小鼠胚胎腭突间充质细胞体外培养及生物学特性研究

目的 研究小鼠胚胎腭突间充质（EPM）细胞的生物学特性。方法 在显微镜下解剖妊娠第13天的母鼠胚胎腭突，用0.25%胰蛋白酶进行消化获得游离分散的EPM细胞，在含10％胎牛血清的DMEM培养基中进行培养。采用免疫组化方法进行细胞鉴定，通过相差显微镜，生长曲线及透射显微镜观察细胞形态，增殖能力及超微结构。结果 EPM细胞为成纤维细胞样细胞，排列无序，细胞核大，核分裂象多，核膜内陷，核呈分叶状，胞浆含大量线粒体及粗面内质网。免疫组化显示角蛋白标记为阴性，S－100蛋白及波形蛋白标记为阳性。EPM细胞呈指数生长，有较强的增殖能力。结论 EPM细胞体外培养生长及增殖良好，可作为腭裂基础研究较理想的研究对象。     

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目的建立BALB／c近交系小鼠腭胚突间充质（EPM）细胞体外培养模型并研究其生物学特性。方法芩研究于2012年11月至2013年7月在山西医科大学医学寄生虫研究所进行。采用机械分离及胰蛋白酶消化法，简便、准确、快速地获得高成活率的BALB／c近交系小鼠EPM细胞，采用免疫组化法鉴定细胞，并进行细胞增殖和形态学观察。结果EPM细胞为成纤维细胞样细胞，呈梭型，单层漩涡状排列，排列无序，细胞核大，核分裂像较多。免疫组化染色显示，角蛋白标记为阴性，波形蛋白标记为阳性。EPM细胞呈指数生长，有较强的增殖能力。结论建立的BALB／c近交系小鼠EPM细胞体外培养技术模型可较好地保持EPM细胞的基本生物学特性。     

地塞米松对小鼠胚胎腭突间充质细胞增殖的影响

目的　探讨地塞米松对小鼠胚胎腭突间充质 (EPM )细胞增殖的影响。方法　在显微镜下解剖妊娠第 13d的鼠胚腭突 ,用 0 .2 5 %胰蛋白酶进行消化获得游离分散的EPM细胞 ,在DMEM培养基中进行培养 ,并采用反复贴壁法纯化EPM细胞。在培养基中加入 10 6M地塞米松 ,采用AgNOR染色、Feulgen染色及透射电子显微镜观察 ,检测地塞米松对EPM细胞增殖能力的影响。结果　地塞米松处理的EPM细胞银染颗粒数减少 (P <0 .0 1) ,核面积比及DNA合成能力下降 (P <0 .0 1) ,胞浆内线粒体数目减少 ,粗面内质网肿胀。结论　地塞米松对EPM细胞的增殖及生物合成有明显的抑制作用 ,影响腭突的正常发育 ,是腭裂发生的机制之一     

叶酸对小鼠腭突间充质细胞增殖的影响

目的:从细胞水平对维A酸致小鼠腭裂畸形作用和叶酸是否有拮抗维A酸的致畸作用及其机制进行研究。方法:给予孕鼠过量维A酸,诱导胚胎腭裂模型,不完全消化法提取孕期第14天、17天(GD14、17)的胚胎腭突间充质细胞(EPM cells)进行培养并鉴定细胞;采用四甲基偶氮唑盐(MTT)法检测加入不同浓度叶酸与未加入叶酸细胞增殖的情况。采用SPSS16.0软件包对数据进行统计学处理。结果:①在GD10、GD12管饲孕鼠50 mg/kg维A酸,可致胎鼠腭裂畸形;②不完全消化法提纯EPM细胞,纯度达到98%;③维A酸导致GD14的EPM细胞增殖受到抑制,20 μg/mL、40 μg/mL浓度叶酸可拮抗这种抑制作用。结论:①过量维A酸可致小鼠腭裂畸形,腭突间充质细胞增殖受抑制是其致畸机制之一;②叶酸可拮抗维A酸对小鼠GD14 EPM细胞增殖的抑制作用。     

Interaction between Smad 3 and Dishevelled in murine embryonic craniofacial mesenchymal cells

OBJECTIVES: To determine the in vivo interaction between Smad 3 and Dishevelled-1. DESIGN: Cell culture transfection followed by immunoprecipitation with specific antibodies. SETTING AND SAMPLE POPULATION: The Department of Molecular, Cellular, and Craniofacial Biology, Birth Defects Center, University of Louisville. EXPERIMENTAL VARIABLE: Overexpression of myc-Smad 3. OUTCOME MEASURE: Western blotting of anti-Dishevelled immunoprecipitates for Smad 3. RESULTS: Smad 3 and Dishevelled isoforms-1, -2, and -3 all bind Smad 3 in glutathione-S-transferase (GST) pull-down assays and Smad 3 binds to Dishevelled-1 in vivo. Stimulation of the transforming growth factor beta (TGFbeta) pathway leads to increased binding of Smad 3 and Dishevelled-1 in vivo. CONCLUSION: Smad 3 binds all three known isoforms of Dishevelled and binds Dishevelled 1 in vivo. TGFbeta signaling modulates the interaction between Smad 3 and Dishevelled-1.     

不同浓度地塞米松对小鼠腭胚突间充质细胞作用规律的研究

目的 研究细胞水平地塞米松(Dex)致腭裂畸形的生物学机制,筛选出影响腭胚突间充质(EPM)细胞生长的地塞米松最适浓度.方法 腭胚突问充质细胞进行原代培养,实验组细胞分别以1×l0-9、1×10-8、1×10-7和1×10-6mol·L-1地塞米松加以干预,MTT比色法检测各组细胞的增殖率,碘化丙啶(PI)染色观察各组...     

亚甲基四氢叶酸还原酶基因在小鼠胚胎腭突间充质细胞中沉默效应的研究

目的构建亚甲基四氢叶酸还原酶（MTHFR）特异性小干扰RNA（siRNA）真核表达载体,体外观察其对MTHFR基因的沉默作用。方法采用基因克隆技术,将合成的特异性MTHFR RNA干扰寡核苷酸序列插入真核表达载体PsiRNA-Hh1neo_G2,构建MTHFR siRNA真核表达载体。应用核转染技术将PsiRNA-MTHFR重组质粒分别导入原代培养的小鼠胚胎腭突间充质（MEPM）细胞。48 h和5 d后用实时定量聚合酶链反应（Real-Time PCR）和Western blot技术检测MEPM细胞内MTHFR mRNA及蛋白水平的表达情况。结果成功构建PsiRNA-MTHFR真核表达载体。经检测48 h和5 d后转染PsiRNA-MTHFR的MEPM细胞MTHFR mRNA及蛋白表达均显著下调。结论构建的RNA干扰真核表达载体能明显干扰MTHFR mRNA及蛋白的表达,为进一步研究MTHFR的功能以及其调控胚胎腭突融合的机制奠定基础。     

MSX-1在腭突间充质细胞中表达的改变及意义

目的　探讨MSX基因对腭突未分化间充质细胞生物学行为的影响 ,明确MSX基因在腭裂发生中的意义。方法　MTT方法观察对照组、RA处理组细胞的增殖变化 ,原位杂交检测两组细胞中MSX基因扩增水平的变化。结果　浓度为 1× 10 -8mol /L的RA明显抑制腭突细胞的增殖 ,MSX基因表达阳性。结论　提示RA通过诱导MSX基因的表达而抑制细胞增殖 ,在腭裂发生过程中MSX基因是RA发挥生物学作用的靶基因     

RNA干扰原代培养胚胎腭间充质细胞Smad2/3基因对atRA阻滞细胞周期作用的影响

目的：证实atRA可以通过Smad2/3作用于p21,从而导致胚胎腭间充质细胞周期受阻而出现腭裂。方法：原代培养胎鼠胚胎腭间充质细胞并进行鉴定。采用RNA干扰原代培养胚胎腭间充质细胞Smad2/3基因后,Western印迹检测Smad2/3、pSmad2、pSmad3和p21的蛋白表达水平及干扰后细胞周期的变化。采用SPSS11.0软件包对数据进行单因素方差分析和t检验。结果：经过免疫组化鉴定,证实原代培养C57BL/6N胎鼠腭突间充质细胞成功,并且Smad2/3siRNA可以有效干扰Smad2/3在原代培养MEPM细胞中的表达。Western印迹检测结果证实,Smad2/3siRNA可以通过敲减atRA诱导的Smad2/3表达增高现象,继而降低atRA诱导的p21表达增高现象。此外,Smad2/3siRNA在一定程度上降低了atRA诱导的胎鼠腭突间充质细胞G1期阻滞现象。结论：Smad2/3通过p21参与atRA诱导的胚胎腭突间充质细胞G1期阻滞现象。     

Interaction between epidermal growth factor and triamcinolone acetonide in mouse palatal mesenchymal cells in vitro

Palatal mesenchymal cells from mouse embryos (MEPM cells) had a high number of specific receptors for epidermal growth factor (EGF). At 37 degrees C, the number of high-affinity receptors (dissociation constant, Kd of 6.0 X 10(-10) M) was 2.0 X 10(5) per cell and of low-affinity receptors (Kd of 2.9 X 10(-9) M), 3.8 X 10(5) per cell. At 2 degrees C, a single class of receptors (Kd of 4.8 X 10(-9) M) was detected at 1.8 X 10(5) per cell. Triamcinolone acetonide, a potent cleft-palate-inducing agent, slightly inhibited the recovery of 125I-labelled EGF binding capacity in MEPM cells after down regulation of EGF receptors; it did not affect the binding properties of 125I-labelled EGF in these cells.     

SD大鼠骨髓间充质干细胞的体外分离培养鉴定及骨向诱导分化研究

目的：建立骨髓间充质干细胞（MSCs）体外分离培养体系,并进行骨向分化诱导以探讨大鼠MSCs的体外培养方法及向成骨细胞分化的条件。方法：采用全骨髓贴壁筛选培养法分离成年SD大鼠MSCs,经条件培养液诱导培养后,进行细胞形态学观察、绘制细胞生长曲线、免疫细胞化学检测及碱性磷酸酶活性、钙结节的形成测定。结果：SD大鼠MSCs在体外分离培养扩增形态呈长梭形,细胞生长曲线呈S形。诱导条件下,细胞碱性磷酸酶活性明显增高,并出现了矿化结节。结论：SD大鼠MSCs体外分离培养体系能够成功建立,培养出的细胞能向成骨细胞分化,可以作为骨组织工程的种子细胞。     

atRA对C57BL/6N小鼠腭突间充质细胞周期分布的影响及其作用机制

目的：在全反式维A酸（all-trans retinoic acid,atRA）诱导建立腭裂小鼠模型的基础上,检测胎鼠体内胚胎腭突间充质细胞的周期分布,并检测相关周期蛋白的变化情况,为进一步阐明维A酸（retinoic acid,RA）诱导腭裂机制提供新的线索。方法：以atRA建立C57BL/6N胎鼠腭裂模型,采用流式细胞术及免疫组化检测小鼠胚胎腭突间充质细胞的周期分布,通过实时定量RT-PCR和Western印记法检测p21和pRb在胎鼠胚胎腭突间充质中的mRNA和蛋白表达水平。采用SPSS11.0软件包对数据进行单因素方差分析和t检验。结果：流式细胞术及免疫组化检测发现,在小鼠妊娠日（gestation day,GD）第10天时给予atRA,可以诱导胎鼠胚胎腭突间充质细胞在一定妊娠阶段出现G1期细胞周期阻滞,同时也使胎鼠胚胎腭突间充质内p21和pRb的表达水平出现改变。而妊娠第12天（GD12）时给予atRA则无此现象。结论：p21与pRb参与了GD10时给予atRA诱导组胎鼠胚胎腭突间充质细胞G1期阻滞的发生。     

Human dentin matrix induces cartilage formation in vitro by mesenchymal cells derived from embryonic muscle

Dentin matrix was assayed for its potential to elicit chondrogenesis of mesenchymal cells in vitro. The substratum was prepared by demineralization of human tooth root dentin, while embryonic thigh muscle was used as a source of mesenchymal cells. Formation of chondrocytes from mesenchymal cells occurred in the presence of dentin matrix, and in the same sequence as previously shown with substrata of demineralized bone.     

Chondrocyte-like colony formation of mesenchymal cells by dentin extracts in agarose gel culture

In this study, the effects of guanidine extracts from demineralized bovine dentin matrix on rat mesenchymal cells were investigated by use of an agarose gel culture. The dentin extracts were divided into water-soluble and -insoluble fractions. Rat mesenchymal cells obtained from the cultivation of skeletal muscle tissue and embedded in agarose gel were treated with these two fractions. After three weeks of cultivation, the treated cells formed colonies that were stained metachromatically with toluidine blue in a dose-dependent manner. The activity necessary to form chondrocyte-like colonies by the water-insoluble fraction was significantly higher than that by the water-soluble fraction. Each chromatographic fraction of the water-insoluble part of dentin extracts on tandem Sephacryl S-200 High-resolution columns was also investigated. Chondrocyte-like colony-forming activity was concentrated in a single fraction. However, the electrophoretic pattern of this fraction revealed that there were still some bands of molecular weight between 18 and 30 kDa. According to the Western blot analysis of this fraction, there was a band corresponding to purified transforming growth factor beta (TGF-beta) under the non-reducing condition. After reduction, this band disappeared and we found a band corresponding to a component of 13 kDa as well as TGF-beta. These findings suggest that TGF-beta is present not only in bone but also in the dentin matrix.