南阳牛骨髓间充质干细胞的分离培养及多向分化

本研究旨在建立南阳牛骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)体外分离培养方法,在此基础上研究其生物学特性和多向分化的能力。采用骨髓穿刺法取3月龄小牛的肋骨骨髓,分离培养BMSCs,传代培养并测定其生长曲线,RT-PCR检测Oct4、Nanog、Sox2基因的表达,然后取P3 BMSCs分别向神经和脂肪细胞进行诱导分化,并利用组织学染色技术和RT-PCR技术进行鉴定。结果表明,分离得到的BMSCs大小均匀,多呈梭形的成纤维细胞样生长;RT-PCR可检测到干细胞因子Oct4、Nanog、Sox2的表达;不同代次细胞生长曲线呈S型,一般在第3天时进入指数生长期,第7天后进入平台期;成神经诱导后,甲苯胺蓝染色可见明显的尼氏体结构,RT-PCR检测ENO2和GFAP基因表达呈阳性;成脂肪诱导后,油红O染色后可见大量的脂滴存在,RT-PCR检测Leptin和PPAR基因表达呈阳性。试验证明,成功分离得到了南阳牛BMSCs,且其具有多向诱导分化潜能。     

崂山奶山羊胎儿骨髓间充质干细胞（BMSCs）的分离培养及成神经诱导分化

旨在建立崂山奶山羊胎儿骨髓间充质干细胞（BMSCs）体外分离培养方法,并研究其生物学特性和成神经分化的能力。取怀孕3个月的崂山奶山羊胎儿股骨,分离培养骨髓间充质干细胞,并进行传代培养,测定其细胞倍增时间,利用RT-PCR技术检测Oct4、Nanog、Sox2基因的表达;取P3 BMSCs分别向成神经细胞进行诱导分化,并从组织学水平和基因水平进行鉴定。结果表明,分离得到的胎儿骨髓间充质干细胞大小较为均匀,呈梭形的成纤维细胞样,可表达Oct4、Nanog、Sox2基因;传代接种后第4天进入指数生长期,第8天进入平台期,前10代BMSCs的平均倍增时间为29.7 h;P3 BMSCs成神经诱导后,尼氏体经甲苯胺蓝染色后可见紫蓝色,其特异性表达基因ENO2和GFAP表达呈阳性。获得的崂山奶山羊BMSCs具有成神经分化潜能。     

Study on Inducing Equine Bone Marrow Mesenchymal Stem Cells Differentiated into Chondrocytes

This study was aimed to establish equine bone marrow mesenchymal stem cells(BMSCs) line and induce it to differentiate into chondrocytes. The BMSCs were collected by cutting the bone and flushing the cutting surface by PBS, and then the bone marrow was washed with PBS,the collected cells were cultured after centrifugation. The cells were purified by passaging,the stem cell properties were tested before the induction. And the cells were also appraised by the expression of the special gene of chondrocytes as well as staining the differentiated cells with Alcian blue to insure the induction was effective. The obtained BMSCs expressed Sox2 and Nanog genes, which were the stem cell special genes, and also expressed CD44, CD90 and CD105 genes, but absent of CD34 and CD45 genes. The shapes of the 3rd passage BMSCs were changed after cultured in inducing medium for a few days. Furthermore, the cells were positive to Alcian blue staining, increased expression of the Col special gene of chondrocyte day by day as well. According to this study, the BMSC line was established, and the BMSCs were induced and differentiate into chondrocytes successfully.     

马骨髓间充质干细胞向软骨细胞的诱导分化

试验旨在建立马骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）细胞系,并诱导其向软骨细胞分化。通过获取马BMSCs,进行细胞培养和纯化,对第3代（P3）细胞进行干细胞特性鉴定,并诱导其向软骨细胞分化,对分化后的细胞染色,并检测其软骨细胞特异性基因的表达。结果显示,获得的马BMSCs表达标记基因Sox2和Nanog,并表达间充质干细胞表面标记因子CD44、CD90和CD105,不表达造血细胞表面标志物CD34和CD45。P3代细胞经诱导培养后形态发生改变,阿尔新蓝染色为阳性,并表达软骨细胞特异基因Col,且其表达量随着诱导分化时间的增加而增高。综上表明,本试验建立了马BMSC细胞系,并成功诱导其分化为软骨细胞,为软骨损伤的干细胞治疗提供了试验依据。     

Molecular and Cellular Characterization of Buffalo Bone Marrow‐Derived Mesenchymal Stem Cells

Immune privileged mesenchymal stem cells (MSCs) can differentiate into multiple cell types and possess great potential for human and veterinary regenerative therapies. This study was designed with an objective to isolate, expand and characterize buffalo bone marrow‐derived MSCs (BM‐MSCs) at molecular and cellular level. Buffalo BM‐MSCs were isolated by Ficoll density gradient method and cultured in Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum (FBS). These cells were characterized through alkaline phosphatase (AP) staining, colony‐forming unit (CFU) assay, mRNA expression analysis (CD 73, CD 90, CD 105, Oct4 and Nanog), immunolocalization along with flow cytometry (Stro 1, CD 73, CD 105, Oct4, Sox2 and Nanog) and in situ hybridization (Oct4 and Sox2). Multilineage differentiation (osteogenic, adipogenic and chondrogenic) was induced in vitro, which was further assessed by specific staining. Buffalo BM‐MSCs have the capacity to form plastic adherent clusters of fibroblast‐like cells and were successfully maintained up to 16^th passage. These cells were AP positive, and further CFU assay confirmed their clonogenic property. RT‐PCR analysis and protein localization study showed that buffalo BM‐MSCs are positive for various cell surface markers and pluripotency markers. Cytoplasmic distribution of mRNA for pluripotency markers in buffalo BM‐MSCs and multilineage differentiation were induced in vitro, which was further assessed by specific staining. To the best of our knowledge, this is the first report of buffalo BM‐MSCs, which suggests that MSCs can be derived and expanded from buffalo bone marrow and can be used after characterization as a novel agent for regenerative therapy.     

干细胞中两个关键细胞因子Oct4和Sox2

胚胎干细胞(ESC)在谱系特异性标志被激活前,Oct4和Sox2蛋白水平是细胞向谱系选择发展过程中的连续临时性标志。Oct4和Sox2转录因子在启动细胞重编程、维持ESC多能性和决定其是否走向分化方面具有关键作用。它通过与靶基因调控区结合,选择性地抑制分化基因或者激活多能性基因的表达而达到调控目的。干细胞共激活复合物(SCC)是Oct4和Sox2在Nanog基因协同激活时所需要的,它直接与Oct4和Sox2相互作用并集中在Nanog和Oct4启动子部位以及大部分被Oct4和Sox2占据的基因组区域,在维持ES细胞多能性和保持基因组完整性方面发挥着重要功能。因此,对Oct4、Sox2这两个关键性细胞因子作用机制深入了解,有助于细胞重编程分子机制的进一步阐明,为干细胞的相关研究奠定基础。     

过表达JHDM2A对猪iPSCs诱导效率的影响

研究旨在探讨组蛋白赖氨酸去甲基化酶(JHDM2A)对猪成纤维细胞诱导为多能干细胞效率的影响,并对其内在的分子机制进行探究。以猪成纤维细胞为材料,采用多西环素(DOX)诱导的慢病毒生产诱导多能干细胞(iPSCs)体系,在此基础上过表达JHDM2A,通过碱性磷酸酶染色和绘制诱导时间轴检测其对诱导效率的影响;普通PCR技术检测克隆的多能性;免疫荧光检测多能因子的蛋白表达;实时荧光定量PCR检测JHDM2A在形成克隆后以及克隆分化过程中对多能因子、组蛋白甲基化相关基因表达的影响。结果表明,JHDM2A过表达组细胞在第3天发生形态变化,第8天形成iPSCs克隆,相比于对照组分别提前了1和2 d。碱性磷酸酶染色结果显示,与对照组相比,JHDM2A过表达组克隆形态明显改善,染色着色更深,诱导效率提高8倍。普通PCR结果显示,JHDM2A过表达组iPSCs、对照组iPSCs以及猪成纤维细胞(PFF)均表达内源性Oct4、Sox2、Klf4、c-Myc、Nanog,且iPSCs的Oct4表达量高于PFF。免疫荧光结果显示,JHDM2A过表达组iPSCs表达Oct4、Sox2、Stat3、JHDM2A,弱表达SSEA1、SSEA4。实时荧光定量PCR结果显示,与对照组和猪成纤维细胞相比,JHDM2A过表达组iPSCs的Sox2、Klf4、c-Myc、Nanog、Oct4、Tcl1的表达显著升高(P<0.05),Tfcp2l1和Zfp57的表达显著降低(P<0.05);JHDM2A过表达组P5代iPSCs培养基去除DOX后,随着代数的增加,Sox2、Nanog的表达逐渐降低,Klf4、c-Myc的表达先升高后降低,Oct4、Tcl1、Tfcp2l1、Zfp57的表达先降低后升高。以上结果表明,过表达JHDM2A通过促进组蛋白去甲基化提高猪成纤维细胞的诱导效率。     

崂山奶山羊脂肪间充质干细胞系的建立及鉴定

旨在建立崂山奶山羊脂肪间充质干细胞系并对其进行初步鉴定.采集崂山奶山羊脂肪组织,在Ⅰ型胶原酶的分解消化作用下,将脂肪组织中的单核细胞进行分离并扩增培养;测定其生长曲线,利用Real time RT-PCR技术对其表达的干细胞因子进行鉴定;将传至第3代的脂肪间充质干细胞进行成骨诱导分化,利用茜素红染色进行鉴定.结果显示,崂山奶山羊脂肪间充质干细胞形态为长梭形、星形等成纤维细胞样细胞形态,但比成纤维细胞饱满,第3代细胞生长至3 d左右时细胞进入指数生长期,生长至6~7 d时进入平台期;经成骨诱导分化后,经茜素红染色可见骨结节被染成深红色.综上提示,崂山奶山羊脂肪间充质干细胞具有诱导分化潜能.     

山羊骨髓间充质干细胞的分离培养

旨在对山羊的骨髓间充质干细胞(BMSCs)进行分离培养。将分离得到的BMSCs进行传代培养,采用RT-PCR法检测其干细胞转录因子oct4和sox2基因,以验证其干细胞特性;在此基础上绘制BMSCs的生长曲线,对其生物学特性进行直观了解。结果表明,分离到的山羊BMSCs原代细胞呈现出成纤维样,并能够表达oct4和sox2基因,证明了其干细胞特性;其生长曲线呈"S"型,在1~2 d时为潜伏期,第3天时进入指数生长期,第7天时进入平台期。结果提示,分离得到的细胞具有BMSCs的特性,能够用于后续的相关试验研究。     

崂山奶山羊永生化骨髓间充质干细胞的诱导分化研究

为研究崂山奶山羊永生化骨髓间充质干细胞(BMSCs)的诱导分化潜能,采用P3代BMSCs和P30代TERT-BMSCs进行体外成脂和成神经诱导分化。结果显示,当细胞传至P30代时,TERT-BMSCs生长旺盛;在成脂诱导后,P3代BMSCs和P30代TERT-BMSCs均能观察到细胞中透明脂滴的形成,油红O染料染色后可见脂滴被染成红色;在成神经诱导后,P3代BMSCs和P30代TERT-BMSCs均能形成树突样或三角形的形态,甲苯胺蓝染色后可观察到细胞中尼氏体的存在。综上提示,永生化的崂山奶山羊BMSCs在多次传代后仍具有较强的多向诱导分化能力,这为永生化MSCs的广泛应用提供了理论依据。     

不同代次大鼠骨髓间充质干细胞和脂肪间充质干细胞成骨分化比较

本研究旨在观察不同代次骨髓间充质干细胞(BMSCs)和脂肪间充质干细胞(ADSCs)体外培养的生长特点和体外诱导成骨能力。通过密度梯度离心和贴壁培养法分离培养大鼠骨髓间充质干细胞和脂肪间充质干细胞,用含地塞米松、抗坏血酸、β-甘油磷酸钠的培养液定向诱导传代细胞向成骨细胞分化,并利用茜素红染色、碱性磷酸酶染色及PCR方法检测成骨细胞。结果表明骨髓及脂肪间充质干细胞呈成纤维细胞样生长,增殖能力强,生长迅速。第5、10、15、20代BMSCs及ADSCs经诱导培养后茜素红染色呈阳性并且出现"矿化"、碱性磷酸酶活性强,随着细胞代次的递增,诱导后细胞碱性磷酸酶活性呈递减趋势;诱导后的两类细胞传代后细胞仍能继续分化,并形成正常的"矿化"结节,且碱性磷酸酶染色均弱于初次诱导。结果提示,BMSCs及ADSCs易于分离培养及体外扩增,诱导条件下成骨能力强且成骨细胞传代培养仍具有成骨能力,适合作为再生医学骨组织工程的种子细胞。     

镉对体外培养鸡骨髓基质细胞成骨分化的毒性作用

骨是镉毒性作用的主要靶器官之一,但其对鸡骨髓基质细胞(bone marrow stromal cells,BMSCs)增殖和成骨分化的毒性作用仍不清楚。本研究利用差速贴壁纯化法获得鸡BMSCs,加入不同浓度镉处理不同时间,采用CCK-8法检测细胞增殖,碱性磷酸酶(alkaline phosphatase,ALP)和茜素红染色鉴定成骨分化,RT-PCR检测成骨相关基因(COL1、OSX、RUNX2、ALP、OCN、OPG、OPN、RANKL)表达变化。结果显示,1~10μmol/L的镉显著促进BMSCs增殖,20μmol/L的镉显著抑制其增殖(P<0.05);5μmol/L以上的镉可显著抑制ALP活性,并以浓度依赖方式极显著抑制成骨相关基因(COL1、OSX、RUNX2、ALP、OCN、OPG、OPN)mRNA表达,上调RANKL mRNA表达(P<0.01)。表明,一定浓度镉可抑制鸡BMSCs体外增殖及其向成骨细胞(OB)的分化。     

Nanog基因的生物学功能研究进展

胚胎干细胞具有无限增殖能力和多向分化潜能决定了它在医学及生物学基础研究中具有巨大的应用潜力。探索维持胚胎干细胞特性的分子机制成为胚胎干细胞的生物学研究中的热点。研究发现与维持胚胎干细胞多能性相关的基因有Oct4、Nanog、Sox2等，其中Nanog是2003年5月末发现的一个基因，它对维持胚胎干细胞多能性起关键性作用，能够独立于L1F/Stats维持ICM和ES细胞的多能性。几年来，Nanog的生物学功能及其与Oct4、Sox2等多能性维持基因之间的相互作用关系已有较为深入的研究。作者在综述Nanog基因的表达特征和功能的基础上，重点探讨Nanog基因表达调控以及Oct4、Sox2等多能性维持基因之间的相互作用关系，并展望其应用前景。     

荷斯坦牛羊水来源干细胞分离培养及其表面标志检测

利用羊水来源干细胞培养技术体系,从胎龄285 d荷斯坦奶牛胎儿羊水中分离培养干细胞;采用RT-PCR技术检测干细胞表面标志或相关基因。成功分离培养出奶牛羊水来源干细胞,干细胞中CD-90、Nanog、Oct4、TERT、Sox2、Desmin和HES1表达阳性。从奶牛胎儿羊水中分离干细胞具有可行性和有效性,为进行转基因研究提供靶细胞奠定了基础。     

Further insights into the characterization of equine adipose tissue-derived mesenchymal stem cells

Adipose tissue-derived stem cells (ADSCs) represent a promising subpopulation of adult stem cells for tissue engineering applications in veterinary medicine. In this study we focused on the morphological and molecular biological properties of the ADSCs. The expression of stem cell markers Oct4, Nanog and the surface markers CD90 and CD105 were detected using RT-PCR. ADSCs showed a proliferative potential and were capable of adipogenic and osteogenic differentiation. Expression of Alkaline phosphatase (AP), phosphoprotein (SPP1), Runx2 and osteocalcin (OC) mRNA were positive in osteogenic lineages and peroxisome proliferator activated receptor (Pparγ2) mRNA was positive in adipogenic lineages. ADSCs show stem cell and surface marker profiles and differentiation characteristics that are similar to but distinct from other adult stem cells, such as bone marrow-derived mesenchymal stem cells (BM-MSCs). The availability of an easily accessible and reproducible cell source may greatly facilitate the development of stem cell based tissue engineering and therapies for regenerative equine medicine.     

羊水来源干细胞基因组特征的研究进展

人和动物存在羊水来源的多潜能干细胞(amniotic fluid-derived stem cells,AFSCs)。AFSCs被公认为是介于胚胎干细胞和成体干细胞之间的一种特殊的细胞类型,因此,AFSCs同时具有成体干细胞和胚胎干细胞的生物学特性。AFSCs干细胞特性的保持是以其标志基因以及基因组的整体特异表达为基础。目前,已经有相关研究对不同胎龄时期的AFSCs的标志基因Oct4、Sox2、Nanog、SSEA系列以及Tra-1-60和Tra-1-81等在基因组中的表达特征进行了分析。对AFSCs的相关研究进展进行综述,以期为羊水干细胞的进一步系统、深入研究提供理论依据。     

Differentiation of canine bone marrow stromal cells into voltage- and glutamate-responsive neuron-like cells by basic fibroblast growth factor

We investigated the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into voltage- and glutamate-responsive neuron-like cells. BMSCs were obtained from the bone marrow of healthy beagle dogs. Canine BMSCs were incubated with the basal medium for neurons containing recombinant human basic fibroblast growth factor (bFGF; 100 ng/ml). The viability of the bFGF-treated cells was assessed by a trypan blue exclusion assay, and the morphology was monitored. Real-time RT-PCR was performed to evaluate mRNA expression of neuronal, neural stem cell and glial markers. Western blotting and immunocytochemical analysis for the neuronal markers were performed to evaluate the protein expression and localization. The Ca²⁺ mobilization of the cells was evaluated using the Ca²⁺ indicator Fluo3 to monitor Ca²⁺ influx. To investigate the mechanism of bFGF-induced neuronal differentiation, the fibroblast growth factor receptor inhibitor, the phosphoinositide 3-kinase inhibitor or the Akt inhibitor was tested. The bFGF treatment resulted in the maintenance of the viability of canine BMSCs for 10 days, in the expression of neuronal marker mRNAs and proteins and in the manifestation of neuron-like morphology. Furthermore, in the bFGF-treated BMSCs, a high concentration of KCl and L-glutamate induced an increase in intracellular Ca²⁺ levels. Each inhibitor significantly attenuated the bFGF-induced increase in neuronal marker mRNA expression. These results suggest that bFGF contributes to the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells and may lead to the development of new cell-based treatments for neuronal diseases.     

Isolation, culture and chondrogenic differentiation of canine adipose tissue- and bone marrow-derived mesenchymal stem cells–a comparative study

In the dog, mesenchymal stem cells (MSCs) have been shown to reside in the bone marrow (bone marrow-derived mesenchymal stem cells: BM-MSCs) as well as in the adipose tissue (adipose tissue-derived stem cells: ADSCs). Potential application fields for these multipotent MSCs in small animal practice are joint diseases as MSCs of both sources have shown to possess chondrogenic differentiation ability. However, it is not clear whether the chondrogenic differentiation potential of cells of these two distinct tissues is truly equal. Therefore, we compared MSCs of both origins in this study in terms of their chondrogenic differentiation ability and suitability for clinical application. BM-MSCs harvested from the femoral neck and ADSCs from intra-abdominal fat tissue were examined for their morphology, population doubling time (PDT) and CD90 surface antigen expression. RT-PCR served to assess expression of pluripotency marker Oct4 and early differentiation marker genes. Chondrogenic differentiation ability was compared and validated using histochemistry, transmission electron microscopy (TEM) and quantitative RT-PCR. Both cell populations presented a highly similar morphology and marker expression in an undifferentiated stage except that freshly isolated ADSCs demonstrated a significantly faster PDT than BM-MSCs. In contrast, BM-MSCs revealed a morphological superior cartilage formation by the production of a more abundant and structured hyaline matrix and higher expression of lineage specific genes under the applied standard differentiation protocol. However, further investigations are necessary in order to find out if chondrogenic differentiation can be improved in canine ADSCs using different protocols and/or supplements.