DC.用流式细胞仪检测各组收获DC的免疫表型及MSC对DC吞噬功能的影响;用酶联免疫吸附试验检测各组DC白细胞介素12(IL-12)的分泌;混合淋巴细胞反应检测MSC对DC介导的异体T淋巴细胞增殖能力的抑制作用.结果与正常DC组相比较,正常诱导组和MSC诱导组培养7 d后未成熟DC的共刺激分子CD1a、CD80、CD83、CD86、CD40和HLA-DR的表达量较低(P＜0.05);与正常DC组相比较,诱导9 d后,正常诱导组和MSC诱导组成熟DC的CD40、CD86和CD83的表达量较低(P＜0.05).与正常DC组相比较,CML诱导组的不成熟DC的吞噬功能显著降低(P＜0.05),CML诱导组DC IL-12的分泌量显著下降(P＜0.05).CML诱导组的DC对T淋巴细胞增殖的刺激作用有所下降(P＜0.05).结论 CML患者骨髓MSC对DC的分化成熟以及其免疫调控能力有一定的抑制作用.

Abstract：

Objective To study the effects and mechanisms of mesenthymal stem cells (MSCs)derived bone marrow of patients with chronic myelogenous leukemia (CML) on function of monocytederived dendritic cells in vitro. Methods Bone marrow mononuclear cells from CML patients were obtained and cultured. Peripheral blood mononuclear cells (PBMCs) derived from normal volunteers were isolated and cultured in DC differentiational condition. Moreover, PBMCs were co-cultured with CML bone marrow-derived MSCs (CML-MSC) or normal volunteers' bone marrow-derived MSC (normal-MSC) in DC differentiational condition. Immunophenotype and the endocytosis of monocytederived DCs were investigated by FACS. The level of IL-12 was evaluated by enzyme linked immunosorbant assay (ELISA). The immunoregulatory ability was detected by mixed lymphocyte culture assay. Results CML-MSCs or normal-MSC inhibited the up-regulation of CD1a,CD40,CD80,CD86,and HLA-DR during DC differentiation and reduced CD40,CD86,and CD83 expression during DC maturation. CML-MSCs inhibited the endocytosis of DCs and decreased their capacity to secret IL-12. CML-MSC could significantly suppress the function of DCs stimulating proliferation of T lymphocytes. Conclusion CML-derived MSCs harbored effect on the differentiation and maturation of DCs in vitro ; CML-MSC could inhibit the immunregulation of DCs.     

体外转染GFP-Bcl-2基因对低氧条件下间充质干细胞细胞周期的影响

目的探讨低氧条件下诱导骨髓来源的间充质干细胞（MSCs）向目标细胞分化为相关疾病提供了潜在的临床治疗途径。方法将表达GFP-Bcl-2的慢病毒载体转染大鼠骨髓MSCs使其过量表达（GFP-Bcl-2-MSCs）。在低氧环境下诱导其分化并检测细胞凋亡和增殖。采用流式细胞术检测MSCs和GFP-Bcl-2-MSCs在低氧条件下MSCs细胞细胞周期分布。采用Western blot检测不同组细胞cyclinD1、cyclinE及PCNA表达变化。结果在体外低氧诱导条件下,Bcl-2基因对MSCs有明显的抗凋亡功能。与空载体对照组相比：实验组（GFP-Bcl-2-MSCs）细胞凋亡率下降27%,细胞增殖率升高了58%。但细胞周期分布及cyclinD1、cyclinE、及PCNA的表达无明显差异。结论研究证实了抗凋亡基因修饰的MSCs（GFP-Bcl-2-MSCs）在低氧环境下可抑制细胞凋亡,但细胞周期的相关机制需要进一步研究。     

硫酸钙对小鼠骨髓间充质干细胞分化和迁移的影响

目的 分析不同浓度的硫酸钙（calcium sulfate , CaSO4）对小鼠骨髓间充质干细胞（mesenchymal stem cells , MSCs）分化和迁移的影响,探讨CaSO4促进骨修复的细胞机制。方法 分离小鼠骨髓MSCs,绘制细胞生长曲线,评价不同浓度的CaSO4对细胞生长的影响,用qRT-PCR方法评价CaSO4作用后的成骨相关基因Osterix因子和骨钙素的表达,通过刮痕合拢实验和琼脂糖斑点实验,评估CaSO4对MSCs迁移能力的影响。结果 中浓度（0.8 g/L）和低浓度（0.1 g/L）的CaSO4对细胞生长无明显影响,高浓度（1.5 g/L）的CaSO4抑制了细胞增殖;中浓度的CaSO4在作用了1~4 d后,显著提高了MSCs成骨基因Osterix和骨钙素的表达,也明显促进了细胞迁移;高浓度（1.5 g/L）的CaSO4反而抑制了MSCs分化和迁移。结论 一定浓度的CaSO4能够诱导MSCs成骨分化,使MSCs细胞迁移能力增强;成骨细胞的出现可能是细胞迁移能力提高的原因之一。 关键词： 硫酸钙;间充质干细胞;迁移;骨再生     

Nonadherent cell population of human marrow culture is a complementary source of mesenchymal stem cells (MSCs).

To obtain enough quantity of osteogenic cells is a challenge for successful cell therapy in bone defect treatment, and cell numbers were usually achieved by culturing bone marrow cells in a relatively long duration. This study reports a simple and cost-effective method to enhance the number of mesenchymal stem cells (MSCs) by collecting and replating the nonadherent cell population of marrow MSCs culture. Bone marrow MSCs were isolated from 11 patients, cultured at a density of 1 x 10(5)/cm(2) to 1 x 10(6)/cm(2) in flasks. For the first three times of media change, the floating cells were centrifuged and replated in separate flasks. The total number of cells in both the primary and replating flasks were counted at day 21. Cell proliferation rate, potentials for osteogenic, chondrognenic, and adipogenic differentiation were examined in both cell types in vitro. In vivo osteogenic potentials of the cells were also tested in mice implantation model. The results showed that MSCs derived from nonadherent cell population of marrow cell cultures have similar cell proliferation and differentiation potentials as the originally attached MSCs in vitro. When implanted with hydroxyapatite/tricalcium phosphate (HA-TCP) materials subcutaneously in serve combined immune deficiency (SCID) mice, newly formed bony tissues were found in both cell type groups with osteocalcin expression. We have obtained 36.6% (20.70%-44.97%) more MSCs in the same culture period when the nonadherent cell populations were collected. The findings confirmed that the nonadherent cell population in the bone marrow culture is a complementary source of MSCs, collecting these cells is a simple and cost-effective way to increase MSCs numbers and reduce the time required for culturing MSCs for clinical applications.     

Native multipotential stromal cell colonization and graft expander potential of a bovine natural bone scaffold

Graft expanders are bone scaffolds used, in combination with autografts, to fill large bone defects in trauma surgery. This study investigates the graft expander potential of a natural bone substitute Orthoss® by studying its ability to support attachment, growth and osteogenic differentiation of neighboring multipotential stromal cells (MSCs). Material consisting of bone marrow (BM) aspirate and reamer‐irrigator‐aspirator (RIA)‐harvested autograft bone was co‐cultured with commercially available Orthoss® granules. Native MSCs attached to Orthoss® were expanded and phenotypically characterized. MSCs egress from neighboring cancelous bone was assessed in 3D Matrigel co‐cultures. MSC differentiation was evaluated using scanning electron microscopy and measuring alkaline phosphatase (ALP) activity per cell. CD45⁺ hematopoietic lineage cells and highly proliferative CD90⁺CD73⁺CD105⁺ MSCs preferentially colonized Orthoss® granules, over RIA bone chips. MSC colonization was followed by their intrinsic osteogenic differentiation, assessed as mineral deposition and gradual rise in ALP activity, even in the absence of osteogenic stimuli. When in contact with mixed cell populations and RIA chips, Orthoss® granules support the attachment, growth and osteogenic differentiation of neighboring MSCs. Therefore, natural bone substitutes similar to Orthoss® can be used as void fillers and graft expanders for repairing large bone defects in conjunction with autologous BM aspirates and autografts. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1950–1958, 2013     

正常成人和非霍奇金淋巴瘤患者的骨髓间充质干细胞生物学特性的比较

目的比较研究非霍奇金淋巴瘤(NHL)患者和正常成人的骨髓间充质干细胞(MSCs)的生物学特性及分化能力,为临床细胞移植提供实验依据。方法获取并分离正常成人和NHL患者的骨髓MSCs,用低血清培养液培养、传代,观察MSCs的形态和生长特性,以流式细胞仪检测其免疫表型和细胞周期,体外诱导MSCs向脂肪和骨分化,行MSCs的染色体分析,电镜观察其超微结构。结果正常成人和NHL患者的骨髓MSCs具有相似的细胞形态、生长特性和免疫表型,而且都可以向骨和脂肪分化;NHL来源的MSCs具有正常的核型和超微结构,超过90%的细胞处于静止期,未发现肿瘤细胞的特征性结构。结论从NHL患者骨髓中获取的MSCs经培养传代,与正常成人骨髓MSCs一样,具有在体外大量扩增并保持低分化状态和多向分化的能力。     

Bone morphogenetic protein 2 induces placental growth factor in mesenchymal stem cells

Bone-forming osteoblasts differentiate from pluripotent mesenchymal stem cells (MSCs) in a multistage process that can be modeled in vitro using MSCs isolated from adult human trabecular bone or bone marrow. To identify new genes involved in osteoblast differentiation, we have performed large-scale gene expression profiling using high-density cDNA microarrays in primary human MSCs treated with the known osteogenic agent bone morphogenetic protein 2 (BMP-2). The vascular endothelial growth factor (VEGF) family member placental growth factor (PlGF) was found as an early regulated gene whose induction was already detected after 2 h treatment with BMP-2. Tissue distribution analysis of PlGF mRNA expression using microarrays revealed a very restricted expression of PlGF only in BMP-2-treated MSCs and in placenta as expected. Ribonuclease protection assay (RPA) confirmed the induction of PlGF and showed preferential expression of the PlGF-1 isoform over PLGF-2 in MSCs and MG63 cells. BMP-2 stimulated PlGF expression in MG63 cells with an EC50 of about 50 ng/ml and mRNA levels peaked between 24 and 32 h after stimulation. Furthermore, induction of PlGF by BMP-2 appeared specific, as other osteogenic agents including vitamin D3, transforming growth factor beta, and basic fibroblast growth factor were inactive. BMP-2 stimulated PlGF secretion from MG63 and MSC cells, but PlGF had no effect on MSC proliferation and osteoblastic differentiation. Based on the known function of PlGF in the recruitment of endothelial and hematopoietic stem cells, these results suggest a paracrine role for MSC-derived PlGF in the angiogenesis and hematopoiesis that accompany BMP-2-induced bone formation.     

Mesenchymal stem cells from the bone marrow stroma: basic biology and potential for cell therapy

Mesenchymal stem cells (MSCs) can be isolated from the bone marrow stroma where they constitute an adult somatic stem cell population distinct from hemapoietic stem cells. MSCs are multipotent cells in that they have the capacity to generate progeny that can differentiate into multiple cell lineages. MSCs can be explanted in vitro from bone marrow aspirates and expanded in culture where they can be induced to terminally differentiate into osteoblasts, chrondrocytes, adipocytes, tenocytes and heamapoteic supporting tissue. This ability to differentiate has also been demonstrated in vivo following transplantation into rodents. Recent work has shown that MSCs may have a broader capacity for differentiation than was previously envisioned. In some circumstances, this increased potential for differentiation may make MSCs viable alternatives to embryonic stem cells. Accordingly, the multipotential capacity of MSCs, their accessible origin, and high ex vivo expansive potential, makes these cells attractive as tools for tissue engineering and cell-based therapy. This review will explore the basic biology of MSCs derived from adult tissues and consider their isolation, culture, and differentiation. In addition, we will examine some of the potential clinical applications of these cells and consider the future perspectives of their use.     

Activation of protease-activated receptor-2 leads to inhibition of osteoclast differentiation.

PAR-2 is expressed by osteoblasts and activated by proteases present during inflammation. PAR-2 activation inhibited osteoclast differentiation induced by hormones and cytokines in mouse bone marrow cultures and may protect bone from uncontrolled resorption. INTRODUCTION: Protease-activated receptor-2 (PAR-2), which is expressed by osteoblasts, is activated specifically by a small number of proteases, including mast cell tryptase and factor Xa. PAR-2 is also activated by a peptide (RAP) that corresponds to the "tethered ligand" created by cleavage of the receptor's extracellular domain. The effect of activating PAR-2 on osteoclast differentiation was investigated. MATERIALS AND METHODS: Mouse bone marrow cultures have been used to investigate the effect of PAR-2 activation on osteoclast differentiation induced by parathyroid hormone (PTH), 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], and interleukin-11 (IL-11). Expression of PAR-2 by mouse bone marrow, mouse bone marrow stromal cell-enriched cultures, and the RAW264.7 osteoclastogenic cell line was demonstrated by RT-PCR. RESULTS: RAP was shown to inhibit osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11. Semiquantitative RT-PCR was used to investigate expression of mediators of osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11 in mouse bone marrow cultures and primary calvarial osteoblast cultures treated simultaneously with RAP. In bone marrow and osteoblast cultures treated with PTH, 1,25(OH)2D3, or IL-11, RAP inhibited expression of RANKL and significantly suppressed the ratio of RANKL:osteoprotegerin expression. Activation of PAR-2 led to reduced expression of prostaglandin G/H synthase-2 in bone marrow cultures treated with PTH, 1,25(OH)2D3, or IL-11. RAP inhibited PTH- or 1,25(OH)2D3-induced expression of IL-6 in bone marrow cultures. RAP had no effect on osteoclast differentiation in RANKL-treated RAW264.7 cells. CONCLUSION: These observations indicate that PAR-2 activation inhibits osteoclast differentiation by acting on cells of the osteoblast lineage to modulate multiple mediators of the effects of PTH, 1,25(OH)2D3, and IL-11. Therefore, the role of PAR-2 in bone may be to protect it from uncontrolled resorption by limiting levels of osteoclast differentiation.