绵羊输卵管上皮细胞的分离与培养

通过建立输卵管上皮细胞的体外培养和传代的方法，进行形态学观察及冻存。取雌性绵羊输卵管上皮细胞，用胶原酶消化和不锈钢网过筛法分离输卵管内膜基质细胞和上皮细胞，进行单细胞原代及传代培养。在输卵管上皮细胞的培养可持续培养4-6周，传3-4代。原代培养细胞7d长成单层，传代细胞4d长成单层。该研究旨在建立一套输卵管上皮细胞体外培养和传代的方法，为进一步研究β-防御素在雌性生殖道的作用方面提供很好的研究模型。     

山羊乳腺上皮细胞培养体系的建立

乳腺是转基因动物技术的一个主要研究对象,因为通过遗传操作可改变乳汁成分,获得具有生物活性成分的重要外源蛋白质产物［１］。近几年来,乳腺生物反应器研究成功的例子屡有报道［２］,但通过乳腺生物反应器生产的蛋白质能达到商品化所需浓度的却为数不多,其中一个重要原因是人们对乳腺上皮细胞的各种生理特性及其基因表达调控的分子机制了解得不够深入［３］。有鉴于此,本研究建立了正常山羊乳腺上皮细胞（ＧＭＥＣ）培养体系,为进一步研究ＧＭＥＣ的增殖和分化以及基因表达调控的分子机制提供了有利条件。１　材料与方法１．１　细…     

绵羊气管上皮细胞的分离培养及绵羊肺炎支原体对其的吸附

为建立简便、高效、稳定的绵羊气管上皮细胞的体外培养及鉴定的方法,并在所培养的细胞上研究绵羊肺炎支原体侵染模型,本研究利用链蛋白酶冷消化法对绵羊气管上皮细胞进行体外分离,通过差速贴壁法纯化气管上皮细胞并进行传代后用液氮保存,细胞复苏后通过测定生长曲线、上皮细胞骨架蛋白角蛋白8和18以及对染色体与微生物的检测等对所培养细胞进行鉴定,并将鉴定纯化的气管上皮细胞用绵羊肺炎支原体进行侵染。结果显示,成功培养出绵羊气管上皮细胞,纯化的细胞在显微镜下排列紧密,形态均一,呈鹅卵石铺路样。细胞生长曲线呈S型,分子生物学手段和增菌试验未检测到气管上皮细胞中含有微生物污染,通过细胞免疫组化检测到了细胞角蛋白8、18,染色体核型数目为2n=54。绵羊肺炎支原体能黏附于分离纯化的绵羊支气管上皮细胞,为进一步研究支原体侵染细胞的机制等奠定了基础。     

牦牛输卵管上皮细胞和卵泡颗粒细胞的培养

从牦牛输卵管壶腹部获取上皮细胞,用抽吸法从牦牛卵泡中获取颗粒细胞,对牦牛输卵管上皮细胞和卵泡颗粒细胞进行体外培养和传代的培养,观察培养过程中2种细胞原代和传代培养的生长方式及形态特点。两者培养144~216 h可形成融合的单层细胞,其传代密度保持105~106/mL为宜。     

牦牛输卵管上皮细胞和卵泡颗粒细胞的培养

分离培养牦牛输卵管上皮细胞和卵泡颗粒细胞的目的是克服体外受精中早期胚胎发育阻断,建立有利于牦牛早期胚胎体外发育的共培养体系.采集牦牛输卵管上皮细胞进行原代及传代培养,同时从卵泡液中获取颗粒细胞进行原代培养,培养过程中观察2种细胞的生长方式和形态特点.输卵管上皮细胞贴壁生长时,呈多边形,且呈单层成簇生长.培养144 h～216 h,可形成细胞单层.颗粒细胞贴壁生长时,呈现聚集生长特性,贴壁细胞形状不规则,呈放射状.培养72 h～96 h,形成颗粒细胞单层.     

牛输卵管上皮细胞培养的研究

用０．７６％的ＥＤＴＡ分离剥取输卵管上皮细胞是一种好的试剂,１０％胎牛血清（ＦＣＳ）ＴＣＭ－１９９培养其上皮细胞,５－６ｄ即可生长成单层,寿命可延至３－４周,共同培养从第７天开始,其囊胚发育率显著高于与颗粒细胞共同培养。     

利用RT-PCR技术扩增绵羊输卵管上皮细胞Ghrelin基因

提取绵羊输卵管上皮细胞总RNA,采用Primer 5.0软件设计引物,运用RT-PCR技术扩增Ghrelin基因。结果显示,经测序和序列比对鉴定,该方法成功地扩增到了大小为200 bp的目的基因,表明RT-PCR技术是检测绵羊输卵管上皮细胞中Ghrelin基因的有效手段,可为今后研究不同激素水平下绵羊输卵管组织中Ghrelin基因的表达量变化及进一步体外试验奠定基础。     

蒙古绵羊原始生殖细胞(PGCs)的分离培养与鉴定

本试验从绵羊胎儿的生殖嵴中分离出原始生殖细胞,经体外培养后进行了初步鉴定。为建立绵羊原始生殖细胞(primordial germ cells,PGCs)的培养体系,试验对比了不同培养条件对蒙古绵羊PGCs生长的影响,将得到的胚胎生殖细胞(EG细胞)作形态学、酶学和免疫荧光鉴定。结果表明:绵羊EG细胞集落隆起,边缘整齐,呈鸟巢状,细胞排列紧密;AKP染色为弱阳性;免疫荧光检测特异标志物Oct3/4呈弱阳性,SSEA-1、SSEA-3、SSEA-4、TRA-1-61、TRA-1-80呈强阳性。     

孕酮对绵羊输卵管上皮细胞β-防御素2表达的影响

研究孕酮(P4)对绵羊输卵管上皮细胞β-防御素2(SBD2)基因表达的影响,探讨P4调节SBD2表达的潜在机制。建立绵羊输卵管上皮细胞体外培养体系,用不同浓度(10^-6、10^-7、10^-8、10^-9、10^-10mol/L)P4分别处理绵羊输卵管上皮细胞0、2、6、12、24、48h,筛选P4诱导绵羊输卵管上皮细胞SBD2mRNA表达的最佳条件。然后使用10-6mol/L孕激素核受体拮抗剂RU486,50μmol/L蛋白激酶C(PKC)信号通路阻断剂H7预处理细胞1h,再使用P4诱导SBD2mRNA表达的最佳条件处理细胞,同时设只添加阻断剂(RU486和H7)处理组、只添加孕酮(P4)处理组和空白对照组,通过RT-qPCR技术检测SBD2mRNA的表达变化。10-9mol/LP4诱导绵羊输卵管上皮细胞6h和24h时SBD2mRNA表达显著高于对照组(P<0.01),且与P4组相比,添加RU486和H7后显著抑制了P4诱导的SBD2mRNA的表达水平(P<0.01)。P4以浓度和时间依赖性方式显著增加SBD2mRNA表达,并且诱导可能通过孕酮核受体(PR)介导的基因组途径以及PKC信号通路来提高绵羊输卵管上皮的先天免疫防御能力。     

水牛输卵管上皮细胞的培养

在水牛体外胚胎生产过程中,胚胎发育阻滞现象较为普遍,严重影响了体外生产胚胎的效率。与输卵管上皮细胞共培养可以促进早期胚胎的体外发育,克服发育中的阻断现象,增加可以发育到囊胚期胚胎的数量,使胚胎能在更合适的时间移植,以提高移植后的着床率。水牛输卵管上皮细胞的培养技术,是随着对水牛的体外受精的研究而开展起来的,如何培养出适用于共培养的输卵管单层上皮细胞,在很多文章中都也只有过概述。本实验就如何培养水牛输卵管上皮细胞,摸索建立了一套较为合适的水牛输卵管上皮细胞培养的模型,为水牛的胚胎工程工作提供参考数据。     

仔猪胰腺细胞体外培养体系的建立

为了研究体外培养仔猪胰腺细胞的适宜条件,试验选取1日龄的仔猪胰腺组织置于冰上清洗、修剪,对消化酶、离心方式、培养皿铺被方式、培养液及添加不同促生长因子等多种因素进行筛选和优化。结果表明:0.25%胰酶消化所获得的细胞强于0.1%胶原酶消化获得的细胞,细胞清亮,易于生长;采用500 r/min离心5 min、多次离心所获得的细胞较Percoll不连续密度梯度离心和5%BSA等密度梯度离心所获得的细胞贴壁率高,生长能力强,易于培养传代;采用0.1%明胶或20μg/mL的层黏连蛋白铺被培养皿均比无铺被的培养皿获得更多的贴壁细胞;以RPMI 1640、M199、F12、L-DMEM多种培养液培养仔猪胰腺细胞,表现出多种细胞形态和生长趋势;在基础培养液中添加上皮生长因子、角质化生长因子、β-巯基乙醇和白血病细胞抑制因子、转铁蛋白与亚硒酸盐混合试剂等促干细胞增殖因子可促进不同形态细胞的增殖。说明按此方式培养可获得大量、多种细胞形态的仔猪胰腺祖/干细胞。     

蒙古绵羊β-防御素基因相对表达水平实时定量PCR方法的建立

为了利用SYBR Green Ⅰ实时荧光定量PCR技术对蒙古绵羊雌性生殖道β-防御素基因表达水平进行相对定量测定,建立蒙古绵羊β-防御素基因相对表达水平的实时荧光定量PCR方法,试验根据GenBank中羊β-防御素基因序列设计合成引物,进行实时荧光定量PCR,同时以β-Actin为内参基因对β-防御素基因进行均一化处理,利用荧光阈值(Ct值)计算β-防御素基因的相对表达量.结果表明:扩增产物为β-防御素;利用SYBR Green Ⅰ实时荧光定量PCR技术可以测定蒙古绵羊β-防御素基因表达水平相对含量.     

新生广西巴马小型猪肾成纤维细胞体外培养体系的建立

利用微量液体组织块贴壁法原代培养分离新生广西巴马小型猪肾成纤维细胞,进行常规传代培养、冷冻和复苏;通过接触抑制和解除抑制研究该类细胞的增殖潜力,免疫荧光进行鉴定;并通过脂质体介导对分离到的新生广西巴马小型猪肾成纤维细胞分别进行EGFP-N1和DsRed-N1基因转染。结果成功分离到新生广西巴马小型猪肾成纤维细胞,体外可以大量传代和冷冻,目前已经传至50代以上,生长良好;冷冻复苏和接触抑制解除后仍可以正常培养和传代;细胞免疫荧光检测,波形蛋白表达阳性,角形蛋白表达阴性;转染48h后荧光显微镜下可以观察到绿色荧光和红色荧光,表明瞬时转染新生广西巴马小型猪肾成纤维细胞可以获得成功,其中转染DsRed-N1基因的新生广西巴马小型猪肾成纤维细胞已经传至60代以上。结果表明,本试验成功建立了新生广西巴马小型猪肾成纤维细胞的体外培养体系,在体外能稳定培养和传代,并可以成功获得转基因的新生广西巴马小型猪肾成纤维细胞。     

Establishment and characterization of a fibroblast cell line derived from Mongolian sheep

The Mongolian sheep ear marginal tissue fibroblast cell line (MSF32) from 32 samples was successfully established by using primary explants technique and cell cryoconservation technology. MSF32 cells were adherent, with a population doubling time of 28.2 h. Chromosome analysis showed that >90.2% of cells were diploid (2n = 54) prior to cell passage 4. Isoenzyme analyses of lactate dehydrogenase and malate dehydrogenase showed that the MSF22 cells had no cross‐contamination with other species. Tests for cell line contamination with bacteria, fungi, viruses and mycoplasmas were also negative. Plasmids encoding the fluorescent proteins pEGFP‐N3, pEGFP‐C1, pECFP‐N1, pECFP‐mito, pDsRed1‐N1 and pEYFP‐N1 were transfected into cells to study exogenous gene expression in the cells. The plasmid transfection efficiency was between 12.3% and 63.3%. Every index of the MSF32 cell line meets all the standard quality controls of American Type Culture Collection (ATCC). Not only has the genetic resources of the Mongolian sheep been preserved at the cell level, but also valuable materials had been provided for genome, postgenome and somacloning research.     

蒙古绵羊卵丘细胞ghrelin部分序列的鉴定

为了研究生长激素释放多肽(ghrelin)mRNA是否在蒙古绵羊卵丘细胞中表达,试验根据Gen-Bank中报道的绵羊ghrelin序列设计1对特异性引物,以蒙古绵羊卵丘细胞中的RNA为模板,采用实时定量RT-PCR技术扩增蒙古绵羊ghrelin的cDNA,并进行测序分析。结果表明:扩增得到的蒙古绵羊ghrelin cDNA为ghrelin的部分序列,将测序结果与已发表的绵羊ghrelin序列进行比对发现,233个碱基中有1个碱基的差异,该差异不影响翻译后多肽的序列;该cDNA片段包含长度为231 bp的开放读码框(ORF),编码77个氨基酸的绵羊ghrelin前原肽。     

苜蓿花药悬浮培养体系的建立

以苜蓿花药愈伤组织为材料建立悬浮细胞系,对悬浮细胞系建立的适宜培养基进行筛选,并对培养液的pH值及细胞活力进行研究.结果表明,参试苜蓿品种在NB液体培养基中愈伤组织诱导率较高;同一激素水平下,NB培养基愈伤组织诱导率明显高于MS液体培养基.培养液pH值出现下降-上升-平稳的趋势.接种第3 d悬浮细胞活力最强.     

Establishment and characterization of a coculture system of equine endometrial epithelial and stromal cells

To investigate the equine endometrium as close to the in vivo situation as possible, we established a coculture system for epithelial and stromal cells (ECs/SCs). ECs and SCs were isolated from nine endometrial tissue specimens. ECs obtained as glandular formations were cultivated on one side of the semipermeable membrane of a Millicell^® insert. After 2 days, SCs (2 × 10⁴ cells/membrane) were seeded onto the other side of the same membrane. During cocultivation, the low serum containing culture medium (Theuß et al., 2010) was supplemented with different concentrations and combinations of 17β‐estradiol (2.0–3.0 pg/ml medium) and progesterone (0.5–15.0 ng/ml medium). Once the cocultures formed continuous cell layers as determined by phase‐contrast microscopy, the membranes were fixed and processed for light microscopical examination. Cytokeratin 19, steroid hormone receptors and the uterine proteins uteroglobin and calbindin_D9k were detected using immunocytochemistry to determine the degree of culture purity and functional cellular differentiation. The culture purity of the EC layer averaged ≥95%. Uteroglobin and calbindin_D9k were consistently expressed in ECs, while hormone receptors were predominantly absent in both cell populations. An explicit cytomorphological epithelial differentiation with formation of round‐oval to polygonal cell forms was encountered in ≤50% of all ECs and independent of supplemented steroids. Based on the findings altogether, and despite the partly absent congruence to the in situ prerequisites, we established a standardized and reproducible coculture system, which offers a basic approach for studies of physiologic and pathophysiologic issues in the mare.     

Use of the phenolsulphonphthalein dye test for fallopian tube patency in cattle

Results of preliminary investigations of fallopian tube patency in cattle using a method based on intrauterine instillation of PSP dye, and detection of the dye in the urine, suggest that this test can provide a useful diagnostic aid in cases of bilateral occlusion. In such cases PSP dye is not evident in the urine two hours later. In normal animals dye is present within 30 minutes. In two animals with unilateral blockage dye appeared at an intermediate time between the normal and bilaterally occluded cases. Repetition of the test and the use of adequate volumes of dye to cause uterine distension may be necessary to eliminate false negatives.     

Palmitate metabolism by isolated sheep rumen epithelial cells.

Ruminal palmitate metabolism was examined using an isolated cell system. Palmitate oxidation to 14CO2 by rumen epithelial cells isolated from the rumens of mature sheep was linear during the course of a 2-h incubation (11.1 nmoles.million cells-1.2 h-1) and 3.6 times the rate of palmitate oxidation by cells isolated from neonatal rumen (3.1 nmoles.million cells-1.min-1). Subsequent experiments were conducted with mature rumen epithelial cells. Neither acetate (50 mM), propionate (10 mM), dibutyryl cAMP (.2 mM), nor insulin (10 mU/mL) altered palmitate oxidation to CO2. However, butyrate (10 mM) addition reduced (P less than .05), and ammonia (15 mM) tended to reduce (P less than .10), palmitate oxidation (51.6 and 82.0% of control, respectively), whereas addition of glucose (2.5 mM) increased (P less than .05) palmitate oxidation (151% of control). Of the compounds tested, only propionate, butyrate, and ammonia reduced palmitate oxidation to total acid-soluble metabolites. Propionate (10 mM) addition completely abolished palmitate oxidation to acid-soluble metabolites. Succinate addition (5 to 50 mM) increased palmitate oxidation to CO2 but exhibited no consistent effect on palmitate oxidation to either acid-soluble metabolites or beta-hydroxybutyrate. Propionate completely abolished palmitate oxidation to beta-hydroxybutyrate, suggesting that propionate-induced inhibition of palmitate oxidation is not mediated via succinate. The data indicate 1) that rumen epithelium is capable of oxidizing palmitate, 2) that ruminal palmitate oxidation may be subject to regulation by developmental factors, and 3) that palmitate metabolism seems to be influenced more by ruminally derived metabolites than by factors derived exclusively from the general circulation.     

荷斯坦奶牛乳腺上皮细胞的分离培养

目的：建立荷斯坦奶牛乳腺上皮细胞的分离培养方法。方法：采用组织块种植法培养奶牛乳腺上皮细胞,利用胰蛋白酶差时消化法分离、纯化上皮细胞。结果：成功培养出奶牛乳腺上皮细胞,显微镜下观察,纯化的乳腺上皮细胞呈典型上皮细胞形态,细胞之间排列紧密,呈鹅卵石铺路样,形态均一,多角形的单层聚集。通过荧光免疫细胞染色方法对细胞骨架蛋白-角蛋白18进行鉴定,呈现阳性反应。乳腺上皮细胞增殖旺盛,经25次以上传代后长势仍然良好。结论：采用组织块种植法结合胰酶差时消化法成功获得纯化的奶牛乳腺上皮细胞。