不同干预时间正弦电磁场对大鼠骨髓基质干细胞成骨性分化的影响

目的:研究不同治疗时间正弦电磁场(50 Hz,1.8mT)对体外培养大鼠骨髓间充质干细胞成骨性分化的影响,筛选出最佳临床治疗时间.方法:采用贴壁筛选法培养原代大鼠骨髓间充质干细胞,每天在频率为50 Hz,强度为1.8 mT的磁场环境中处理0.5 h、1.0h、1.5 h、2.0h和2.5h;同时设立未经电磁场处理的细胞作为对照组.于处理后的第3 d、6d、9d和12 d分别测定细胞碱性磷酸酶活性、骨钙素分泌量、钙化结节数以及Ⅰ型胶原表达量,并比较各组间差异;于处理后12 h提取细胞总RNA,用RT Real-Time PCR法检测成骨性分化基因Osterix表达情况.结果:正弦电磁场干预1.0h能明显促进骨髓间充质干细胞的成骨性分化,表现在该组的碱性磷酸酶活性、骨钙素分泌量、钙化结节数、Ⅰ型胶原表达量以及成骨性分化基因的表达量最高,亦显著高于对照组(P＜0.05).结论:50Hz、1.8mT强度的正弦电磁场能促进骨髓间充质干细胞的成骨性分化,以作用1.0h成骨效果最为明显.     

50Hz正弦交变电磁场促进体外培养成骨细胞分化成熟的双“强度窗”效应

为研究频率为50 Hz不同强度正弦交变电磁场对体外培养成骨细胞分化及基因表达的影响,体外分离培养大鼠颅骨成骨细胞,传代后随机分为15组,分别用频率50 Hz、强度为0 mT(对照)和0.9～4.8 mT(每组间隔0.3 mT)的正弦交变电磁场处理。发现:正弦交变电磁场处理3～5 d后,成骨细胞呈漩涡样分布;第9 d,1.8和3.6 mT组的碱性磷酸酶活性极显著高于对照组;第0、12、24和96 h,1.5、1.8、3.0和3.6 mT组碱性磷酸酶、骨形态发生蛋白-2和Osterix基因表达水平显著高于对照组;第10 d,1.8和3.6 mT组钙化结节数明显多于对照组。说明50 Hz、0.9～4.8 mT的正弦交变电磁场能促进体外培养成骨细胞分化成熟,并且具有较明显的双强度窗效应,其中1.8和3.6 mT作用最为明显。     

50Hz 1．8mT电磁场对成骨细胞增殖与分化成熟影响的波形比较研究

在50 Hz 1.8 mT的4种不同波形电磁场(electromagnetic fields,EMFs)中筛选促进体外培养大鼠成骨细胞(rat osteoblasts,ROB)增殖与分化成熟的最佳波形.体外分离培养大鼠颅骨成骨细胞,传代后随机分为5组,分别用频率50 Hz,EMFs强度为0 mT(对照组)和1.8 mT的正弦波、三角波、方波和锯齿波处理ROB,30 min/(次.天).在磁场处理后4～8天细胞呈现特征样分布.方波促进成骨细胞增殖,正弦波抑制成骨细胞增殖.三角波和正弦波增加ALP活性,其中ALP染色、茜素红钙化结节染色和胶原Ⅰ(collagen-Ⅰ)免疫组织化学检测结果与ALP活性一致.在EMFs处理后的24 h、96 h和72 h后EMFs分别提高Runx-2、Opg和Igf基因表达水平,其中尤以正弦波和三角波作用最为显著.上述结果表明:50 Hz 1.8 mT方波促进成骨细胞增殖,正弦波抑制成骨细胞增殖.50 Hz 1.8 mT EMFs能促进体外培养成骨细胞分化成熟,其中尤以正弦波和三角波促进成骨细胞分化成熟作用最为显著.     

3种间充质干细胞成软骨分化潜能比较

目的:比较骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞3种间充质干细胞的成软骨分化潜能,为软骨组织工程中种子细胞的选择提供实验依据。方法:采用贴壁法分别分离提取兔骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞3种间充质干细胞,并进行传代培养,绘制3种间充质干细胞的生长曲线并比较其倍增时间。将3种间充质干细胞成软骨诱导14 d后,行甲苯胺蓝染色及II型胶原免疫组化染色以观测3种细胞成软骨分化能力。结果:脂肪间充质干细胞的倍增时间短于骨髓间充质干细胞,滑膜间充质干细胞的倍增时间最短;3种细胞成软骨诱导14 d后均产生糖胺聚糖和II型胶原,且组与组之间II型胶原表达水平的差异有统计学意义,骨髓间充质干细胞组高于脂肪间充质干细胞组(P0.01),滑膜间充质干细胞组高于骨髓间充质干细胞组(P0.01)。结论:在一定的培养条件下,3种间充质干细胞均有一定的成软骨细胞分化潜能,滑膜间充质干细胞最快的增殖速度及最强的成软骨分化潜能。     

丙二醛对小鼠骨髓间充质干细胞成骨分化的影响

为了探索氧化应激活性中间产物丙二醛对小鼠骨髓间充质干细胞成骨分化的影响及机制,体外培养的间充质干细胞经丙二醛处理后,在第4、7、14、21 d分别提取等份细胞进行碱性磷酸酶活性检测,第7 d时提取RNA通过实时定量RT-PCR测定ALP和Runx2/Cbfal mRNA表达,并在第21 d进行von Kossa染色,茜素红染色.研究发现:丙二醛通过降低碱性磷酸酶活性及ALP和Runx2/Cbfal mRNA的表达,抑制矿化骨节形成.这些结果表明:丙二醛可通过抑制ALP和Runx2/Cbfal通路,抑制小鼠骨髓间充质干细胞的成骨分化.     

不同强度50 Hz脉冲电磁场促进大鼠颅骨成骨细胞矿化成熟最佳参数的筛选

为研究不同强度脉冲电磁场(pulse electromagnetic fields,PEMFs)对大鼠颅骨成骨细胞（rat skull osteoblasts,OB）增殖及成熟矿化的影响,将大鼠颅骨成骨细胞随机分为 7 组. 检测大鼠颅骨成骨细胞的增殖,细胞内碱性磷酸酶（ALP）活性变化,细胞沉积钙盐的情况,组织化学染色以及成骨细胞内标志性分子表达量的改变.结果显示,0.6 mT组促细胞增殖作用最强（P <0.01）;0.6 mT、1.8 mT、3.0 mT和3.6 mT均能提高ALP活性,其中0.6 mT ALP活性最高（P＜0.01）;在磁场处理4 ～12 d时细胞沉积钙盐逐渐增加,6种强度的脉冲电磁场均能促进钙盐沉积,尤以0.6 mT水平最高; ALP 染色、茜素红染色0.6 mT 组均显著高于对照组（P<0.01）;0.6 mT组 Bmp-2和Collagen-1 mRNA 的表达明显（P<0.01）高于对照组,磁场处理组Rankl mRNA 的表达均比对照组低. 0.6 mT 50 Hz 脉冲电磁场是促进成骨细胞增殖和矿化成熟的最佳参数,这为采用脉冲电磁场治疗骨质疏松症提供了治疗参数的基础支持.     

正弦电磁场促进成骨细胞成熟分化依赖于BMP-Smad信号通路

正弦电磁场（SEMFs）能够显著促进大鼠成骨细胞（ROBs）成熟分化,但其作用机制未知。本研究旨在阐明BMP-Smad信号通路对SEMFs促进ROBs成熟分化的影响。取新生SD 大鼠颅骨,多次酶消化体外分离培养得到ROBs传代培养后,利用50 Hz 1.8 mTs SEMFs处理0,5,15,30和60 min,Western印迹检测BMP-2表达和Smad1/5/8磷酸化水平,免疫荧光染色检测P-Smad1/5/8核转位。加入BMP-Smad信号通路的阻断剂noggin后,50 Hz 1.8 mTs SEMFs分别处理3 d和6 d（2 h/d）后,检测胞内碱性磷酸酶（ALP）活性,磁场处理2 d后（2 h/d）,real-time PCR和Western印迹分别检测Ⅰ型胶原（collagen1）和成骨相关转录因子RUNX-2基因和蛋白质表达量。结果发现,50 Hz 1.8 mTs SEMFs处理ROBs后,BMP-2的表达量显著增加,胞内Smad1/5/8快速磷酸化,而对非磷酸化的Smad1/5/8表达无影响。同时,SEMFs处理30 min后引起P-Smad1/5/8发生核转位。50 Hz 1.8mTs SEMFs能够显著促进ALP活性增加,促进成骨相关因子collagen1和RUNX-2基因和蛋白质表达。加入BMP-Smad信号通路的阻断剂noggin后,SEMFs促进ALP活性增加,collagen1和RUNX-2基因和蛋白质表达水平被显著抑制。上述结果说明,50 Hz 1.8 mTs SEMFs促进成骨细胞成骨性分化依赖于BMP-Smad信号通路。     

龟板脂肪酸调控鼠骨髓间质干细胞增殖作用

为了解龟板浸膏中对鼠骨髓间质干细胞体外增殖起促进作用的化学成分,用石油醚提取促进鼠骨髓间充质干细胞增殖的龟板有效部位,用MTT比色法及流式细胞仪研究了提取物调控鼠骨髓间充质干细胞活性,采用GC-MS技术研究了石油醚提取物的化学成分。初步结果表明,石油醚提取物能明显促进干细胞增殖,其主要成分是脂肪酸、甾醇和甾酮,且十八烷酸、十六烷酸和甾酮能起调控鼠骨髓间充质干细胞活性。龟板浸膏中,脂肪酸起调控鼠骨髓间充质干细胞增殖作用,这为龟板浸膏促进鼠骨髓间充质干细胞增殖又不引起干细胞过度生长的分子机制提供实验依据,也为中医药调控干细胞的研究提供重要的参考。     

龟板脂肪酸调控鼠骨髓间质干细胞增殖作用

为了解龟板浸膏中对鼠骨髓间质干细胞体外增殖起促进作用的化学成分,用石油醚提取促进鼠骨髓间充质干细胞增殖的龟板有效部位,用MTT比色法及流式细胞仪研究了提取物调控鼠骨髓间充质干细胞活性,采用GC-MS技术研究了石油醚提取物的化学成分。初步结果表明,石油醚提取物能明显促进干细胞增殖,其主要成分是脂肪酸、甾醇和甾酮,且十八烷酸、十六烷酸和甾酮能起调控鼠骨髓间充质干细胞活性。龟板浸膏中,脂肪酸起调控鼠骨髓间充质干细胞增殖作用,这为龟板浸膏促进鼠骨髓间充质干细胞增殖又不引起干细胞过度生长的分子机制提供实验依据,也为中医药调控干细胞的研究提供重要的参考。     

骨髓间充质干细胞分化为心肌细胞过程中Notch表达的研究

目的研究骨髓间充质干细胞分化为心肌细胞过程中Notch表达的研究。方法用密度梯度离心法分离培养犬骨髓间充质干细胞,按照酶法及差速贴壁法分离培养心肌细胞。观察干细胞增殖及传代情况。单独培养的干细胞为对照组,实验组将骨髓间充质干细胞与心肌细胞共培养,用RT-PCR、免疫细胞化学、MTT等方法检测干细胞分化为心肌细胞的情况,及干细胞在增殖与分化为心肌细胞过程中Notch信号系统的表达情况。结果骨髓间充质干细胞呈梭形、旋涡样生长,增殖及传代能力强,并可诱导分化为心肌样细胞,免疫荧光示心肌细胞标志物的表达。RT-PCR及免疫细胞化学显示实验组有Notch信号通路受体及配体的表达,而对照组表达微弱。结论骨髓间充质干细胞在增殖及分化过程中存在Notch信号通路,在干细胞分化为心肌细胞过程中Notch信号系统的表达上调。     

The frequency window effect of sinusoidal electromagnetic fields in promoting osteogenic differentiation and bone formation involves extension of osteoblastic primary cilia and activation of protein kinase A

Electromagnetic fields (EMFs) have emerged as a versatile means for osteoporosis treatment and prevention. However, its optimal application parameters are still elusive. Here, we optimized the frequency parameter first by cell culture screening and then by animal experiment validation. Osteoblasts isolated from newborn rats (ROBs) were exposed 90 min/day to 1.8 mT SEMFs at different frequencies (ranging from 10 to 100 Hz, interval of 10 Hz). SEMFs of 1.8 mT inhibited ROB proliferation at 30, 40, 50, 60 Hz, but increased proliferation at 10, 70, 80 Hz. SEMFs of 10, 50, and 70 Hz promoted ROB osteogenic differentiation and mineralization as shown by alkaline phosphatase (ALP) activity, calcium content, and osteogenesis-related molecule expression analyses, with 50 Hz showing greater effects than 10 and 70 Hz. Treatment of young rats with 1.8 mT SEMFs at 10, 50, or 100 Hz for 2 months significantly increased whole-body bone mineral density (BMD) and femur microarchitecture, with the 50 Hz group showing the greatest effect. Furthermore, 1.8 mT SEMFs extended primary cilia lengths of ROBs and increased protein kinase A (PKA) activation also in a frequency-dependent manner, again with 50 Hz SEMFs showing the greatest effect. Pretreatment of ROBs with the PKA inhibitor KT5720 abolished the effects of SEMFs to increase primary cilia length and promote osteogenic differentiation/mineralization. These results indicate that 1.8 mT SEMFs have a frequency window effect in promoting osteogenic differentiation/mineralization in ROBs and bone formation in growing rats, which involve osteoblast primary cilia length extension and PKA activation.     

The Time-Dependent Manner of Sinusoidal Electromagnetic Fields on Rat Bone Marrow Mesenchymal Stem Cells Proliferation,Differentiation, and Mineralization

Electromagnetic fields (EMFs) are used clinically to promote fracture healing and slow down osteoporosis without knowledge of optimal parameters and underlying principles. In the present study, we investigate the effects of irritation for different durations with 15 Hz 1 mT sinusoidal EMFs (SEMFs) on rat bone marrow mesenchymal stem cells (BMSCs) proliferation, differentiation, and mineralization potentials. Our results show that SEMFs irritation promote rat BMSCs proliferation in a time-dependent manner, and the expression of osteogenic gen [Cbfa 1/RUNX2, bone sialoprotein (BSP), osteopontin (OPN)], alkaline phosphatase activity, and calcium deposition were enhanced after SEMFs treatment depending on the time duration of treatment. To determine the role of MEK/ERK signaling pathway, U0126, a MEK/ERK inhibitor was used. It can suppress rat BMSCs’ proliferation with or without SEMF exposure, and partly attenuate the expression of osteogenesis related proteins (RUNX2, BSP, OPN) which were improved by SEMF. This finding suggests that the effects of SEMF on rat BMSCs’ proliferation differentiation and mineralization are time duration dependent and MEK/ERK signaling pathway plays important role.     

Comparison of gingiva‐derived and bone marrow mesenchymal stem cells for osteogenesis

Presently, bone marrow is considered as a prime source of mesenchymal stem cells; however, there are some drawbacks and limitations. Compared with other mesenchymal stem cell (MSC) sources, gingiva‐derived mesenchymal stem cells (GMSCs) are abundant and easy to obtain through minimally invasive cell isolation techniques. In this study, MSCs derived from gingiva and bone marrow were isolated and cultured from mice. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometry. Compared with bone marrow MSCs (BMSCs), the proliferation capacity was judged by CCK‐8 proliferation assay. Osteogenic differentiation was assessed by ALP staining, ALP assay and Alizarin red staining. RT‐qPCR was performed for ALP, OCN, OSX and Runx2. The results indicated that GMSCs showed higher proliferative capacity than BMSCs. GMSCs turned more positive for ALP and formed a more number of mineralized nodules than BMSCs after osteogenic induction. RT‐qPCR revealed that the expression of ALP, OCN, OSX and Runx2 was significantly increased in the GMSCs compared with that in BMSCs. Moreover, it was found that the number of CD90‐positive cells in GMSCs elevated more than that of BMSCs during osteogenic induction. Taking these results together, it was indicated that GMSCs might be a promising source in the future bone tissue engineering.     

Uric Acid Promotes Osteogenic Differentiation and Inhibits Adipogenic Differentiation of Human Bone Mesenchymal Stem Cells

To investigate the effect of uric acid on the osteogenic and adipogenic differentiation of human bone mesenchymal stem cells (hBMSCs). The hBMSCs were isolated from bone marrow of six healthy donors. Cell morphology was observed by microscopy and cell surface markers (CD44 and CD34) of hBMSCs were analyzed by immunofluorescence. Cell morphology and immunofluorescence analysis showed that hBMSCs were successfully isolated from bone marrow. The number of hBMSCs in uric acid groups was higher than that in the control group on day 3, 4, and 5. Alizarin red staining showed that number of calcium nodules in uric acid groups was more than that of the control group. Oil red‐O staining showed that the number of red fat vacuoles decreased with the increased concentration of uric acid. In summary, uric acid could promote the proliferation and osteogenic differentiation of hBMSCs while inhibit adipogenic differentiation of hBMSCs.     

牙源性间充质干细胞对成骨前体细胞成骨分化的影响

目的:探讨牙源性间充质干细胞对成骨前体细胞成骨分化的影响。方法:将小鼠成骨前体细胞MC3T3-E1分为两组,观察组为牙源性间充质干细胞与MC3T3-E1细胞共培养,对照组为单一MC3T3-E1细胞培养。采用CCK-8法检测细胞增殖水平,采用酶联免疫法检测碱性磷酸酶(Alkaline phosphatase,ALP)活性并进行茜素红染色,采用qRT-PCR、Western blot检测ALP与骨桥素(osteopontin,OPN) m RNA与蛋白表达水平。结果:细胞共培养1 d与3 d后,观察组的细胞增殖指数、ALP活性显著高于对照组(P<0.05)。与对照组相比,观察组的矿化结节显著增加,经茜素红染色呈红褐色。细胞共培养1 d与3 d后,观察组的ALP、OPN m RNA与蛋白相对表达水平显著高于对照组(P<0.05)。结论:牙源性间充质干细胞能促进成骨前体细胞的ALP、OPN表达,提高ALP活性,增加细胞增殖能力,诱发矿化,从而促进成骨分化。     

阿司匹林对骨髓间充质干细胞移植治疗缺血性脑卒中的影响研究进展

阿司匹林是缺血性脑卒中患者急性期治疗药物及卒中再发的二级预防常用药物,骨髓间充质干细胞(BMSCs)移植是治疗缺血性脑血管疾病的新的新兴技术。已证实阿司匹林可抑制骨髓间充质干细胞的增殖及影响骨髓间充质干细胞的分化。本文就阿司匹林对骨髓间充质干细胞移植治疗缺血性脑卒中的影响等进行综述。     

二甲双胍调控糖尿病大鼠骨髓间充质干细胞的增殖和分化

虽然二甲双胍广泛用于治疗2型糖尿病,但是其对骨骼的潜在影响知之甚少。因此,本研究评估了二甲双胍对培养的大鼠骨髓间充质干细胞(MSCs)和脂肪细胞两者的分化以及增殖的影响。首先随机组形成对照实验,其中对照组为在不经二甲双胍处理培养基中培养MSCs细胞21 d,而二甲双胍组则在用100μmol/L二甲双胍处理培养基中培养MSCs 21 d。结果表明,二甲双胍增强了大鼠MSCs的成骨细胞分化细胞中ALP的活性,抑制了培养中MSCs脂肪形成分化的过程,但是增强了MSCs细胞的增殖能力。