Estrogen regulation of growth and alkaline phosphatase expression by cultured human bone marrow stromal cells.

Estrogen has been reported to regulate the growth and differentiation of cultured murine osteoprogenitor cells in bone marrow stroma. This study tested the ability of 17beta-estradiol (E2) to regulate growth and expression of alkaline phosphatase (ALP), an osteoblastic differentiation marker, in strains of normal human bone marrow stromal cells derived from different donors. In eight strains examined, E2 at 1 and 10 nM produced at most modest effectxs on growth and ALP activity. Growth inhibition, seen in 4 of the 8 strains, was more common than stimulation (2 of the 8 strains); the greatest observed E2 effect was an inhibition of ca. 50%. E2 altered ALP activity less dramatically than cell growth. Differences from control in total ALP per culture were seen in only two strains: one was a reduction, one an increase. Colony forming assays were used to determine if E2 changed the proportion of ALP-expressing cells in marrow stromal cell cultures. In contrast to growth experiments, ALP expression under colony forming conditions (200 cells per 35 mm-diameter well) was dependent on the type of serum supplementation used. Under permissive conditions using medium supplemented with 10% charcoal-treated fetal bovine serum, 10 nM E2 increased the number of ALP-positive colonies (cfu-ap) but not the total number of colonies formed (cfu-f). When cells cultured in the presence or absence of 10 nM E2 were replated at colony forming densities, significantly higher proportions of cfu-ap were found in 2 of 6 strains examined, while pretreatment with E2 affected the number of cfu-f in only 1 of the 6 strains. Similar results were obtained when colony formation was carried out in the presence of dexamethasone and ascorbate, although these agents themselves increased the formation of both cfu-f and cfu-ap. These results show that the direct effects of E2 on human marrow stromal cells are small and vary depending on the cell strain and on the experimental conditions; however, the E2 actions observed in this study were consistent with reports that E2 exerts direct actions on osteoblasts and osteoblast progenitor cells that favor rather than suppress their phenotypic expression.     

自制脱蛋白骨与骨髓基质细胞的生物相容性研究

目的 探讨表面脱钙的脱蛋白异体骨与骨髓基质细胞的生物柑容性，为骨组织工程提供合适的支架材料。方法 用经物理与化学方法处理而制得的脱蛋白骨与骨髓基质细胞（MSCs)复合培养，进行形态学观察、细胞增殖、ALP的活性、骨钙素的分泌与蛋白质含量检测；扫描电镜观察细胞在脱蛋白骨材料上的情况。结果 骨髓基质细胞能在脱蛋白骨上贴附、增殖，其生长及功能不受影响。结论 本方法所制的脱蛋白骨与MSCs具有良好的生物相容性，可作为MSCs的载体应用于骨组织工程。     

去卵巢大鼠骨髓基质细胞beta-catenin蛋白表达变化观察

目的观察卵巢切除大鼠骨髓基质细胞（BMSC）beta-catenin蛋白表达的变化。方法将18只健康3月龄未经产雌性SD大鼠随机平分为去卵巢和假手术两个组。术后14周,取左侧股骨,新鲜分离骨髓细胞,静置贴壁法体外培养9d后,采用间接免疫荧光方法检测BMSC中beta-catenin蛋白表达和定位的变化。取右侧股骨,在双能直线χ-射线骨密度仪上检测骨密度的变化。结果大鼠去卵巢14周后,股骨近端和远端的骨密度显著下降（P〈0.05）,而股骨干的骨密度没有变化（P〉0.05）。在体外培养的假手术组BMSC中,beta-catenin蛋白主要在胞核中表达。与假手术组相比,去卵巢组BMSC核中beta-catenin蛋白表达减少,而胞膜中表达增加。结论beta-catenin蛋白在卵巢切除大鼠BMSC核中表达的下降可能与绝经后骨质疏松症的发生有关。     

煅烧骨与诱导的MSCs复合的实验研究

目的：观察大鼠骨髓基质干细胞（MSCs）与经胶原涂层处理的煅烧骨（CCB）复合行体外培养时贴附、增殖能力的变化,探讨构建组织工程骨体外培养的最适时间。方法：制备鼠尾胶原（I型胶原）,无菌条件下,将煅烧骨置于胶原液中,2h后取出骨块,置滤网中将孔内外的胶原溶液沥干。体外培养的大鼠MSCs经矿化液诱导向成骨细胞分化。将分化后的成骨细胞滴加到涂胶原的煅烧骨块上,同时以未涂胶原的煅烧骨块为对照组。3天,7天时取材,扫描电镜下观察实验组与对照组成骨细胞在煅烧骨表面的贴附数量及形态,用细胞计数法测定细胞在两组材料的生长曲线,组织化学方法检测成骨细胞的ALP活性。结果：扫描电镜发现实验组成骨细胞的贴附数量显著高于对照组（P〈0．01）。细胞计数认为实验组细胞在材料上粘附数量多,增殖速度快。实验组ALP活性明显高于对照组（P〈0．01）。结论：胶原修饰的煅烧骨具有良好的组织相容性,能明显提高成骨细胞的贴附,并且促进成骨细胞活性维持,体外培养7～8天时,材料上的细胞数达到最大,应尽快植入体内。     

骨康方含药血清对骨髓基质细胞成骨分化过程中 I型胶原基因表达的影响

目的观察骨康方含药血清对体外培养骨髓源性骨髓基质细胞（Bone Marrow Stromal Cells,BMSCs )中I型胶原的影 响。方法采用梯度离心法获取大鼠骨髓源性MSCs,随机将实验用大白兔分为骨康方高、中、低3个剂量组,对实验用大白兔 给药干预7 d后,通过血清药理学的方法使用各组大白兔血清联合诱导剂培养传6代BMS&2周后,RT-PCR方法检测I型胶 原mRNA表达。结果高、中浓度骨康方含药血清加诱导剂作用于体外培养的BMSCs2周后,能使BMSCs中I型胶原mRNA 的表达升高。结论具有补肾填精壮骨作用的骨康方可以升高大鼠体外BMSCs向成骨细胞分化过程中I型胶原mRNA的表 达,这可能是防治骨质疏松症的机理之一。     

大鼠骨髓基质细胞体外成脂、成骨分化的研究

目的　观察体外定向诱导大鼠骨髓基质细胞分化为脂肪细胞和成骨细胞。方法　全骨髓法分离大鼠骨髓基质细胞 ,传 3代后分别在成脂、成骨诱导条件下继续培养 ,油红O染色、碱性磷酸酶染色和VonKossa染色判定其分化结果。结果　传 3代大鼠骨髓基质细胞成脂诱导 2 1d后 ,分化为脂肪细胞的阳性率为 (83. 6± 2 . 8) % ,成骨诱导 12d后碱性磷酸酶染色阳性率达 (87. 6± 2. 8) % ,连续诱导35d可见矿化结节形成。结论　随着诱导条件的不同 ,大鼠骨髓基质细胞在体外可定向分化为脂肪细胞或成骨细胞。     

转录因子cbfa1在骨髓干细胞向成骨细胞分化过程中的表达

目的 通过观察转录因子cbfal的表达方式，探讨骨髓干细胞向成骨细胞定向分化的机制。方法 在诱导小鼠骨髓干细胞向成骨细胞分化的过程中，以双重荧光免疫方法在细胞水平发现和定位转录因子cbfa1，并观察其表达方式。结果 转录因子cbfa1在骨髓干细胞分化时于原代培养第3天开始出现，并持续表达于成骨细胞成熟过程中，其表达特异性地定位于细胞核内。结论 转录因子cbfa1与成骨细胞分化密切相关；定位观察cbfa1的表达为进一步研究其调控机制建立了有利的方法。     

成骨生长肽诱导的大鼠骨髓基质细胞基因表达谱变化中骨折愈合相关基因的筛选

目的基因表达谱芯片筛选成骨生长肽诱导大鼠骨髓基质细胞骨折愈合相关基因的表达变化及其促进骨折愈合的机制。方法密度梯度离心法分离大鼠骨髓基质细胞,以10-9mol/L的成骨生长肽诱导24h的第三代大鼠骨髓基质细胞作为实验组,以第三代大鼠骨髓基质细胞为对照组,提取诱导组与实验组总RNA,分别用cy5和cy3探针逆转录标记,与博奥27KRat Genome Array芯片杂交,荧光扫描分析。结果芯片结果显示大鼠骨髓基质细胞经成骨生长肽刺激24h后共有145个差异表达的基因,其中91个上调表达的基因,54个下调表达的基因。差异表达的基因中可能与骨折愈合相关的基因共有27个:包括4个血管发生相关的基因、9个骨代谢相关的基因、14个炎症与免疫相关的基因。结论基因表达谱芯片有助于研究成骨生长肽诱导大鼠骨髓基质细胞促进骨折愈合的分子机制。     

Hyaluronan increases RANKL expression in bone marrow stromal cells through CD44.

HA activates CD44 to stimulate RANKL expression in bone marrow stromal cells. HA stimulation of RANKL is blocked by anti-CD44 antibody and is absent in cells from CD44(-/-) mice. CD44(-/-) mice exhibit thicker cortical bone and a smaller medullary cavity, but indices of bone resorption are not affected. INTRODUCTION: Hyaluronan (HA), the major nonprotein glycosaminoglycan component of the extracellular matrix in mammalian bone marrow, functions in part through its receptor, CD44, to stimulate a series of intracellular signaling events that lead to cell migration, adhesion, and activation. To determine whether HA activation of CD44 influences RANKL and osteoprotegerin (OPG) expression and whether CD44 is functionally important in bone metabolism, we studied whole bone and bone marrow stromal cells (BMSCs) from wildtype and CD44(-/-) mice. MATERIALS AND METHODS: BMSCs from wildtype and CD44(-/-) mice at 7 weeks of age were cultured and treated with either HA or anti-CD44 antibody. The levels of mRNA of RANKL, OPG, CD44, alkaline phosphatase (ALP), osteocalcin (OC), and alphaI collagen (COLL) were determined by quantitative real-time RT-PCR. Levels of RANKL and CD44 protein were measured by immunoblotting, and expression of CD44 in whole bone was determined by immunohistochemical staining. Double immunofluorescence staining and confocal microscopy were used to study colocalization of Cbfa1, CD44, and HA. Tibias were imaged using muCT, and cancellous and cortical parameters were measured. Osteoblast and osteoclast surface in the distal femoral metaphysis and osteoclast on the endocortical surface at the tibio-fibular junction were measured using quantitative histomorphometry. Differences were analyzed using ANOVA and the Newman-Keuls test. RESULTS: Addition of HA dose-dependently increased RANKL mRNA (3.6-fold) and protein (3-fold) levels in BMSCs. Stimulation of RANKL by HA could be blocked with anti-CD44 antibody. Treatment of cells with HA or anti-CD44 antibody had no significant effect on OPG mRNA levels. Both CD44 and HA localized on the plasma membrane in cells expressing Cbfa1. HA localization on the cell membrane disappeared when cells were preincubated with anti-CD44 antibody. Compared with control mice, cortical bone of CD44(-/-) was thicker, and medullary area was smaller at both 7 and 17 weeks, but at 7 weeks, indices of bone resorption were normal. At 17 weeks of age, tibial mass of CD44(-/-) mice was higher than control mice. CD44(-/-) animals expressed less RANKL in whole bone (-30%) and in BMSCs (-50%). Cells from CD44(-/-) animals failed to respond to either HA or CD44 antibody treatment. CONCLUSIONS: HA can increase RANKL expression in BMSCs through CD44.     

无血清培养骨髓基质干细胞向神经细胞诱导分化条件的优化

目的：探索无血清培养的骨髓基质干细胞（BMSCs）向神经细胞诱导分化条件的优化方案,为BMSCs应用于脊髓损伤的临床治疗创造条件.方法：用含2% Utroser G的UltraCULTURE无血清培养体系体外扩增人BMSCs,采用流式细胞仪检测培养细胞的表面标志,再以全反式维甲酸、β-巯基乙醇和神经生长因子为主要成分组成6种诱导液诱导BMSCs向神经细胞分化,镜下观察细胞形态学变化,用抗人β微管蛋白（β-Tubulin）抗体和抗人胶质纤维酸性蛋白（GFAP）抗体进行免疫荧光染色鉴定诱导后的神经细胞,通过流式细胞仪检测诱导后细胞的凋亡率.结果：无血清培养的BMSCs在6种诱导条件下均可不同程度地分化为神经样细胞,镜下可见诱导后细胞表现为神经细胞形态特征,免疫荧光染色显示β-Tubulin和GFAP均有阳性表达,诱导后细胞发生不同程度的凋亡,其中全反式维甲酸和神经生长因子组成的复合诱导液的诱导效率较高,诱导后细胞凋亡率较低.结论：全反式维甲酸与神经生长因子联合应用可在体外高效、稳定地诱导无血清培养的BMSCs分化为神经样细胞,是较佳的诱导条件.     

Canine distemper virus localised in bone cells of patients with Paget's disease

The technique of in situ hybridisation was applied using radioactively labelled riboprobes to examine for the presence of canine distemper (CDV) and measles (MV) RNA in Paget's disease of bone. The results indicate that in 41% of Paget's patients, CDV RNA could be detected in osteoclasts, osteoblasts, and osteocytes, but not in controls. In contrast, RNA to the measles virus was not detected. We suggest that CDV may in some cases play a role in the aetiology of Paget's disease.     

间歇性压力培养环境对兔骨髓基质干细胞增殖的影响

目的:研究间歇性压力培养环境对兔骨髓基质干细胞(MSC)增殖的影响,探讨假体置换术后活动所需的最适宜压力。方法:以新西兰兔的骨髓基质干细胞为实验材料,通过噻唑盐(MTT)比色试验,观察间歇性压力对骨髓基质干细胞增殖的影响。结果:MSC在60、100 kPa间歇性压力培养环境下MTT反应的OD值小于对照组,压力值越大,OD值越小(P<0.05)。而20 kPa间歇性压力情况可显著促进MSC增殖,并随时间增加而增强。在实验第7天时,实验组OD值高于对照组(P<0.05),差异有显著性意义。结论:关节置换术后早期下地活动产生的较大应力,会明显抑制骨髓腔内MSC的增殖,不利于骨的重建愈合,临床应当避免。然而,较小的间歇性压力对MSC增殖有明显的促进作用,提示关节置换患者可在较小压力范围(<20 kPa)内进行早期关节被动活动训练,有利于关节术后骨的重建愈合。     

Long-term culture of cells from bone affected by Paget's disease.

Cells obtained from surgical bone specimens of eight patients with Paget's disease of bone were maintained in culture for up to 8 months and seven passages. The doubling time during the period of maximal cell growth ranged from 4 to 12 days. Evidence consistent with the hypothesis that many of the cells were bone cells included the following: (a) histochemical techniques demonstrated staining of some cells for alkaline phosphatase or acid phosphatase and succinic dehydrogenase; (b) parathyroid extract stimulated increased uptake of 3H-thymidine and 3H-uridine; (c) parathyroid extract suppressed and salmon calcitonin stimulated uptake of 3H-proline; and (d) crystalline calcium deposits were found within cells and extracellularly. Ultrastructural analysis revealed that three of the eight cultures contained cells whose nuclei had inclusions which were almost identical to those found in the osteoclast nuclei of all patients with Paget's disease. The maintenance of cells derived from pagetic bone in long-term culture should aid in testing the hypothesis that Paget's disease represents a slow virus infection of bone.     

成人骨髓源成骨细胞体内异位成骨的实验研究

目的　观察成人骨髓源成骨细胞与珊瑚羟基磷灰石 (CHA)复合构建的组织工程化骨组织的体内异位成骨能力 ,探讨适宜的组织工程化骨组织的构建方式。　方法　抽取健康成人骨髓 ,采用全骨髓法培养 ,使成人骨髓基质干细胞 (hBMSCs)定向诱导分化为成骨细胞 ,然后种植于CHA上 ,复合培养 5d后植入裸鼠股部肌袋内 ,以未种植细胞的CHA作为对照。术后 4,8,12周取材作一般观察、X线摄片、组织学检查和源于成人的碱性磷酸酶 (ALP)及骨钙素 (OCN)的RT PCR检测。　结果　原代和传代培养的细胞具有活跃的增殖能力 ,成骨细胞与CHA复合生长良好。实验组术后 4、8、12周均有新骨形成 ,随着时间延长 ,新骨生成量增多 ;对照组则均无新骨形成。术后 4周实验组RT PCR检测源于人的ALP及OCN表达均为阳性 ,对照组阴性。　结论　成人骨髓源成骨细胞与CHA复合培养后构建的组织工程化骨组织 ,具有良好的异位成骨能力 ,可望应用于临床修复骨缺损。     

慢病毒介导骨形态发生蛋白-7基因的干细胞用于组织工程骨构建的实验研究

目的 建立能够稳定表达骨形态发生蛋白-7(BMP-7)的骨髓基质干细胞(BMSCs),观察其成骨分化,并与纳米羟基磷灰石胶原(nHAC)材料复合培养体外构建组织工程骨的可行性.方法 实验分4组:BMP-7和eGFI基因转染组(A组)、eGFP基因转染组(B组)、常规成骨诱导组(C组)、未转染组(D组).用G418筛选获得阳性细胞后接种于nHAC支架体外复合培养.荧光显微镜下观察eGFP表达,判断转染效率;以逆转录-聚合酶链反应(RTPCR)、ELISA检测目的基因表达,四唑盐(MTT)实验检测细胞活力,碱性磷酸酶(ALP)活性和Gomori染色榆测成骨情况;扫描电镜观察细胞在支架材料中的黏附、生长状况.结果 慢病毒24 h对BMSCs的转染率约为90%.RTPCR检测到A组在1300 bp处出现特异性条带,其他组结果阴性;ELISA检测24 h BMP-7蛋白含量为(150.2±18.3)pg/mL,种植支架复合培养8周后BMP-7含量为(76.6±7.4)pg/mL;MTT实验显示细胞活性与未转染组比较差异无统计学意义(P>0.05);ALP活性以16 d最强;扫描电镜见细胞种植nHAC支架后黏附、生长良好.结论 BMP-7可在BMSCs内稳定表达,并诱导其向成骨分化;与nHAC复合培养可构建组织工程骨.     

Differential proteome of bone marrow mesenchymal stem cells from osteoarthritis patients

OBJECTIVE: Adult mesenchymal stem cells (MSCs) are multipotent cells whose primary reservoir is bone marrow (BM). Following situations of extensive tissue damage, MSCs are mobilized and migrate to the site of injury. Osteoarthritis (OA) is a condition that involves extensive cartilage and bone damage. To gain insight into the pathogenesis of OA, we have analyzed the differential BM-MSCs proteome of OA patients. METHODS: MSCs protein extracts were prepared from BM aspirates from six patients with OA and from six hip fracture subjects without OA, and analyzed by Two-dimensional gels, using the differential in-gel electrophoresis approach. Differentially expressed proteins were identified by mass spectrometry. In addition, the chemotactic responses of OA and control MSCs were assessed. RESULTS: The majority of proteins that changed at least 1.5-fold (P<0.05) belonged to the following three categories: metabolic enzymes (14 proteins, 36%), cytoskeleton/motility (12 proteins, 32%), and transporters (three proteins, 8%). In OA MSCs, a high percentage of metabolic enzymes (n=8, 57%) were up-regulated and most of the proteins related to cytoskeleton/motility (n=9, 75%) were down-regulated. There was a significant increase in the migration response of OA MSCs to platelet-derived growth factor-BB (chemotaxis index CI: 5.13+/-1.19 vs 3.35+/-0.42, P=0.043). CONCLUSIONS: In this study, we have described the differential proteome of BM-MSCs from OA patients together with an increased chemotactic response of these cells in the context of OA. These results could indicate an activation of OA BM-MSCs in response to chemotactic signals sent by the altered subchondral bone in an attempt to heal damaged tissue.