Cbfa1对骨髓间充质干细胞分化的定向调控研究

目的研究Cbfa1调控间充质干细胞(MSCs)分化为成骨细胞的分子机制。方法用Percoll密度梯度离心法分离获得人、大鼠和小鼠的MSCs,通过苏木精-伊红染色、细胞化学、免疫细胞化学、免疫荧光及透射电镜等方法进行鉴定。传代培养后的MSCs分别用重组真核表达载体pcD-NA3.1(+)/Cbfa1-II通过脂质体系统转染,用NorthernBlot检测三种成骨细胞特异性细胞外基质蛋白mRNA的表达情况。结果转染后人、大鼠和小鼠的MSCs都有骨钙素、骨桥接蛋白和I型胶原的表达,三者间无明显差别。结论Cbfa1在转录水平定向调控间充质干细胞分化为成骨细胞,可应用于骨科疾患的基因治疗。     

Cbfal定向调控骨髓间充质干细胞向成骨细胞分化的实验研究

目的 研究核心结合因子(Cbfal)调控骨髓问充质干细胞(MSCs)分化为成骨细胞的分子机制。方法 用Percoll密度梯度离心法分离获得人、大鼠和小鼠的MSCs，通过HE、细胞化学、免疫细胞化学、免疫荧光及透射电镜等方法进行鉴定。传代培养后的MSCs分别用重组真核表达载体pcDNA3．1( )／Cbfal-Ⅱ通过脂质体系统转染，用Northern Blot检测三种成骨细胞特异性细胞外基质蛋白mRNA的表达情况。结果 转染后人、大鼠和小鼠的MSCs都有骨钙素、骨桥接蛋白和Ⅰ型胶原的表达，三者间无明显差别。结论 Cbfal在转录水平定向调控间充质干细胞分化为成骨细胞，可应用于骨科疾患的基因治疗。     

Cbfal对骨髓间充质干细胞分化的定向调控研究

目的 研究Cbfal调控间充质干细胞(MSCs)分化为成骨细胞的分子机制。方法 用Percoll密度梯度离心法分离获得人、大鼠和小鼠的MSCs，通过苏木精-伊红染色、细胞化学、免疫细胞化学、免疫荧光及透射电镜等方法进行鉴定。传代培养后的MSCs分别用重组真核表达载体pcD-NA3．1( )／Cbfal-Ⅱ通过脂质体系统转染，用Northern Blot检测三种成骨细胞特异性细胞外基质蛋白mRNA的表达情况。结果 转染后人、大鼠和小鼠的MSCs都有骨钙素、骨桥接蛋白和Ⅰ型胶原的表达，三间无明显差别。结论 Cbfal在转录水平定向调控间充质干细胞分化为成骨细胞．可应用于骨科疾患的基因治疗。     

Osf2／Cbfal对间充质干细胞的促成骨效应

目的：观察重组腺病毒介导Osf2／cbfal对间充质干细胞(MSCs)的促成骨效应。方法：分离培养兔MSCs，用重组Osf2／cbfal腺病毒感染MSCs，蛋白印迹检测目的基因表达，对硝基苯磷酸二钠法和放免法分别检测成骨细胞标志基因碱性磷酸酶(ALP)和骨钙素表达。结果：感染后MSCs检测到Osf2／cbfal表达，从第4天开始。ATP活性和骨钙素含量较单纯诱导组和对照组显著增高。结论：Osf2／Cbfal可明显促进MSCs向成骨细胞定向分化。     

Osf2/Cbfa1对间充质干细胞的促成骨效应

目的:观察重组腺病毒介导Osf2/Cbfa1对间充质干细胞(MSCs)的促成骨效应。方法:分离培养兔MSCs,用重组Osf2/Cbfa1腺病毒感染MSCs,蛋白印迹检测目的基因表达,对硝基苯磷酸二钠法和放免法分别检测成骨细胞标志基因碱性磷酸酶(ALP)和骨钙素表达。结果:感染后MSCs检测到Osf2/Cbfa1表达,从第4天开始,ATP活性和骨钙素含量较单纯诱导组和对照组显著增高。结论:Osf2/Cbfa1可明显促进MSCs向成骨细胞定向分化。     

脐血与骨髓间充质干细胞的成骨细胞定向诱导及其成骨活性比较

目的:研究脐血与成人骨髓间充质干细胞(mesenchymalstemcells,MSCs)的定向分化为成骨细胞的条件及其成骨活性,比较两种不同MSCs定向成骨细胞诱导后的成骨活性。方法:采用标准Ficoll-Hypaque技术分离脐血和成人骨髓MSCs,以地塞米松、β-磷酸甘油和维生素C为辅剂定向诱导成骨细胞,碱性磷酸酶、骨矿化结节和I型胶原作为成骨细胞鉴定与活性评价的指标。结果:两种MSCs的细胞形态和生物学特性无明显差异。定向成骨细胞诱导后,成骨细胞标志性产物碱性磷酸酶、骨钙素、上清钙、骨矿化结节和I型胶原均得到了稳定的表达,并显著高于未诱导MSCs,而且两种细胞的成骨活性差异无显著性意义。结论:脐血和成人骨髓MSCs在适当的条件下均可向成骨细胞定向转化,两种细胞均可作为骨组织工程的种子细胞。     

脐血与骨髓间充质干细胞的成骨细胞定向诱导及其成骨活性比较

目的：研究脐血与成人骨髓间充质干细胞(mesenchymal stem ceus，Mscs)的定向分化为成骨细胞的条件及其成骨活性，比较两种不同Mscs定向成骨细胞诱导后的成骨活性。方法：采用标准Ficou-Hypque技术分离脐血和成人骨髓Mscs，以地塞米松、β-磷酸甘油和维生素C为辅剂定向诱导成骨细胞，碱性磷酸酶、骨矿化结节和Ⅰ型胶原作为成骨细胞鉴定与活性评价的指标。结果：两种MSCs的细胞形态和生物学特性无明显差异。定向成骨细胞诱导后，成骨细胞标志性产物碱性磷酸酶、骨钙素、上清钙、骨矿化结节和Ⅰ型胶原均得到了稳定的表达，并显高于未诱导MSCs，而且两种细胞的成骨活性差异无显性意义。结论：脐血和成人骨髓MSCs在适当的条件下均可向成骨细胞定向转化，两种细胞均可作为骨组织工程的种子细胞。     

骨折愈合刺激素(金葡液)对体外培养成骨细胞的影响

目的观察骨折愈合刺激素(金葡液)对体外培养成骨细胞的作用。方法在新生SD大鼠头颅骨第二继代成骨细胞(OB2)培养液中分别加入不同浓度(20000～200U/L)骨折愈合刺激素(金葡液),分别观察OB2的增殖功能(用波长570nm处OD值表示)、分化功能犤用碱性磷酸酶(ALP)活性表示犦和矿化功能(用矿化结节数量/视野表示)。结果OD值实验组为(0.336±0.073)～(0.359±0.051),对照组为0.347±0.035;ALP(U/g蛋白质)活性实验组为83±9,对照组为81±4;矿化结节数量/视野(个)实验组为6.000±1.826,对照组为1.5±1.0。结论骨折愈合刺激素(金葡液)各浓度对OB2的增殖和分化功能均无明显作用(P>0.05),而对其矿化功能具有明显的刺激作用(P<0.01)。     

兔骨髓间充质干细胞向成骨分化对血管内皮细胞形成的可能影响

背景：种子细胞的成骨功能及局部血管形成是骨组织工程修复重要影响因素。目的：体外研究兔骨髓间充质干细胞(mesenchymal stem cells，MSCs)诱导培养分化为成骨细胞的功能表现及血管内皮生长因子(vascular endothelia gmwth factor，VEGF)的表达。探讨MSCs向成骨分化及对血管内皮细胞形成的可能影响。设计：以细胞为研究对象，单一样本研究，重复观察测量。单位：一所大学组织胚胎学教研室。材料：本实验于2002-03／12在广东医学院组织胚胎学教研室完成。对象为兔骨髓间充质干细胞。方法：分离、培养新生兔骨髓间充质干细胞，用含地塞米松、β-甘油磷酸钠和维生素C的条件培养液诱导MSCs向成骨细胞分化，观察诱导细胞碱性磷酸酶(ALP)染色、钙化结节形成以及VEGF的表达。主要观察指标：①细胞形态学观察。②培养细胞的成骨分化鉴定。③诱导培养的成骨细胞VEGF表达。结果：MSCs诱导培养后，细胞ALP呈强阳性染色，形成矿化结节，且VEGF表达增强，其灰度值为132．3&;#177;4．6，与MSCs的VEGF表达灰度值(148．5&;#177;5．3)比较，差异有显著性意义(P&;lt;0．05)。结论：兔MSCs经诱导可分化为成骨细胞，且VEGF表达增强，可能参与内皮细胞的形成。     

兔骨髓间充质干细胞向成骨分化对血管内皮细胞形成的可能影响

背景种子细胞的成骨功能及局部血管形成是骨组织工程修复重要影响因素.目的体外研究兔骨髓间充质干细胞(mesenchymal stem cells,MSCs)诱导培养分化为成骨细胞的功能表现及血管内皮生长因子(vascular endothelia growthfactor,VEGF)的表达.探讨MSCs向成骨分化及对血管内皮细胞形成的可能影响.设计以细胞为研究对象,单一样本研究,重复观察测量.单位一所大学组织胚胎学教研室.材料本实验于2002-03/12在广东医学院组织胚胎学教研室完成.对象为兔骨髓间充质干细胞.方法分离、培养新生兔骨髓间充质干细胞,用含地塞米松、β-甘油磷酸钠和维生素C的条件培养液诱导MSCs向成骨细胞分化,观察诱导细胞碱性磷酸酶(ALP)染色、钙化结节形成以及VEGF的表达.主要观察指标①细胞形态学观察.②培养细胞的成骨分化鉴定.③诱导培养的成骨细胞VEGF表达.结果MSCs诱导培养后,细胞ALP呈强阳性染色,形成矿化结节,且VEGF表达增强,其灰度值为132.3±4.6,与MSCs的VEGF表达灰度值(148.5±5.3)比较,差异有显著性意义(P＜0.05).结论兔MSCs经诱导可分化为成骨细胞,且VEGF表达增强,可能参与内皮细胞的形成.     

人骨形态发生蛋白质7基因转染兔骨髓基质干细胞向成骨细胞的分化

背景骨形态发生蛋白质是目前发现惟一的一类具有成骨能力的细胞因子,它能刺激基质干细胞向成软骨细胞和成骨细胞方向分化.目的应用基因工程方法,观察人骨形态发生蛋白质7基因转染兔骨髓基质干细胞向成骨细胞的定向分化.设计单一样本观察.单位中山大学附属第一医院骨科. 材料pcDNA1.1/AMP-hBMP7质粒.方法实验于2004-05/2005-03在中山大学附属第一医院外科实验室完成.全骨髓培养法培养兔骨髓基质干细胞,在体外分别转染pcD-NA1.1/AMP-hBMP7和pcDNA1.1/AMP并留置空白对照.检测转染基因的转录和表达,观察细胞形态和生长情况,通过对感染细胞的碱性磷酸酶染色、钙盐染色、电镜观察胞质中钙质成分以及检测骨钙素鉴定其成骨分化表型.主要观察指标①兔骨髓基质干细胞的形态及生长.②表达产物的鉴定.③碱性磷酸酶染色(钙钴法)结果.④茜素红染色结果.⑤透射电镜观察结果.⑥扫描电镜观察结果.⑦骨钙素的测定结果.结果人骨形态发生蛋白质7基因存在于转染后的骨髓基质干细胞中并表达相应的mRNA.目的基因表达后的细胞形态略有变化,但生长曲线与未转染组无明显变化.骨钙素表达较未转染组明显增强.转染组细胞的碱性磷酸酶染色和钙盐染色均为阳性.结论人骨形态发生蛋白质7基因转染可促进兔骨髓基质干细胞向成骨细胞表型分化.     

脉冲电磁场诱导人骨髓间充质干细胞向成骨细胞分化的研究

目的 观察脉冲电磁场(PEMFs)对人骨髓间充质干细胞(hMSCs)向成骨细胞分化的影响。方法 分离并培养hMSCs，取第3代细胞，分为对照组与处理组，处理组细胞接受频率12Hz、场强1．1mT的PEMFs刺激，每日8h；对照组细胞不接受PEMFs刺激。应用透射电镜观察2组细胞超微结构，采用碱性磷酸酶染色、骨钙素免疫组织化学染色、四环素荧光标记等方法进行观测。结果 细胞培养5～6d即可传代，经PEMFs连续刺激3d后，hMSCs细胞形态发生变化，3周后聚集成细胞钙化结节。透射电镜下观察显示：细胞比较成熟，内质网扩张，其碱性磷酸酶染色、骨钙素免疫组织化学染色和四环素荧光标记均为阳性。结论 hMSCs经特定频率和场强的PEMFs体外刺激后，可向成骨细胞分化，符合成骨细胞的形态学和生物学特性，为体外构建组织工程化骨提供自体种子细胞。     

Enhancement of osteoblastic differentiation of mesenchymal stromal cells cultured by selective combination of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2)

It is well known that bone marrow contains mesenchymal stromal cells (MSCs), which can show osteoblastic differentiation when cultured in osteogenic medium containing ascorbic acid, beta-glycerophosphate and dexamethasone. The differentiation results in the appearance of osteoblasts, together with the formation of bone matrix; thus, in vitro cultured bone (osteoblasts/bone matrix) could be fabricated by MSC culture. This type of cultured bone has already been used in clinical cases involving orthopaedic problems. To improve the therapeutic effect of the cultured bone, we investigated the culture conditions that contributed to extensive osteoblastic differentiation. Rat bone marrow was primarily cultured to expand the number of MSCs and further cultured in osteogenic medium for 12 days. The culture was also conducted in a medium supplemented with either bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor (FGF-2), or with sequential combinations of both supplements. Among them, the sequential supplementation of FGF-2 followed by BMP-2 showed high alkaline phosphatase activity, sufficient bone-specific osteocalcein expression and abundant bone matrix formation of the MSC culture. These data implied that the number of responding cells or immature osteoblasts was increased by the supplementation of FGF-2 in the early phase of the culture and that these cells can show osteoblastic differentiation, of which capability was augmented by BMP-2 in the late phase. The sequential supplementation of these cytokines into MSC culture might be suitable for the fabrication of ideal cultured bone for use in bone tissue engineering.     

脂质体介导的rhIGF-1基因对成骨细胞增殖分化的影响

目的探讨重组人胰岛素生长因子（rhIGF-1）基因对体外培养大鼠成骨细胞增殖分化的影响。方法采用酶消化法获取大鼠乳鼠颅骨成骨细胞，传至第3代进行成骨细胞鉴定，钙化结节用茜素红（ARS）染色，钙钴法显示成骨细胞中碱性磷酸酶（ALP）的表达。用脂质体法将pcDNA3．1-rhIGF-1质粒转染成骨细胞。倒置显微镜下观察细胞的生长形态，MTT法检测成骨细胞增殖情况，碱性磷酸酶及骨钙素（OCN）测定成骨细胞活性。结果建立了稳定的大鼠成骨细胞模型；pcDNA3．1-rhIGF-1质粒转染组与对照组及空质粒pcDNA3．1组比较成骨细胞增殖旺盛、ALP和OGN的分泌增加，有显著性差异（P〈0．01）。结论外源性rhIGF-1基因能够刺激大鼠成骨细胞增殖、分化，促进骨形成。     

Gene transfer of the Runx2 transcription factor enhances osteogenic activity of bone marrow stromal cells in vitro and in vivo.

Marrow stromal cells (MSCs) have the potential to differentiate into multiple mesenchymal cell types. To harness the power of MSCs for bone regeneration, methods must be developed to direct their differentiation selectively to the osteoblast lineage. The objective of this study was to examine the feasibility of using ex vivo Runx2 gene transfer to enhance the osteogenic activity of MSCs. Primary MSCs isolated from C57BL6 mice were transduced with adenoviral vectors encoding beta-galactosidase or Runx2. Cells transduced with Ad-Runx2 expressed Runx2 protein and underwent osteoblast differentiation as measured by increases in alkaline phosphatase activity and mineralization. Time-course studies revealed that Runx2 protein was highest 1 day after transduction and declined below the limits of detection by 15 days. Osteoblast marker mRNA expression paralleled Runx2 levels. In contrast, Runx2-dependent mineralization persisted for the duration of the experiment. To assess in vivo osteogenic activity, Ad-Runx2-transduced and control MSCs were adsorbed to two different carrier scaffolds and subcutaneously implanted into C57BL6 mice. In both cases, MSCs expressing Runx2 formed substantially more bone than cells transduced with control virus. Taken together, these studies indicate that Runx2 gene transfer may be an effective route to enhance the osteogenic potential of MSCs.     

pcDNA3-hBMP2转染兔骨髓基质细胞的体外表达及体内成骨能力

背景：能否通过病毒或者非病毒载体将骨形态发生蛋白2转导到骨髓基质细胞发挥成骨作用？目的：观察真核表达载体pcDNA3-hBMP2转染兔骨髓基质细胞后体外表达，同时将MSC转染后但未筛选兔骨髓基质细胞自体回植入肌组织，利用X线观察其成骨情况。设计、时间及地点：观察对比实验。实验于2004—11／2005-04在辽宁医学院骨科研究室完成。材料：6只成年新西兰大白兔，雌雄不拘，体质量2．0～3．0kg。BMP2抗体为美国Sanaka公司产品，pcDNA3-hBMP2由解放军第四军医大学生化教研室蒲勤教授提供，限制性内切酶购于大连宝生物工程有限公司。方法：从大肠杆菌提取超纯质粒pcDNA3-hBMP2，从成年兔股骨抽取骨髓，密度梯度分离法分离培养骨髓基质干细胞，将细胞分成4组；A组：pcDNA3-hBMP2转染进行G418筛选；B组：pcDNA3-hBMP2转染未用G418筛选；C组：给予pcDNA3空载体转染；D组：仅加入脂质体转染试剂Fugene 6。主要观察指标：①应用免疫组织化学法检测转染后瞬时表达。②应用免疫组织化学法检测细胞骨钙素表达，分别应用原位杂交法检测细胞Ⅰ型胶原表达。③转染2周后将B组细胞自体回植入兔后腿肌组织中，移植4周后应用X射线观察成骨情况。结果：①pcDNA3-hBMP2成功转染入骨髓基质干细胞内并100％瞬间表达BMP2。②基因转染4周后，A组细胞骨钙素及Ⅰ型胶原表达高于C组及D组。③B组细胞回植肌组织4周后，X射线可显示新骨形成。结论：pcDNA3-hBMP2能安全有效转染兔骨髓基质干细胞，通过其分泌物BMP2来作用诱导细胞加速分化为成骨细胞。     

Bone morphogenetic protein-2 in biodegradable gelatin and β-tricalcium phosphate sponges enhances the in vivo bone-forming capability of bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) have been used for bone tissue engineering due to their osteogenic differentiation capability, but their application is controversial. To enhance their capability, we prepared biodegradable gelatin sponges incorporating β-tricalcium phosphate ceramics (GT sponge), which has been shown to possess excellent controlled drug-release properties. The GT sponge was used as a carrier for both rat MSCs and bone morphogenetic protein-2 (BMP-2) and osteogenic differentiation was assessed by subcutaneous implantation of four different kinds of implants, i.e. GT-alone, MSC-GT composites, BMP-GT composites and BMP-GT composites supplemented with MSCs (BMP-MSC-GT) in rats. Two weeks after implantation, histological sections showed new bone formation in the peripheral parts of the BMP-GT and in almost the total volume of the BMP-MSC-GT implants. After 4 weeks, histology as well as microCT analyses demonstrated extensive bone formation in BMP-MSC-GT implants. Gene expression and biochemical analyses of both alkaline phosphatase and bone-specific osteocalcin confirmed the histological findings. These results indicate that the combination of MSCs, GT and BMP synergistically enhances osteogenic capability and provides a rational basis for their clinical application in bone reconstruction.     

Conditioned media cause increases in select osteogenic and adipogenic differentiation markers in mesenchymal stem cell cultures

The optimal mesenchymal stem cell (MSC) culture conditions that would allow clinically viable tissue-engineered devices are still yet to be determined. Most MSCs are found in the bone marrow, an area that also contains numerous osteoblasts and adipocytes. Paracrine signalling may be leveraged to modulate MSC differentiation in the preparation of a tissue-engineered device. Thus, the objectives of this study were to determine the effects of adipocyte-conditioned medium (CM) on MSC differentiation to osteoblasts and to determine the effects of osteoblast CM on MSC differentiation to adipocytes. Two groups of murine MSCs were given either an osteogenic differentiation medium or an adipogenic differentiation medium. CM was taken from one group and administered to the opposite group in concentrations of 25% or 50%. Metabolic activity, total protein and alkaline phosphatase (ALP) assays were conducted on the osteogenic group at predefined time points throughout the 21 day study, while metabolic activity, triglyceride and oil red O assays were conducted on the adipogenic group at predefined time points. Adipocyte CM administered at a concentration of 50% increased the ALP production of MSCs undergoing osteogenic differentiation. Additionally, osteoblast CM increased the triglyceride production of MSCs undergoing adipogenic differentiation and enlarged the lipid vesicles that were produced by the cells. The effects of the osteoblast CM were seen at both concentrations, but were greatest at the 50% CM level.     

Fucoidan‐induced osteogenic differentiation promotes angiogenesis by inducing vascular endothelial growth factor secretion and accelerates bone repair

Osteogenesis and angiogenesis, including cell–cell communication between blood vessel cells and bone cells, are essential for bone repair. Fucoidan is a chemical compound that has a variety of biological activities. It stimulates osteoblast differentiation in human mesenchymal stem cells (MSCs), which in turn induces angiogenesis. However, the mechanism by which this communication between osteoblasts and endothelial cells is mediated remains unclear. Thus, the aim of this study was to clarify the relationship between fucoidan‐induced osteoblastic differentiation in MSCs and angiogenesis in endothelial cells. First, the effect was confirmed of fucoidan on osteoblast differentiation in MSCs and obtained conditioned media from these cells (Fucoidan‐MSC‐CM). Next, the angiogenic activity of Fucoidan‐MSC‐CM was investigated and it was found that it stimulated angiogenesis, demonstrated by proliferation, tube formation, migration and sprout capillary formation in human umbilical vein endothelial cells. Messenger ribonucleic acid expression and protein secretion of vascular endothelial growth factor (VEGF) were dramatically increased during fucoidan‐induced osteoblast differentiation and that its angiogenic activities were reduced by a VEGF/VEGF receptor‐specific binding inhibitor. Furthermore, Fucoidan‐MSC‐CM increased the phosphorylation of mitogen‐activated protein kinase and PI3K/AKT/eNOS signalling pathway, and that its angiogenic effects were markedly suppressed by SB203580 and AKT 1/2 inhibitor. Finally, an in vivo study was conducted and it was found that fucoidan accelerated new blood vessel formation and partially promoted bone formation in a rabbit model of a calvarial bone defect. This is the first study to investigate the angiogenic effect of fucoidan‐induced osteoblastic differentiation through VEGF secretion, suggesting the therapeutic potential of fucoidan for enhancing bone repair.