应用脉冲电磁场刺激人骨髓间充质干细胞体外培养的生物学特性的研究

目的　应用脉冲电磁场 (PEMFs)刺激体外分离培养的人骨髓间充质干细胞 (hMSCs) ,对其生物学特性进行研究。方法　PEMFs (1 2Hz、 1 1mT、 8h/d)刺激hMSCs体外培养 ,计算细胞的倍增时间 ,观察细胞超微结构 ,放免法测定骨钙素的分泌情况和钙化结节VonKossa染色。结果　脉冲电磁场作用后的细胞增殖能力提高 ,细胞形态发生变化 ,透射电镜观察提示细胞成熟 ,VonKossa钙染色阳性 ,骨钙素的分泌活性增高。结论　hMSCs经合理的PEMFs体外刺激培养后 ,不仅能够大量扩增细胞 ,同时能够向成骨细胞转化 ,具有一定的成骨活性 ,是一种较为理想的骨种子细胞来源     

应用脉冲电磁场刺激人骨髓间充质干细胞体外培养的生物学特性的研究

目的应用脉冲电磁场(PEMFs)刺激体外分离培养的人骨髓间充质干细胞(hMSCs)，对其生物学特性进行研究。方法PEMFs(12Hz、1．1mT、8h／d)刺激hMSCs体外培养，计算细胞的倍增时间，观察细胞超微结构，放免法测定骨钙素的分泌情况和钙化结节Von Kossa染色。结果脉冲电磁场作用后的细胞增殖能力提高，细胞形态发生变化，透射电镜观察提示细胞成熟，Von Kossa钙染色阳性，骨钙素的分泌活性增高。结论hMSCs经合理的PEMFs体外刺激培养后，不仅能够大量扩增细胞，同时能够向成骨细胞转化，具有一定的成骨活性，是一种较为理想的骨种子细胞来源。     

脉冲电磁场对骨髓间充质干细胞作用研究进展

目的 旨在探究脉冲电磁场对骨髓间充质干细胞作用条件及机制,以促进其临床应用.方法 由第一作者应用计算机检索PubMed、中国期刊全文数据库(CNKI)、维普数据库和万方数据库1997-05/2012-03相关文献.在标题、摘要、关键词中以"pulsed electromagnetic field(PEMFs),bone marrow mesenchymal stem cells(BMMSCs),differentiation,proliferation"或"脉冲电磁场,骨髓间充质干细胞,分化,增殖"为检索词进行检索.选择文章内容与脉冲电磁场有关者,同一领域文献则选择近期发表在权威杂志文章.初检得到184篇文献,根据纳入标准选择41篇文献进行综述.结果 与结论 在一定条件下,脉冲电磁场对骨髓间充质干细胞的增殖及分化有促进作用,对于骨质疏松、骨不连等临床应用有一定意义.     

骨髓间充质干细胞及其诱导成骨的研究进展

间充质干细胞(MSCs)是一种多潜能成体干细胞,主要存在于骨髓,还存在于胚胎时期间充质来源的骨外组织[1],如皮肤成纤维细胞、脂肪干细胞、骨骼肌的卫星细胞和血管内皮细胞等。骨髓MSCs(BMSCs)是骨髓基质的组成成分,体外分离培养后,在一定的诱导条件下,BMSCs具有向成骨细胞、软骨细胞、神经细胞、脂肪细胞、心肌细胞等多向分化的能力[2-3],这些细胞经过20～30个培养周期,仍能保持多向分化潜能。无论是自体的还是同种异源的BMSCs,一般都不会引起宿主的免疫反应。BMSCs以其来源充足、取材简便、对供体损伤小、易于分离培养、体外增殖能…     

不同频率脉冲电磁场促人骨髓间充质干细胞增殖的实验研究

目的探求脉冲电磁场(pulsed electromagnetic fields,PEMF)促进人骨髓间充质干细胞(human mesenchymal stem cells,hMHCs)增殖的适合频率.方法选用场强1.1mT,频率分别为5、25、50、75、100、150Hz的PEMF刺激hMSCs,30min/d,光镜观察细胞形态,MTT法和流式细胞技术检测细胞增殖水平及生长周期.结果各频率组PEMF刺激均能促进MSCs增殖;频率不同,促增殖的效应不一,其中50Hz频段效果最为显著,生长曲线比对照组明显前移,S期细胞百分比(28.89±0.25)%较对照组(13.57±0.55)%增高107%.结论频率是PEMF促进hMSCs增殖的重要影响因素之一,50Hz为促进hMSCs增殖的适合频率.     

雌激素受体a介导小鼠骨髓间充质干细胞成骨分化

目的通过研究17β-雌二醇干预小鼠骨髓间充质干细胞(MSCs)体外成骨分化,揭示雌激素预防和治疗绝经后骨质疏松症(PMO)的机制。方法贴壁法分离培养小鼠的MSCs,并通过流式细胞仪鉴定其细胞表面标志物,同时体外诱导分化,鉴定MSCs多向分化潜能;免疫荧光、RT-PCR和Western Blot检测MSCs表达雌激素受体α和β(ERα和ERβ);在雌激素干预MSCs成骨诱导分化过程中,添加10-11~10-7M的17β雌二醇和/或ER拮抗剂ICI182,780和ERα选择性拮抗剂MPP,采用冯·科萨染色和定量钙离子测定方法,检测矿化结节数量及钙离子浓度。结果体外培养的小鼠MSCs表达ERα和ERβ。与对照组相比,17β雌二醇干预能显著增加矿化结节数量增加及钙离子浓度,ER拮抗剂ICI182,780和ERα选择拮抗剂MPP能逆转17β雌二醇的成骨诱导效应。结论 17β雌二醇主要通过ERα能提高MSCs体外成骨诱导分化,可能是雌激素替代治疗PMO的实验依据。     

体外负压培养对骨髓间充质干细胞成骨活性的影响

目的：探讨体外负压培养对骨髓间充质干细胞（bone marrow derived stroma cells,BMSCs）成骨活性的影响。方法：取第3代BMSCs分为实验组和对照组,实验组进行间歇性负压培养,设置压力为17kPa,每次30min,每日4次,干预2周;对照组于普通CO2培养箱中常规培养。倒置显微镜下观察细胞形态,检测ALP活性,免疫组织化学检测Ⅰ型胶原的表达,RT-PCR检测2周后以及终止负压后1、2、3d骨保护素（osteoprotegerin,OPG）mRNA和骨保护素配体（osteoprotegerin ligand,OPGL）mRNA表达水平。结果：诱导2周后,BMSCs呈现出显著的成骨细胞特性,与对照组比较,ALP活性显著增加,Ⅰ型胶原表达阳性,实验组细胞OPG mRNA表达水平显著提高,OPGL mRNA表达水平显著降低,且差异均有统计学意义（P〈0.05）。负压终止后3d,两组细胞OPG mRNA和OPGL mRNA表达水平差异无统计学意义（P〉0.05）。结论：体外负压培养可以提高BMSCs成骨活性。     

骨髓间充质干细胞成骨分化的研究与进展

超临界大面积或大段骨缺损仍是临床面临的难题,研究者们致力于研发人工骨材料,但为了解决人工骨材料成骨效应不良的问题,人们越来越关注骨髓间充质干细胞在骨组织工程中的应用。本综述以骨髓间充质干细胞、成骨分化、成骨细胞、临床应用"Bone marrow mesenchymal stem cells,osteogenesis differentiation,osteoblasts cell,clinical application"为检索词,应用计算机搜索CNKI数据库、万方数据库、维普数据库、PUBMED数据库,全面归纳总结骨髓间充质干细胞的分离培养、骨髓间充质干细胞鉴定方法、成骨诱导方法、成骨分化鉴定及其在临床中的应用,为证实其作为种子细胞治疗骨组织疾病提供理论依据。学者们初步研究了骨髓间充质干细胞结合移植、组织工程等技术来治疗骨和软骨缺损、骨关节炎、股骨头坏死等疾病,均取得了较好的临床疗效。但是骨髓间充质干细胞会有一定的优缺点,其临床试验及远期疗效的验证还需进一步深入研究。     

17β-雌二醇对大鼠骨髓间充质干细胞成骨分化的作用

目的观察17β-雌二醇（E2）对大鼠骨髓来源的间充质干细胞（MSCs）成骨分化的作用，探索雌激素替代治疗绝经后骨质疏松的实验机制。方法体外分离培养大鼠骨髓间充质干细胞，利用贴壁法纯化细胞。连续传代3次后，进行成骨细胞的诱导分化，将细胞分为4个实验组，分别添加含有0．13001—0．1nmol·L^-1 17β-E2的成骨细胞诱导分化液，同时设空白对照组。各组细胞培养第7天、14天和21天时利用放免法、酶联免疫吸附法分别测定碱性磷酸酶活性（alkaline phosphatase，ALP）、骨钙素（bone gla protein，BGP）以及Ⅰ型胶原含量，同时在培养21天时用茜素红染色法计数各组细胞的矿化结节数。结果与对照组相比，各实验组表达ALP活性、BGP与Ⅰ型胶原含量、形成矿化结节能力随17β-E2浓度的增加而增强，呈剂量依赖性。差异均有显著性，P〈0．05。结论17β-E2能增强MSCs体外诱导成骨分化，是17β-E2在绝经后骨质疏松预防和治疗中发挥作用的实验依据。     

Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation

Human mesenchymal stem cells (hMSCs) are a promising candidate cell type for regenerative medicine and tissue engineering applications by virtue of their capacity for self‐renewal and multipotent differentiation. Our intent was to characterize the effect of pulsed electromagnetic fields (PEMFs) on the proliferation and osteogenic differentiation of hMSCs in vitro. hMSCs isolated from the bone marrow of adult patients were cultured with osteogenic medium for up to 28 days and exposed to daily PEMF stimulation with single, narrow 300 µs quasi‐rectangular pulses with a repetition rate of 7.5 Hz. Relatively greater cell numbers were observed at late stages of osteogenic culture with PEMF exposure. The production of alkaline phosphatase (ALP), an early marker of osteogenesis, was significantly enhanced at day 7 with PEMF treatment in both basal and osteogenic cultures as compared to untreated controls. Furthermore, the expressions of other early osteogenic genes, including Runx2/Cbfa1 and ALP, were also partially modulated by PEMF exposure, indicating that osteogenesis in hMSCs was associated with the specific PEMF stimulation. Based on ALP and alizarin red S staining, the accumulation of ALP protein produced by the hMSCs as well as calcium deposits reached their highest levels at day 28. Our results indicate that extremely low‐frequency PEMF stimulation may play a modulating role in hMSC osteogenesis. Taken together, these findings provide insights on the development of PEMF as an effective technology for regenerative medicine. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res     

脉冲电磁场对MC3T3-E1细胞增殖与分化的影响

目的研究不同强度50Hz脉冲电磁场(Pulse electromagnetic fields,PEMFs)对小鼠成骨细胞系MC3T3-E1增殖与分化的影响。方法 MC3T3-E1传代培养后分成七组,分别采用0.0 m T、0.6 m T、1.2 m T、1.8 m T、2.4 m T、3.0 m T和3.6 m T 50 Hz脉冲电磁场处理,90 min/d。MTT法检测细胞增殖情况,成骨性分化检测处理3 d、6 d、9 d、12 d后ALP活性和首次处理12 h后BMP-2、Runx-2、OPG基因表达情况。结果 6种强度的脉冲电磁场均促进细胞增殖,其中0.6 m T的促增殖效应最强(P<0.01)。在成骨性诱导培养条件下,细胞内ALP活性随电磁场处理时间的延长而逐渐增加,第9 d达到峰值,之后开始回落。0.6 m T、3.0 m T和3.6 m T均能提高ALP活性,其中0.6 m T始终处于最高水平(P<0.01)。三种基因的表达水平在首次处理12 h,在0.6 m T、1.2 m T、3 m T和3.6 m T组均显著提高,但仍然是0.6 m T的增强效应最为明显。结论 0.6 m T 50 Hz脉冲电磁场具有最佳的促进MC3T3-E1细胞增殖和成骨性分化活性,这可能为采用脉冲电磁场治疗骨质疏松症提供了理想的治疗参数。     

Pulsed electromagnetic fields enhance BMP‐2 dependent osteoblastic differentiation of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) express an osteoblastic phenotype when treated with BMP‐2, and BMP‐2 is used clinically to induce bone formation although high doses are required. Pulsed electromagnetic fields (PEMF) also promote osteogenesis in vivo, in part through direct action on osteoblasts. We tested the hypothesis that PEMF enhances osteogenesis of MSCs in the presence of an inductive stimulus like BMP‐2. Confluent cultures of human MSCs were grown on calcium phosphate disks and were treated with osteogenic media (OM), OM containing 40 ng/mL rhBMP‐2, OM + PEMF (8 h/day), or OM + BMP‐2 + PEMF. MSCs demonstrated minor increases in alkaline phosphatase (ALP) during 24 days in culture and no change in osteocalcin. OM increased ALP and osteocalcin by day 6, but PEMF had no additional effect at any time. BMP‐2 was stimulatory over OM, and PEMF + BMP‐2 synergistically increased ALP and osteocalcin. PEMF also enhanced the effects of BMP‐2 on PGE2, latent and active TGF‐β1, and osteoprotegerin. Effects of PEMF on BMP‐2–treated cells were greatest at days 12 to 20. These results demonstrate that PEMF enhances osteogenic effects of BMP‐2 on MSCs cultured on calcium phosphate substrates, suggesting that PEMF will improve MSC response to BMP‐2 in vivo in a bone environment. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1250–1255, 2008     

人骨髓间质干细胞向表皮细胞分化的研究

目的探讨人骨髓间质干细胞（MSC）在体外培养条件下能否定向诱导分化为表皮细胞。方法抽取无造血系统恶性疾病的成人骨髓标本，采用Percoll细胞分离液（1．073g／ml）分离MSC，在体外传代培养至第3代，分为对照组和实验组。两组分别用普通L-DMEM培养基和含表皮生长因子、胰岛素、维甲酸、氯化钙的L-DMEM培养基诱导7d。在倒置相差显微镜下每天观察两组细胞的形态；采用免疫组织化学法检测P63和广谱细胞角蛋白（PCK）的表达情况。结果实验组细胞由长梭形逐渐转变为扁圆形或形态不规则，部分细胞P63和PCK呈阳性表达；对照组细胞持续呈长梭形，P63和PCK未见表达。结论人骨髓MSC在体外可诱导分化为表皮细胞。     

脉冲电磁场影响人骨髓基质干细胞缝隙连接通讯的实验研究

目的研究脉冲电磁场(PEMFs)对人骨髓基质干细胞(hBMSCs)缝隙连接所介导细胞间通讯(GJIC)的影响。方法透射电镜观察BMSCs超微结构,应用荧光漂白恢复(FRAP)技术,通过激光共聚焦显微镜检测hBM-SCs经PEMFs刺激后GJIC的功能变化。结果经PEMFs刺激后的hBMSCs,平均荧光漂白恢复率为(64.12±0.83)%,较对照组犤(35.26±0.76)%犦有显著性增加(P<0.05)。结论PEMFs刺激能促进hBMSCs的缝隙连接通讯功能。     

成骨细胞特异性钙黏蛋白基因转染对人骨髓基质干细胞成骨分化影响的研究

目的 观测成骨细胞特异性钙黏蛋白(Cad-Ⅱ)基因转染对人骨髓基质干细胞(hBMSCs)成骨分化的影响. 方法把脂质体介导的Cad-Ⅱ cDNA转染体外分离培养的hBMSCs,检测Cad-Ⅱ蛋白表达变化,并对比观测转染组和单纯成骨诱导组在培养3,7、14、21 d时,hBMSCs碱性磷酸酶(ALP)和骨钙索的表达变化. 结果 转染组和成骨诱导组3 d后开始有ALP的阳性染色,呈棕黑色,7 d开始有骨钙索的阳性染色并随培养时间延长逐渐增多,但各时间点转染组ALP浓度(P=0.008)和骨钙素染色阳性数(P=0.023)均显著高于单纯成骨诱导组.转染组和成骨诱导组从14 d后开始有红色的刚件染色矿化结节出现,结节数目随时间推移而增多. 结论 Cad-Ⅱ基因转染可促进hBMSCs向成骨细胞的分化.     

Demonstration of the presence of independent pre-osteoblastic and pre-adipocytic cell populations in bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSC) are defined as plastic-adherent, clonal cells that are common progenitors for osteoblasts and adipocytes. An inverse relationship between bone and fat has been observed in several clinical conditions and has been suggested to be caused by re-directing MSC differentiation into one particular lineage. However, this inverse relationship between bone and fat is not consistent and under certain in vivo conditions, bone and fat can change independently suggesting separate precursor cell populations. In order to test for this hypothesis, we extensively characterized two plastic-adherent clonal MSC lines (mMSC1 and mMSC2) derived from murine bone marrow. The two cell lines grew readily in culture and have undergone more than 100 population doublings with no apparent differences in their growth rates. Both cell lines were positive for the murine MSC marker Sca-1 and mMSC1 was also positive for CD13. Both cell lines were exposed to in vitro culture induction of osteogenesis and adipogenesis. mMSC1 and not mMSC2 were only able to differentiate to adipocytes evidenced by the expression of adipocyte markers (aP2, adiponectin, adipsin, PPARgamma2 and C/EBPa) and the presence of mature adipocytes visualized by Oil Red O staining. On the other hand, mMSC2 and not mMSC1 differentiated to osteoblast lineage as demonstrated by up-regulation of osteoblastic makers (CBFA1/RUNX2, Osterix, alkaline phosphatase, bone sialoprotein and osteopontin) and formation of alizarin red stained mineralized matrix in vitro. Consistent with the in vitro results, mMSC2 and not mMSC1, were able to form bone in vivo after subcutaneous implantation in immune-deficient (NOD/SCID) mice. Our data suggest that contrary to the current belief, bone marrow contains clonal subpopulations of cells that are committed to either osteoblast or adipocyte lineage. These cell populations may undergo independent changes during aging and in bone diseases and thus represent important targets for therapy.     

脉冲电磁场对接骨板性骨质疏松的影响

我们对钢板固定家兔胫骨用ＰＥＭＦｓ进行治疗 ,观察固定骨段的骨孔隙率 ,骨矿含量和矿化沉积率的变化 ,以期为临床应用ＰＥＭＦｓ防治接骨板性骨质疏松提供依据。1　材料与方法1 1　动物　健康成年大耳白家兔 18只 ,雌雄不限 ,体重在 2 5～ 3 0ｋｇ(河南医科大学实验动物中心提供 )。标准全价营养饲料分笼喂养。1 2　接骨板　采用天津产 4孔 316Ｌ不锈钢掌骨接骨板。1 3　仪器　ＴＦ ＶＩ型中医多功能治疗仪由郑州市颈肩腰腿痛医院研究所提供 (专利号 :992 385 4 2 3)。1 4　分组与处理　动物随机分为Ａ、Ｂ、Ｃ三组 ,每组各 6只。Ａ组为…