Ι型骨质疏松山羊骨髓基质细胞体外成脂、成骨分化的研究

观察体外定向诱导Ι型骨质疏松山羊骨髓基质细胞(BMSCs)分化为脂肪细胞和成骨细胞。建立去卵巢骨质疏松山羊动物模型,全骨髓法分离培养其骨髓基质细胞。第二代细胞分别成脂、成骨诱导培养,油红O染色、碱性磷酸酶染色、茜素红染色判定其分化结果。成脂诱导21d,油红O染色有脂滴形成;成骨诱导14d,碱性磷酸酶染色阳性;连续诱导35d,茜素红染色证实有钙化结节形成。Ι型骨质疏松山羊骨髓基质细胞在体外可以定向诱导分化为脂肪细胞和成骨细胞。     

HA/Ti梯度薄膜材料的体外细胞相容性研究

用射频磁控溅射与水蒸气处理相结合的方法，在医用Ti-6A1-4V合金表面制备HA／Ti梯度薄膜．通过材料与成骨细胞体外共培养，研究实验材料的细胞相容性．结果表明：随着细胞培养时间的延长，两组试样表面细胞均呈典型的S型生长，增殖显著，形态正常．相比之下，HA／Ti梯度薄膜周边和表面的细胞数量更多，贴附更加紧密，显示了良好的细胞亲和力。     

Physiological responses of osteoblasts to cyclic stretching and the change of intracellular calcium concentration

The development of bone tissues is regulated by mechanical stimulation.Cyclic stretching was applied to the osteoblasts that were delivered from rat calvarie,The results showed that stretching at 500με increased the cell proliferation while loading at 1000με and 1500με inhabited cell growth ,Loading also increased the adhesive force between cells and substrate as well as spreading areas of osteobalsts.Furthermore,the fluorescence probe Fluo-3/AM was used to investigate the effect of stretching stimulation on the intracellular calcium concentration of osteoblasts.The intracellular calcium concentration of osteoblasts that were stretched at 500 με for 5 min was 92.9% higher than the control ,After being treated with the panax ontoginseng saponins,the streteched osteoblasts still expressed 28.6% higher intracellular calcium concentration than that of the control ,which proved that both the influx of extracellualr calcium and the release of intracellular calcium store were involved in the increase of intracellular calcium concentration when osteoblasts responded to the cyclic stretching And the influx of extracellular calcium through transmembrance channel played a main role.     

成骨细胞诱导骨髓基质细胞体外成骨的初步研究

目的:探讨在不使用细胞因子或化学药物的情况下,成骨细胞(Osteoblast,OB)与骨髓基质细胞(Bone Marrow Stromal Cells,BMSCs)混合培养时,成骨细胞提供的成骨微环境能否在体外诱导BMSCs向成骨细胞分化,并复合支架形成成熟的骨组织.研究成骨细胞诱导BMSCs有效成骨的最小比值(指成骨细胞与骨髓基质干细胞数量的比值).方法:SY别培养SD乳鼠的成骨细胞与SD大鼠的BMSCs,将成骨细胞和BMSCs以1:9、2:8、3:7、1:0的不同比例进行混合培养,通过测定第3、6、9天培养液上清中的碱性磷酸酶(ALP)的含量,研究成骨细胞促BMSCs有效成骨的最小比值.将两种细胞以该最小浓度比混匀接种于涂附Ⅰ型胶原壳聚糖材料支架上(直径9 mm,高3mm)作为混合培养组,相同终浓度的单纯成骨细胞和单纯BMSCs分别接种于相同支架作为阳性对照及阴性对照.另设置低比值成骨细胞对照组(仅含有共培养组中相同的成骨细胞数,但不含有共培养组中的BMSCs).全部标本均于体外培养8周后取材,通过大体观察、组织学及免疫组织化学等相关检测对新生骨进行评价.结果:成骨细胞和BMSCs以3:7的比例进行混合培养时已可实现有效成骨.3:7比例的混合培养组及阳性对照组(成骨细胞组)体外培养8周后大体观察和苏木素-伊红染色(HE)、ALP染色基本相同,均表达骨特异性细胞外基质Ⅰ型胶原,形成了较成熟的骨组织.阴性对照组(单纯BMSCs组)和低比值成骨细胞组,原细胞支架复合物变小、变形.低比值成骨细胞组在局部形成了少量的骨组织,阴性对照组(单纯BMSCs组)未能发现骨样组织形成.结论:在不使用细胞因子或化学药物的情况下,成骨细胞提供的成骨微环境能够在体外诱导BMSCs向成骨细胞分化并形成成熟的骨组织.混合细胞中成骨细胞与BMSCs的比例为3:7时是有效成骨的最小比值.     

Notch signaling stimulates osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

Notch signaling is one of the most important pathways mediating cell determination and differentiation.In this study, the roles of Notch signal in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) were investigated. The expression of Notch1, Jaggedl and DTXI detected by reverse transcrip-tion polymerase chain reaction (RT-PCR) suggested that Notch signal might exhibit a physiological regulatory role in the differentiation of MSCs. Constitutive expression of the intracellular domain of Notchl (ICN), the active form of Notchl protein, can activate Notch signal in cells without ligands‘ binding, hMSCs were isolated, expanded, and infected with retrovirus carrying green fluorescent protein (GFP) gene or ICN. Overexpression of ICN in hMSCs resulted in enhanced osteogenic differentiation induced by dexamethasone (Dex), which was characterized by an increase of cellular alkaline phosphatase (ALP) activity and calcium deposition. These results indicate that Notch stimulates differentiation of MSCs into osteoblasts.     

组织工程骨在生物反应器内的构建与检测

用经绿色荧光蛋白标记的新西兰兔颅骨来源的成骨细胞复合到生物衍生骨支架材料上,在新型旋转壁式生物反应器内三维构建组织工程骨,同时与静态培养环境中构建的结果进行比较.每12 h取样一次,培养至1周后结束.分别进行组织学、免疫组织化学和代谢产物等分析.结果显示反应器中以两种密度接种和构建的组织生长良好,细胞仍保持正常的染色体形态,并且反应器中构建物的细胞扩增是静态培养的5倍.研究结果还表明,通过反应器内部流体对流所产生的应力刺激及其提供的三维培养环境,可提高成骨细胞碱性磷酸酶的活性表达,从而完成成骨细胞的快速增殖与分化并缩短工程化组织的构建时间.本实验进一步证实了骨组织工程产业化的现实可行性.     

利用漂浮组织块获得高纯度兔成骨细胞的方法

建立了一种能够获得高纯度成骨细胞的简便可靠的培养方法,为成骨细胞的相关研究提供稳定的种子细胞来源。在常规培养方法的基础上进行改良,建立了组织块漂浮培养法,从新生兔颅骨分离、培养成骨细胞。观察细胞的生长状态及形态,绘制生长曲线并计算细胞倍增时间,并进行碱性磷酸酶染色和矿化能力的检测。该方法分离培养的细胞显示典型的成骨细胞生物学特性,获得的成骨细胞纯度高、性状稳定、增殖能力强。说明组织块漂浮培养法是一种可行的培养方法,能够为成骨细胞的相关研究提供稳定可靠的种子细胞来源。     

大鼠骨髓基质干细胞分离培养及向成骨、成脂诱导分化的研究

目的利用细胞原代及传代培养技术将大鼠骨髓基质干细胞诱导分化为成骨细胞和脂肪细胞,为其进一步应用奠定基础.方法全骨髓法分离大鼠骨髓基质干细胞,传代后分别在成骨、成脂诱导条件下继续培养,采用碱性磷酸酶染色、茜素红染色及油红"O"染色观察其成骨及成脂分化结果.结果第2代大鼠骨髓基质干细胞成骨诱导9 d后碱性磷酸酶染色呈阳性细胞,连续诱导14 d后可见矿化结节形成,成脂诱导14 d后可见脂肪细胞形成.结论随着诱导条件的不同,大鼠骨髓基质干细胞在体外可定向分化为成骨细胞和脂肪细胞.     

Effects of genistein, daidzein and glycitein on the osteogenic and adipogenic differentiation of bone marrow stromal cells and on the adipogenic trans-differentiation of osteoblasts

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), alkaline phosphatase (ALP) activity and oil red O assays were used to examine the effects of genistein, daidzein and glycitein on the osteogenic and adipogenic differentiation of primary mouse bone marrow stromal cells (MSCs) and the adipogenic trans-differentiation of primary mouse osteoblasts. The results indicated that daidzein, genistein and glycitein at concentrations from 1×10^-8 mol/L to 1×10^-5 mol/L promoted the proliferation of MSCs and osteoblasts; genistein, daidzein and glycitein promoted osteogenic differentiation and inhibited adipogenic differentiation of MSCs, and inhibited adipocytic transdifferentiation of osteoblasts at appropriate concentrations as 17β-estradiol. It suggests that genistein, daidzein and glycitein regulate a dual differentiational process of MSCs into the osteogenic and adipogenic lineages, and trans-differentiational process of primary osteoblasts into the adipocyte lineages, causing a lineage shift toward osteoblast. Protective effects of them on bone may be mediated by a reversal of adipogenesis which may promote the proliferation, differentiation and mineralization of osteoblasts, and make adipocytes secrete less cytokines which may promote osteoclast formation and activation. In addition, the results also indicated that genistein, daidzein and glycitein may be helpful in preventing the development of steroid induced osteonecrosis.     

不同应变水平拉伸对成骨细胞生理功能的影响

采用四点弯曲加载装置对大鼠颅盖骨中分离出的成骨细胞施加周期性拉伸刺激，研究成骨细胞对不同应变水平的拉伸刺激的生理响应。结果表明，500微应变的周期性拉伸刺激促进了成骨细胞的增殖，细胞的^3H－脯氨酸掺入量增加，碱性磷酸酶（ALP）活性和向胞外分泌钙均升高；而1000微应变的周期性拉伸抑制了成骨细胞的增殖、^3H－脯氨酸掺入量、碱性磷酸酶活力和向胞外分泌钙都降低，说明成骨细胞能够分辨不同变水平的力学刺激，500微应变的周期性拉伸刺激有利于细胞的生长和分化，而1000微应变的周期性拉伸对细胞的生理活性有抑制作用。而且细胞在增殖、基质合成和分化、矿化上表现出一致的变化趋势。