猪雄性生殖干细胞的分离培养及鉴定

本试验旨在探索猪雄性生殖干细胞(mGSCs)体外分离、培养的适宜条件,建立猪雄性生殖干细胞体外培养体系。采用两步酶消化法对新生小猪睾丸生殖干细胞进行了体外分离和初步的培养鉴定,并利用层黏连蛋白和明胶的不同贴壁特性,比较2种差易贴壁分选方法的富集效果,并对传代后的干细胞培养1周后进行碱性磷酸酶染色鉴定,通过免疫荧光技术检测培养细胞是否表达干细胞标志蛋白OCT-4。试验结果表明,层黏连蛋白更适用于猪生殖干细胞的富集、培养,细胞分选效率及增殖生长明显优于采用明胶分选的方法。培养的mGSCs拥有与小鼠mGSCs相同的形态、增殖及表达特征。鉴定结果显示,生长细胞克隆碱性磷酸酶染色呈阳性,支持细胞碱性磷酸酶染色呈阴性;培养的生殖干细胞克隆表达转录蛋白OCT-4,而饲养层支持细胞OCT-4抗体染色则呈阴性。结果表明培养的干细胞克隆仍保持较好的干细胞活性,保持正常的自我复制和分化潜能,初步建立了生殖干细胞培养体系。     

无血清培养关中奶山羊胚胎生殖细胞的研究

山羊胚胎生殖细胞是一种来源于胎儿原始性腺的多能干细胞,建立该细胞体外稳定分离培养体系对研究山羊繁殖育种具有重要价值。本试验通过酶消化法和组织培养法分离培养关中奶山羊胚胎生殖细胞,检测无血清培养基对细胞体外增殖的影响。结果发现,该培养基可以分离得到山羊胚胎生殖细胞,细胞集落形态典型,表达AKP、Oct4、TERT及SSEA-1。经体外分化试验表明,细胞可以分化为类胚体、成纤维样细胞、成脂细胞和卵母细胞样形态。无血清培养基可以用于山羊胚胎生殖细胞的分离与培养,本试验对进一步建立山羊胚胎生殖细胞长期培养体系提供了新的参考。     

崂山奶山羊骨髓间充质干细胞永生化细胞系的建立

为建立崂山奶山羊骨髓间充质干细胞(BMSCs)永生化细胞系,将已构建的pcDNA3.1-EGFPTERT转入崂山奶山羊BMSCs中,利用含G418的培养基筛选获得稳定转染的TERT-BMSCs;通过多次传代,测定其生长曲线;选取高代次TERT-BMSCs的分裂中期相的细胞,检测其染色体及核型的稳定性。结果表明,转入TERT基因的崂山奶山羊BMSCs能够稳定表达该基因,其生长曲线呈"S"形;在多次传代后,P40代细胞的核型正常率仍能达到88.24%。综上提示,转染得到的TERT-BMSCs具有良好的生长状态,其细胞生长稳定,符合永生化细胞特征,为间充质干细胞应用于组织修复及基因工程种子细胞的制备提供了理论基础。     

抑制miR-142-3p对奶山羊化学诱导乳腺上皮细胞泌乳功能的影响

【目的】探索miR-142-3p对奶山羊化学诱导乳腺上皮细胞(chemical induced mammary epithelial cells, CiMECs)体外泌乳功能的调节作用,旨在为促进转基因乳腺生物反应器的发展以及“培养皿奶”的商业化生产奠定基础和提供新的思路。【方法】选取奶山羊耳缘成纤维细胞(GEFs)为研究材料,经单一小分子化合物TGFβR-1/ALK5的抑制剂(RepSox)诱导8 d后,分别从细胞的形态变化、特异性标记物免疫荧光染色以及油红O染色对其进行鉴定。之后运用脂质体转染技术在诱导成功的奶山羊CiMECs中分别转染miR-142-3p的抑制物(miR-142-3p inhibitor)及抑制物对照(inhibitor-NC),不转染的细胞作为空白对照(BC),转染24 h后,采用实时荧光定量PCR检测miR-142-3p的表达水平,CCK-8法检测细胞的增殖率,Western blotting检测β-酪蛋白(β-casein)的表达量,甘油三酯酶法测定甘油三酯的含量。【结果】GEFs诱导8 d后形成岛屿状多核仁样的上皮样聚集;免疫荧光染色结果显示,诱导后的GE...     

Myogenic and adipogenic properties of goat skeletal muscle stem cells

The aims of the present study were to establish a culture system for goat skeletal muscle stem cells and to examine their myogenic and adipogenic properties in vitro. Cells were isolated from the skeletal muscle of the Shiba goat and cultured in vitro. Most of the cells were positive for myogenic markers, such as Pax7, MyoD, and desmin, and immunocytochemistry revealed they differentiated to form myotubes expressing myosin heavy chain, indicating they were highly myogenic. Myogenic differentiation was strongly suppressed by the addition of basic fibroblast growth factor, while proliferation was unaffected. When the cells were cultured in adipogenic differentiation medium, some of the cells differentiated into mature adipocytes that stained with Oil Red-O. These cells were immunocytochemically positive for adipogenic markers, including peroxisome proliferator-activated receptor-gamma (PPAR gamma) and CCAAT/enhancer-binding protein-alpha (C/EBP alpha). These results clearly demonstrate the presence of both myogenic and adipogenic stem cells in goat skeletal muscle.     

山羊类ES细胞的分离与克隆

采集山羊交配后6～8d的桑椹胚、囊胚和孵化囊胚,将桑椹胚和囊胚分别放在小鼠原代胎儿成纤维细胞(PMEF)饲养层和同源原代胎儿成纤维细胞(PGEF)饲养层上比较其脱带时间及脱带率。脱带后,将各自一半胚胎切割,把含ICM的半胚分别放在相应饲养层上进行培养,另一半整胚在各自饲养层上继续培养,而孵化囊胚直接于PGEF饲养层上培养。当ICM增殖一定程度时进行传代,以比较其类ES细胞分离与克隆的效果。结果表明,在2种不同饲养层上,囊胚的脱带时间均短于桑椹胚,囊胚的脱带率均高于桑椹胚,而饲养层的种类对胚胎的脱带时间以及脱带率影响不大。脱带切割囊胚不论在PMEF还是在PGEF饲养层上,其贴壁时间均短于脱带整胚及孵化囊胚,而贴壁率高于脱带整胚,与孵化囊胚相似。脱带整胚及脱带切割胚在PMEF饲养层上所获类ES细胞只能维持3代,而在PGEF饲养层上,脱带切割半胚和孵化囊胚所获类ES细胞传至5代。由此认为,对脱带后的胚胎进行切割处理,有利于ICM的贴壁和增殖;应用同源原代胎儿成纤维细胞饲养层培养系统,有利于类ES细胞的分离与克隆。     

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.     

不同泌乳相关激素和生长因子对奶牛乳腺上皮细胞增殖的影响

本研究旨在探讨不同泌乳相关激素和生长因子对奶牛乳腺上皮细胞增殖的影响及其与细胞外基质主要成分层黏连蛋白的关系。将正常的荷斯坦泌乳期奶牛乳腺上皮细胞进行体外培养,在未包被或包被层黏连蛋白的条件下,以MTT法检测催乳素(PRL)、牛生长激素(GH)、类胰岛素生长因子-1(IGF-Ⅰ)、类胰岛素生长因子-2(IGF-Ⅱ)对细胞增殖作用的影响。在层黏连蛋白包被条件下,进行血清恢复的同时添加不同泌乳相关激素和生长因子,GH、IGF-Ⅰ有促进细胞增殖的作用(P<0.05),PRL、IGF-Ⅱ有维持细胞存活的作用(P<0.05);无血清时,几种激素和生长因子单独添加均无明显促增殖效应(P>0.05)。无基质条件下,与血清联合使用时,PRL、GH、IGF-Ⅰ、IGF-Ⅱ均对细胞生长有不同程度促进作用(P<0.05);无血清时,仅IGF-Ⅰ使细胞增殖速率显著加快(P<0.05)。层黏连蛋白作为培养基质对于体外培养的泌乳乳腺上皮细胞生长速度没有显著促进作用,但有利于PRL和IGF-Ⅱ发挥促存活作用。PRL、GH、IGF-Ⅰ、IGF-Ⅱ对细胞增殖和存活有促进作用,但需要与血清中其他成分协同才能充分发挥作用,其中IGF-Ⅰ促增殖能力最强。     

新生牛雄性生殖细胞源类ES的培养及检测

采用贴壁速率差法分离纯化的新生牛雄性生殖干细胞（Male germ stem cell，mGSCs）和支持细胞（Calf sertoli cell，CSCs）；CSCs饲养层细胞一般维持时间不超过8d；新生牛雄性生殖干细胞在CSCs饲养层上1代培养至5～6d可形成类ES细胞集落，采用机械离散集落传代法有利于集落形成或存在，传代培养至少在前2代细胞集落存在；部分典型细胞集落的中心细胞AKP强阳性，周边细胞AKP弱阳性或阴性；免疫组化检测显示，细胞集落SSEA1强阳性、SSEA3弱阳性和Oct-4呈现阳性染色。表明mGSCs具有ES细胞的某些细胞特性。     

miR-142-3p对奶山羊乳腺上皮细胞泌乳功能的影响

为确定miR-142-3p对奶山羊乳腺上皮细胞泌乳功能的调节作用,试验选取泌乳期奶山羊乳腺上皮细胞为研究材料,利用脂质体转染技术抑制miR-142-3p的表达,采用实时荧光定量PCR、Western blotting、试剂盒等检测miR-142-3p基因沉寂后其对奶山羊乳腺上皮细胞泌乳功能的影响。结果显示,miR-142-3p基因沉寂后,奶山羊乳腺上皮细胞增殖能力增强,β-酪蛋白及甘油三酯分泌增加。由此可知,miR-142-3p通过抑制靶基因作用,影响奶山羊乳腺上皮细胞增殖、分泌β-酪蛋白及甘油三酯等泌乳功能。     

Effect of miR-142-3p on Lactation in Diary Goat Mammary Epithelial Cells

To determine the regulative role of miR-142-3p in lactation of dairy goat mammary epithelia cells (DGMECs),we selected the DGMECs in lactating dairy goat for material,used liposomal transfection techniques to silence miR-142-3p expression,and used Real-time quantitative PCR,Western blotting and Assay Kits to explore the changes of lactation after miR-142-3p inhibition.The results showed that when miR-142-3p was inhibited,the cell proliferation ability of DGMECs was increased,the secretion amount of β-casein and triglyceride were up-regulated.So we concluded that miR-142-3p could influence the lactation function of DGMECs,such as cell proliferation,β-casein and triglyceride secretion,by inhibiting the function of its target genes.     

Maintenance of mouse trophoblast stem cells in KSR-based medium allows conventional 3D culture

Mouse trophoblast stem cells (TSCs) can differentiate into trophoblast cells, which constitute the placenta. Under conventional culture conditions, in a medium supplemented with 20% fetal bovine serum (FBS), fibroblast growth factor 4 (FGF4), and heparin and in the presence of mouse embryonic fibroblast cells (MEFs) as feeder cells, TSCs maintain their undifferentiated, proliferative status. MEFs can be replaced by a 70% MEF-conditioned medium (MEF-CM) or by TGF-ß/activin A. To find out if KnockOut^TM Serum Replacement (KSR) can replace FBS for TSC maintenance, we cultured mouse TSCs in KSR-based, FBS-free medium and investigated their proliferation capacity, stemness, and differentiation potential. The results indicated that fibronectin, vitronectin, or laminin coating was necessary for adhesion of TSCs under KSR-based conditions but not for their survival or proliferation. While the presence of FGF4, heparin, and activin A was not sufficient to support the proliferation of TSCs, the addition of a pan-retinoic acid receptor inverse agonist and a ROCK-inhibitor yielded a proliferation rate comparable to that obtained under the conventional FBS-based conditions. TSCs cultured under the KSR-based conditions had a gene expression and DNA methylation profile characteristic of TSCs and exhibited a differentiation potential. Moreover, under KSR-based conditions, we could obtain a suspension culture of TSCs using extracellular matrix (ECM) coating-free dishes. Thus, we have established here, KSR-based culture conditions for the maintenance of TSCs, which should be useful for future studies.     

奶山羊胎儿成纤维细胞的分离培养及SRY基因性别鉴定

为了探索和建立奶山羊胎儿成纤维细胞体外分离培养及性别鉴定的技术方法,获得转基因克隆羊的供体细胞,本试验用组织块培养法分离纯化得到两株奶山羊胎儿成纤维细胞系,进行细胞形态观察、生长曲线及细胞周期和倍性分析。同时根据GenBank上发布的山羊SRY基因设计合成一对PCR引物作为性别鉴定引物,另外根据山羊BLG基因序列设计一对引物作为内参引物,建立PCR反应体系对两株胎儿成纤维细胞系进行性别鉴定。结果表明,分离的胎儿成纤维细胞活力良好,可在体外快速生长、增殖、稳定培养;阳性对照和山羊胎儿成纤维细胞系2经PCR扩增得到337 bp片段和498 bp的BLG基因片段,而阴性对照和山羊胎儿成纤维细胞系1经PCR扩增得到498 bp的β-乳球蛋白基因片段。将337 bp片段和pMD19-T载体连接,构建重组载体pSRY,通过测序证明337 bp片段为SRY基因片段。这说明有337 bp扩增带的细胞系为雄性,无337 bp扩增带的细胞系为雌性。本试验为转基因奶山羊新品种的培育奠定了基础。     

Primary Cilia in Chondrogenic Differentiation of Equine Bone Marrow Mesenchymal Stem Cells: Ultrastructural Study

Locomotor system disorders in equine species, such as tendon lesions, osteoarthritis, or ligament injuries, are some of the most frequent causes of dramatic reduction in horse performance. Traditional therapies are aimed at the inflammatory process and pain, but they do not regenerate normal tendon or ligament matrix and do not reduce re-injured rates. Mesenchymal stem cells have started to use as therapeutic option to repair these injured tissues. Most studies have focused on their isolation, in vitro culture and phenotyping. However, mesenchymal stem cell ultrastructure has been disregarded in the last years. We investigate the ultrastructural characteristics of these cells once differentiated into chondrocytes. Ultrastructural analysis was conducted on suspension cultures of differentiated chondrocytes from bone marrow mesenchymal stem cells by means of transmission electron microscopy. The morphologic characteristics of these cells, their ability to produce the extracellular matrix, and the presence of a single cilium could be indicative of the mesenchymal cells differentiation into chondrocyte phenotype. This study provides essential data to evaluate the degree of suitable phenotypic stability, for these cells can be used with repair purposes.     

Impact of the source and serial passaging of goat mesenchymal stem cells on osteogenic differentiation potential:implications for bone tissue engineering

Background: Adult mesenchymal stem cells(MSCs) can be conveniently sampled from bone marrow, peripheral blood, muscle, adipose and connective tissue, harvested from various species, including, rodents, dogs, cats, horses,sheep, goats and human beings. The MSCs isolated from adult tissues vary in their morphological and functional properties. These variations are further complicated when cells are expanded by passaging in culture. These differences and changes in MSCs must be considered prior to their application in the clinic or in a basic research study. Goats are commonly used as animal models for bone tissue engineering to test the potential of stem cells for bone regeneration. As a result, goat MSCs isolated from bone marrow or adipose tissue should be evaluated using in vitro assays, prior to their application in a tissue engineering project.Results: In this study, we compared the stem cell properties of MSCs isolated from goat bone marrow and adipose tissue. We used quantitative and qualitative assays with a focus on osteogenesis, including, colony forming unit, rate of cell proliferation, tri-lineage differentiation and expression profiling of key signal transduction proteins to compare MSCs from low and high passages. Primary cultures generated from each source displayed the stem cell characteristics,with variations in their osteogenic potentials. Most importantly, low passaged bone marrow MSCs displayed a significantly higher and superior osteogenic potential, and hence, will be the preferred choice for bone tissue engineering in future in vivo experiments. In the bone marrow MSCs, this process is potentially mediated by the p38 MAPK pathway. On the other hand, osteogenic differentiation in the adipose tissue MSCs may involve the p44/42 MAPK pathway.Conclusions: Based on these data, we can conclude that bone marrow and fat-derived MSCs undergo osteogenesis via two distinct signaling pathways. Even though the bone marrow MSCs are the preferred source for bone tissue engineering, the adipose tissue MSCs are an attractive alternative source and undergo osteo-differentiation differently from the bone marrow MSCs and hence, might require a cell-based enhancer/inducer to improve their osteogenic regenerative capacity.     

Effect of matrigel on the osteogenic potential of canine adipose tissue-derived mesenchymal stem cells

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) are a promising source of cells for bone tissue engineering. Matrigel is a basement membrane extract containing multiple extracellular components. This mixture may promote the osteogenic differentiation of MSCs and provide a more appropriate microenvironment for transplanted cells. Here, we investigated the effect of Matrigel on the osteogenic potential of Ad-MSCs. Canine Ad-MSCs were cultured in 2D and 3D matrices and implanted into subcutaneous pouches of dogs either with or without Matrigel. Culture mineralization, cell adhesion efficiency, cell proliferation, osteoid matrix production and alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase activities were quantified and compared. Ad-MSCs grown in 2D cultures with Matrigel showed higher levels of calcium deposition and ALP activity than those grown in the absence of Matrigel under osteogenic conditions. In 3D cultures, the cells cultivated with Matrigel showed greater attachment, proliferation and osteogenic differentiation than those grown without Matrigel. In vivo, Ad-MSCs implanted with Matrigel showed higher osteogenic potential than those without Matrigel. In conclusion, these data suggest that the use of Matrigel can increase the osteogenic potential of canine Ad-MSCs.     

山羊雄性胎儿生殖细胞建立干细胞系初探

体外培养山羊50~68日龄雄性胎儿生殖细胞,并检测它们的碱性磷酸酶(AP)活性和Oct-4蛋白,探讨性别分化后的生殖细胞用于建立干细胞系的可行性及检测指标。当山羊胎儿睾丸细胞体外培养时,生殖细胞及其来源的细胞克隆均呈AP阴性和Oct-4蛋白阴性,其中有部分细胞克隆表现为AP假阳性。山羊胎儿生殖细胞克隆呈隆突状生长,多为圆形,与周围细胞界限分明,但克隆内细胞间界限不清。细胞克隆至少可以培养3代以上。研究结果显示,山羊雄性胎儿生殖细胞可以用于建立生殖系来源的干细胞系;AP和Oct-4蛋白不适宜用来检测体外培养的山羊胎儿生殖细胞及其来源的细胞系。     

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。     

荷斯坦奶牛子宫内膜上皮细胞的体外培养

为建立和完善奶牛子宫内膜上皮细胞的体外培养技术,采用组织培养和胶原酶消化相结合的方法,对中国荷斯坦奶牛的子宫内膜上皮细胞进行了原代分离培养,经胰酶差时消化进行纯化,并以免疫组化法检测角蛋白表达。结果显示,所采用的方法可高效获得呈铺路石样的上皮细胞,角蛋白表达呈阳性,第2~8代的传代细胞在形态特征和增殖状态上保持着与原代细胞相近的生物学特征。     

塔里木马鹿茸间充质干细胞体外培养及细胞特性的研究

鹿茸间充质干细胞是一类新发现的干细胞。为获得鹿茸间充质干细胞的生物学特征,建立鹿茸间充质干细胞体外培养模式,通过采用改良组织块细胞培养法对生长30 d的塔里木马鹿茸间充质层细胞体外分离培养,观察细胞形态特征,MTT比色法检测细胞生长特点。结果表明:间充质层样品组织块于1 d完全贴壁,贴壁后培养2 d即可观察到组织块边缘迁出少量细胞,贴壁后培养3 d,大量的间充质干细胞从组织块中迁出,并不断增殖,在组织块贴壁后5 d细胞生长融合。传代培养的2~6代细胞在培养到96 h细胞增殖达到高峰。原代培养细胞呈梭形,细胞核呈椭圆形,细胞呈菊花状排列生长;传代细胞形态呈梭形、不规则形,且排列无规则。本研究结果将为鹿茸间充质干细胞的后续研究奠定基础。