RNA干扰沉默PPARγ基因对小鼠骨髓基质干细胞成脂与成骨分化潜能的影响

目的 观察RNA干扰沉默PPAγ基因对小鼠骨髓基质干细胞成脂与成骨分化潜能的影响.方法 针对小鼠PPARγ基因,构建短发夹RNA(shRNA)真核表达载体并转染小鼠骨髓基质干细胞,逆转录-聚合酶链反应(RT-PCR)和免疫印迹沉淀(Wetem-blot)观察PPARγ的基因沉默效果.分别在成脂及成骨诱导培养基条件下,诱导培养14 d,采用油红O(Oil Red O)染色及茜素红染色鉴定分化结果.并与空载体转染组和未转染组比较.结果 测序证实成功构建PPARγ的shRNA真核表达载体pSilencer-shPPARγ,转染小鼠骨髓基质干细胞后,PPARγ基因表达在转录水平和翻译水平都显著受到抑制,与两对照组相比,抑制率分别为92%和90%.成脂及成骨诱导分化后,shPPARγ转染组小鼠骨髓基质干细胞的成骨分化率为56.46%±1.92%,明显高于pSilencer-neo空载体转染组和未转染组(P<0.01);成脂分化率为19.24%±0.98%,显著低于pSilencer-shPPARγ-neo空载体转染组和未转染组(P<0.01).结论 RNA干扰沉默PPARγ基因后,可增强小鼠骨髓基质干细胞的成骨分化潜能,抑制其成脂分化潜能.     

地塞米松和1，25(OH)2D3对体外人骨髓基质细胞成骨及成脂分化的影响

目的 观察地塞米松（Dex）和1，25（OH）2D3（D3）对骨髓基质细胞（MSCs）成骨及成脂分化的影响。方法 以离心法分离培养人MSCs，以10^-7mol／LDex和，或10^-8mol／Ll，25（OH）2D3作为分化诱导剂对细胞进行干预，分别用细胞碱性磷酸酶（ALP）染液试剂盒及苏丹Ⅲ染液对成骨细胞和脂肪细胞进行组织化学染色，计数；使用RT-PCR技术在转录水平检测成骨细胞标记物骨桥蛋白（OPN）及脂肪细胞标记物过氧化酶体增殖激活受体72（PPARγ2）mRNA的表达。结果细胞染色结果表明各干预组ALP^＋细胞百分比均较对照组增加，与对照组相比有显著性差异（P〈0．05），苏丹Ⅲ^＋细胞百分比Dex组较对照组增多，耽组较对照组减少，差异有显著性（P〈0．05），Dex＋D3组较Dex组苏丹Ⅲ^＋细胞数明显减少，两者相比差异有显著性（P〈0．05）；OPNmRNA及PPARγ2mRNA表达未在对照组测得，Dex诱导了OPNmRNA及PPARγ2mRNA表达，1，25（OH）2D3诱导OPNmRNA表达，并抑制Dex诱导的PPARγ2mRNA的表达。结论 Dex促进MSCs的成骨分化及成脂分化，1，25（OH），D，促进MSCs的成骨分化的同时抑制其成脂分化，与Dex合用抑制Dex成脂分化作用，强化了其成骨分化作用，反映了成骨细胞与脂肪细胞间存在的反变关系，表明两者来源于同一前体细胞的可能性。     

地塞米松对人骨髓间充质干细胞成脂分化的基因调控

目的观察地塞米松对人骨髓间充质干细胞（hBMSCs）内成脂转录因子PPARγmR—NA和成骨基因Osteocalcin mRNA表达的影响。方法取健康自愿者骨髓，通过梯度离心、贴壁分离培养获得hBMSCs。传代培养第2代hBMSCs8d，随机分为两组，实验组给予10^-7mol／L地塞米松，对照组不给予地塞米松，5d后收集细胞，采用逆转录．聚合酶链反应（RT-PCR）技术检测两组细胞中PPARγmRNA和Osteocalcin mRNA的表达。结果用地塞米松处理细胞5d，RT-PCR检测结果显示，实验组hBMSCs内PPARγmRNA呈高表达，对照组hBMSCs内PPARγmRNA呈低表达，两组差异有统计学意义（P〈0．01）。实验组hBMSCs内Osteocalcin mRNA呈低表达，对照组hBMSCs内Osteocalcin mRNA呈高表达，两组间差异有统计学意义（P〈0．01）。结论地塞米松能够调控hBMSCs内成脂转录因子高表达，而抑制其成骨表达，这可能与激素性骨坏死的发生机制有关。     

地塞米松对人骨髓基质干细胞成脂与成骨分化的影响的初步报告

目的探讨不同浓度的地塞米松对骨髓基质干细胞成脂与成骨分化的影响。方法取人骨髓分离培养骨髓基质干细胞,经传代后分别置入地塞米松1×10-8mol/L(A组)、1×10-7mol/L(B组),应用Rt-PCR技术分别扩增成脂基因mPNA(PPARγmRNA)、成骨基因mRNA(Osteocalcin mRNA)对扩增产物进行琼脂糖凝胶电泳。通过凝胶成像扫描系统做半定量分析,以PPARγmRNA和Osteocalcin mRNA与β-actin的吸光度比值表示上述产物mRNA的相对含量。结果对A组和B组通过凝胶成像扫描系统作PCR产物半定量分析,显示B组细胞中的Osteocalcin mRNA表达降低,而A组细胞中的表达增加。而且B组中的PPARγmRNA表达升高,A组降低。两者之间的差异均有统计学意义。结论地塞米松可以从分子水平调控骨髓基质干细胞的的分化。地塞米松浓度为1×10-7mol/L时,诱导骨髓基质干细胞向脂肪细胞分化,同时减少、抑制其向成骨细胞分化,这可能是激素骨坏死发生的机制之一。诱导体外培养的骨髓基质干细胞分化为成骨细胞,地塞米松浓度为1×10-8mol/L是比较适宜的。     

激活BMP信号的骨细胞对骨髓基质细胞成骨及成脂分化的作用研究

目的激活骨细胞系MLO-Y4细胞中BMP信号检测培养上清对骨髓基质细胞系ST2成骨、成脂分化的影响,进一步探讨其机制。方法 0.5‰DMSO,0.5μmol/L BMP激动剂FK506分别处理MLO-Y4 24 h后,使用CCK-8检测细胞活力变化,实时荧光定量PCR检测BMP下游靶基因ID1及ID2 mRNA表达变化;用20%MLO-Y4培养上清与80%新鲜培养基混合后培养ST2细胞,分为DMSO组、FK506组。碱性磷酸酶染色代表其成骨分化能力,通过实时荧光定量PCR检测碱性磷酸酶(ALP)、骨钙蛋白(OCN)、骨唾液酸蛋白(BSP)、Runx2等成骨细胞标志基因,过氧化物酶体增殖剂激活受体γ(PPARγ)和C/EBP成脂分化标志基因。免疫印迹试验(Western blotting)检测ST2细胞内Wnt信号下游β-catenin、BMP信号下游p-smad5蛋白表达水平。结果与DMSO作用的MLO-Y4细胞相比,FK506激动的MLOY4细胞内BMP信号靶基因ID1、ID2上调,但不影响细胞活力。FK506组ST2细胞同DMSO组对比,成骨分化相关标志物,包括ALP、OCN、BSP、Runx2(P0.001)均显著升高;成脂分化标志物PPARγ及C/EBP表达则显著降低(P0.001); ST2细胞内β-catenin蛋白表达量上调(P0.05)。结论 BMP信号激动后MLO-Y4细胞上清可以促进ST2细胞成骨分化、抑制成脂分化,其成骨能力增强与细胞内Wnt信号增强有关。     

酒精对人骨髓间充质干细胞成脂与成骨分化的影响初步报告

目的观察酒精对人骨髓间充质干细胞中成脂转录因子PPARγmRNA和成骨基因osteocalcin(骨钙素)mRNA表达的影响。方法取人骨髓分离培养间充质干细胞,经传代后以0.09mol/L的酒精浓度作为诱导剂处理细胞,应用Rt-PCR技术检测实验组和对照组细胞中PPARγmRNA和骨钙素的表达。结果对实验组和对照组通过凝胶成像扫描系统作PCR产物半定量分析,显示实验组细胞中的osteocalcin(骨钙素)mRNA表达降低,而对照组细胞中的表达增加。而且实验组中的PPARγmRNA表达增加,对照组降低,两者之间的差异均有统计学意义。结论酒精能诱导人骨髓间充质干细胞向脂肪细胞分化,而减少骨髓间充质干细胞向成骨分化,这可能与酒精性坏死的发生机制有关。     

Follistatin 表达抑制促进 BMP-2诱导人类骨髓间充质干细胞成骨分化的实验研究

目的探讨Follistatin表达抑制对人类骨髓间充质干细胞（BMSC）细胞活性及骨形态发生蛋白（BMP）‐2诱导人类BMSC成骨分化的影响。方法采用 Follistatin特异性 siRNA 转染人类BMSC ,检测线粒体脱氢酶活性、细胞DNA含量及蛋白含量。采用定量逆转录‐聚合酶链式反应（qRT‐PCR）检测成骨相关基因表达,采用碱性磷酸酶（ALP）染色和茜素红染色检测 Follistatin对BMP‐2诱导人类BMSC成骨分化的影响。结果转染后3、7 d ,人类BMSC代谢、DNA含量显著升高;转染后14 d ,总蛋白含量增加。Follistatin表达抑制后成骨相关基因如ALP、OCN、OSX、OC、OPN、RUNX2等表达升高,且ALP活性及钙沉积量显著增加。结论抑制Follistatin表达可促进人类BMSC细胞活性和BMP‐2诱导人类BMSC成骨分化能力,Follistatin可抑制人类BMSC成骨分化。     

辛伐他汀对老年大鼠骨髓基质干细胞成骨和成脂分化的影响

目的 从细胞和基因水平探讨辛伐他汀对老年大鼠骨髓基质干细胞成骨和成脂分化的影响.方法 18月龄雄性SD大鼠的骨髓基质干细胞进行成骨和成脂诱导培养,培养介质中加入辛伐他汀(10-6、10-7、10-8 mol/L和10-9 mol/L),同时设溶剂对照组.成骨检测:碱性磷酸酶(ALP)染色和定量,茜素红矿化染色和定量,ALP和骨钙素(OC)基因分析;成脂检测:油红脂肪细胞染色和定量,脂蛋白脂酶(LPL)和过氧化物酶增殖活化受体(PPARγ2) 基因分析.结果 在含有低剂量地塞米松的成骨诱导条件下,辛伐他汀随浓度增加促进了细胞基质的矿化,增强了ALP的活性及染色,提高了ALP和OC的基因表达(若无地塞米松,辛伐他汀则无法单独诱导成骨,此结果 未展示);同时,辛伐他汀随浓度增加减弱了脂肪细胞的油红染色,抑制了LPL 和PPARγ2的基因表达.显著性差异皆发生于10-6 mol/L和10-7 mol/L辛伐他汀组.结论 辛伐他汀随浓度增加抑制了老年骨髓基质干细胞的成脂分化,并中等强度地促进了老年骨髓基质干细胞的成骨分化.这表明辛伐他汀具有促进骨合成代谢的作用,可以用于治疗常见的骨代谢疾病,如增龄性的骨质疏松症.     

正弦电磁场对高糖环境下大鼠骨髓间充质干细胞分化的影响

目的探讨正弦电磁场对高糖环境下大鼠BMSCs分化的影响,并探讨其中可能的分子机制。方法贴壁筛选法分离、培养大鼠BMSCs,将第三代细胞分为暴磁组、高糖组、高糖暴磁组及对照组。高糖组采用高糖培养基(葡萄糖浓度为15 mmol/L),电磁场组每天给予1 mT、50 Hz正弦电磁场刺激2 h。刺激21 d后采用茜素红染色、油红O染色观察各组在成骨和成脂分化上的差异,刺激7 d后Real-time PCR检测成骨/成脂相关基因Runx2、ALP、Id4、PPARγ、aP2的表达变化。结果暴磁组钙结节形成能力较对照组增强、成骨相关基因表达增高,高糖组形成脂滴能力增强、成脂相关基因表达增高,高糖暴磁组成骨和成脂基因表达均有增高,但前者更为明显。结论正弦电磁场对高糖环境下的大鼠骨髓间充质干细胞有促进成骨、间接抑制成脂分化的作用,其机制可能与通过上调关键分子Id4有关。     

17-β雌二醇对人成骨样细胞系MG 63 Cbfa 1表达的影响

目的 研究17-β雌二醇对人成骨肉瘤MG 63细胞株Cbfa 1表达的影响,了解雌激素预防骨质疏松的机制中有无Cbfa 1 的参加.方法 17-β雌二醇10-mol/L、10-8mol/L、10-10 mol/L浓度干预人成骨肉瘤MG 63细胞株,24、48 h后抽提RNA、蛋白,半定量RT-PCR,Westem blot检测Cbfa 1 mRNA、蛋白表达的变化.结果 人成骨肉瘤MG 63细胞中有Cbfa 1 mRNA和蛋白的表达.17-β雌二醇10-6mol/L、10-8 mol/ L、10-10 mol/L浓度作用24或48 h,对MG 63细胞的Cbfa 1 mRNA和蛋白表达无影响.结论 人成骨肉瘤MG 63细胞株中有Cbfa 1表达,17-β雌二醇对MG 63细胞Cbfa 1基因的表达无影响.     

Dynamic expression of DKK1 protein in the process whereby Epimedium‐derived flavonoids up‐regulate osteogenic and down‐regulate adipogenic differentiation of bone marrow stromal cells in ovariectomized rats

Objective: To observe the dynamic expression of DKK1 protein in the process whereby Epimedium‐derived flavonoids (EFs) regulate the balance between osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats, and to provide experimental evidence for the mechanism of EFs in the treatment of postmenopausal osteoporosis. Methods: Bone marrow stromal cells from ovariectomized rats were separated and cultivated in osteoinductive or liquid medium for 15 days in vitro. EFs (10 µg/mL) were applied to both cultures. Alkaline phosphatase (ALP) staining, ALP activity determination, Oil Red O staining and fluorescence quantitative polymerase chain reaction were used to determine the influence of EFs on osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats. Moreover, in order to explore the exact mechanism of EFs on osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats, enzyme linked immunosorbent assay was used to determine the dynamic expression of DKK1 protein in this process. Results: EFs increased activity of ALP and mRNA expression of Runx2 (early osteoblast differentiation factor) and decreased mRNA expression of PPARγ‐2 (key factor of fat generation). Importantly, EFs down‐regulated expression of DKK1 protein in an osteogenic induction medium and inhibited up‐regulation of DKK1 protein in an adipogenic induction medium. Conclusion: EFs regulate the balance between osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats by down‐regulating expression of DKK1 protein. This may be an important molecular mechanism of EFs in the context of treatment of postmenopausal osteoporosis.     

Heparin affects human bone marrow stromal cell fate: Promoting osteogenic and reducing adipogenic differentiation and conversion

Heparins are broadly used for the prevention and treatment of thrombosis and embolism. Yet, osteoporosis is considered to be a severe side effect in up to one third of all patients on long-term treatment. However, the mechanisms underlying this clinical problem are only partially understood. To investigate if heparin affects differentiation of skeletal precursors, we examined the effects of heparin on the osteogenic and adipogenic lineage commitment and differentiation of primary human bone marrow stromal cells (hBMSCs). Due to the known inverse relationship between adipogenesis and osteogenesis and the capacity of pre-differentiated cells to convert into the respective other lineage, we also determined heparin effects on osteogenic conversion and adipogenic differentiation/conversion. Interestingly, heparin did not only significantly increase mRNA expression and enzyme activity of the osteogenic marker alkaline phosphatase (ALP), but it also promoted mineralization during osteogenic differentiation and conversion. Furthermore, the mRNA expression of the osteogenic marker bone morphogenic protein 4 (BMP4) was enhanced. In addition, heparin administration partly prevented adipogenic differentiation and conversion demonstrated by reduced lipid droplet formation along with a decreased expression of adipogenic markers. Moreover, luciferase reporter assays, inhibitor experiments and gene expression analyses revealed that heparin had putative permissive effects on osteogenic signaling via the BMP pathway and reduced the mRNA expression of the Wnt pathway inhibitors dickkopf 1 (DKK1) and sclerostin (SOST). Taken together, our data show a rather supportive than inhibitory effect of heparin on osteogenic hBMSC differentiation and conversion in vitro. Further studies will have to investigate the net effects of heparin administration on bone formation versus bone resorption in vivo to unravel the molecular mechanisms of heparin-associated osteoporosis and reconcile conflicting experimental data with clinical observations.     

从少火生气理论探讨肾气丸促巨噬细胞M2型极化影响成骨的作用

目的 基于少火生气理论探讨肾气丸原组方（SQW）和肾气丸去除桂枝、附子（SQQ）诱导巨噬细胞M2型极化对小鼠骨髓间充质干细胞（BMSC）成骨分化作用的效果差异。方法 采用CCK8法分别检测SQW、SQQ对RAW264.7细胞存活率的影响，以确定药物干预的安全作用浓度。将RAW264.7细胞分为CON组、IL-4组、IL-4+SQW组、IL-4+SQQ组。将不同药物组诱导巨噬细胞M2型极化后分泌的细胞上清液作为条件培养基培养BMSC，评估BMSC成骨分化的能力。用碱性磷酸酶染色法（ALP）及茜素红染色法（ARS）检测各组BMSC成骨分化能力；使用免疫荧光技术（IF）检测BMSC成骨标志蛋白骨形态发生蛋白4（BMP4）、骨保护素（OPG）的分布和表达；使用蛋白免疫印迹法（Western Blot）检测M2型巨噬细胞极化相关的精氨酸酶1（ARG1）、白细胞介素10（IL-10）的表达以及BMSC成骨相关的BMP4、RUNT相关转录因子2（RUNX2）的表达。结果 与IL-4+SQQ组相比，IL-4+SQW组更加显著促进M2型巨噬细胞极化相关因子ARG1、IL-10上调（P＜0.01），且其条件培养基培养的BMSC的ALP及ARS染色增强，成骨蛋白BMP4、OPG、RUNX2表达升高（P＜0.05）。结论 肾气丸原组方与肾气丸去除桂枝、附子两味药相比，具备更强的促进巨噬细胞M2型极化与增强BMSC成骨分化能力，为少火生气理论提供了实验依据。     

淫羊藿在MSCs成脂分化过程中对PPARγ基因表达影响的实验研究进展

骨髓基质干细胞具有多向分化的能力,研究发现其成脂分化与骨质疏松症存在着不可忽视的关系.PPAR γ是脂肪分化的主要调控因子,已有研究发现其对MSCs分化方向起关键性调控作用.淫羊藿对MSCs成骨分化具有促进作用,但其在MSCs成脂分化过程中对PPAR γ基因表达影响目前尚无研究报道.     

Differentiation of human marrow stromal precursor cells: bone morphogenetic protein-2 increases OSF2/CBFA1, enhances osteoblast commitment, and inhibits late adipocyte maturation.

Because regulation of the differentiation to osteoblasts and adipocytes from a common progenitor in bone marrow stroma is poorly understood, we assessed effects of bone morphogenetic protein-2 (BMP-2) on a conditionally immortalized human marrow stromal cell line, hMS(2-6), which is capable of differentiation to either lineage. BMP-2 did not affect hMS(2-6) cell proliferation but enhanced osteoblast differentiation as assessed by a 1.8-fold increase in expression of OSF2/CBFA1 (a gene involved in commitment to the osteoblast pathway), by increased mRNA expression and protein secretion for alkaline phosphatase (ALP), type I procollagen and osteocalcin (OC) (except for OC protein), and by increased mineralized nodule formation. Transient transfection with Osf2/Cbfa1 antisense oligonucleotide substantially reduced BMP-2-stimulated expression of ALP mRNA and protein. The effects of BMP-2 on adipocyte differentiation varied: expression of peroxisome proliferator-activated receptor gamma2 (a gene involved in commitment to the adipocyte pathway) was unchanged, mRNA expression of the early differentiation marker, lipoprotein lipase, was increased, and mRNA and protein levels of the late differentiation marker, leptin, and the formation of cytoplasmic lipid droplets were decreased. Thus, by enhancing osteoblast commitment and by inhibiting late adipocyte maturation, BMP-2 acts to shunt uncommitted marrow stromal precursor cells from the adipocyte to the osteoblast differentiation pathway.