鹿茸再生干细胞体外培养及核结合因子a1的检测

为了建立鹿茸再生干细胞的体外分离培养方法,进一步了解鹿茸再生、骨化等生理过程,本试验采用酶消化和组织块培养相结合的手段,成功进行了鹿茸再生干细胞的体外培养及传代扩增,对其生长曲线进行了初步分析,并对核结合因子a1(cbfa1)基因的表达情况进行了免疫细胞化学的分析,结果表明用酶消化和组织块培养法能够很好地获得鹿茸再生干细胞;鹿茸再生干细胞表达Cbfa1基因。     

体外培养小鼠精原干细胞的研究

探讨一种新的体外培养精原干细胞的方法,为进一步研究精原干细胞增殖与凋亡提供依据。采用组合酶消化、差速贴壁法分离精原干细胞,并设计了支持细胞条件培养基、胶质细胞源性神经营养因子(GDNF)处理培养基以及对照培养基3种培养体系,应用细胞活力分析仪检测精原干细胞数量以及活性,精原干细胞标记蛋白c-kit受体和磷酸酶染色技术进行鉴定,激光共聚焦显微镜技术检测细胞增殖核抗原(PCNA)和BCL-2家族蛋白Bax和Bcl2表达的变化。结果:应用组合酶消化和差速贴d壁法得到了纯度70%以上的精原干细胞;在体外培养7 d以内,PCNA的表达条件培养基组明显好于GDNF处理组以及对照组,Bcl2的表达条件培养基组高于GDNF组和对照组,Bax的表达条件培养基组低于GDNF组和对照组。研究表明在7 d内体外培养精原干细胞,条件培养基明显促进细胞增殖,抑制细胞的凋亡。     

山羊毛囊干细胞的分离及体外培养

试验采用2．4U／mL Dispase酶消化和机械切割法分离毛囊隆突部,经胰酶（0．5mg／mL胰酶＋0．2mg／mL EDTA）消化从山羊耳部皮肤分离得到的毛囊干细胞,然后进行形态学观察、细胞生长曲线测定、克隆形成率及免疫组化染色检测,并对毛囊干细胞的基础培养基进行了筛选。结果表明：DMEM／F12是一种适合毛囊干细胞体外增殖培养的培养基;在以DMEM／F12为基础培养液的培养体系中细胞可传代至19代。     

细毛羊毛囊干细胞分离培养方法比较研究

试验旨在对细毛羊毛囊干细胞的分离培养方法进行初步研究,建立细毛羊毛囊干细胞体外培养体系。采用两步酶消化法、机械分离法和两步酶消化法+机械分离法3种方法分离培养细毛羊毛囊干细胞,通过测定毛囊干细胞的数量、克隆形成率及毛囊干细胞的增殖能力等综合评估3种培养方法的优劣,寻求既能方便获得大量毛囊干细胞,又能减少其分化的理想培养条件。倒置显微镜下观察3种培养方法获得的细胞均为铺路石状,均表达角蛋白K19和整合素β1,表明成功获得细毛羊毛囊干细胞,建立体外培养体系。采用"两步酶消化法和机械分离法"相结合的方法获得毛囊干细胞体外培养效果最优。     

昆明小白鼠胚胎干细胞分离培养的影响因素

为了研究影响胚胎干细胞(ESC)体外分离克隆的因素,试验采用3~5日龄大鼠心肌细胞制成心肌细胞条件培养基(RH-CM),比较了4种培养体系对小鼠胚胎干细胞体外培养的影响,并分析比较了不同饲养层对胚胎干细胞分离克隆造成的影响。结果表明:添加重组鼠白血病抑制因子和心肌细胞条件培养基在胚胎贴壁率、内细胞团(ICM)形成率和原代胚胎干细胞集落形成率以及抑制分化的过程中起到的作用显著优于未添加任何诱导成分的培养基;作为饲养层,小鼠胚胎成纤维细胞(MEF)分离培养胚胎干细胞的效果优于小鼠睾丸支持细胞(MS)。     

马鹿茸间充质干细胞传代培养研究

马鹿茸间充质干细胞对鹿茸的生长发育有重要的作用,为研究鹿茸的生长,是否可以通过体外培养获得马鹿茸间充质干细胞,提取细胞蛋白。结果表明:传代马鹿茸间充质干细胞1d内完全贴壁,细胞呈多边形,3～5d细胞生长达到高峰,细胞呈纤维状,7d细胞生长趋于稳定,14d细胞长满。将5.0×10~6个细胞裂解,获得蛋白的浓度为7.3μg/ml。     

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。     

梅花鹿鹿茸间充质层细胞的体外培养和冷冻保存

为了研究梅花鹿鹿茸间充质层细胞的生物学特性,取梅花鹿鹿茸生长顶端间充质层组织,分离间充质层细胞进行体外培养及冷冻保存。结果表明:在含10%胎牛血清(FBS)的DMEM培养基中,鹿茸间充质层细胞能进行短期体外培养,培养的细胞呈成纤维细胞样,培养7 d可长至汇合。以含5%二甲基亚砜(DMSO)和10%FBS的DMEM为冻存液,经梯度降温后冻存,间充质层细胞复苏后存活率较高。在4℃条件下,间充质层组织在含10%FBS的DMEM中可保存7 d。     

牛精原干细胞的分离和纯化及体外培养的一般特性

采用两步酶消化法制备5月龄的牛生殖细胞悬液,用Percoll不连续密度梯度法分离精原细胞,接种于含10%胎牛血清的DMEM/F12培养基中,37℃,5%CO2饱和湿度培养,观察培养细胞的生长和形态变化。结果5月龄牛的曲细精管主要包含细胞为精原细胞、Sertoli细胞,每克睾丸实质收获生精上皮细胞总数平均为3.18×106个细胞,精原细胞纯化后纯度达69.27%,精原细胞主要分布于27%~35%的Percoll梯度中。牛精原干细胞体外培养6~7 d后开始分裂,20 d后精原干细胞形成小集落。结果表明用两步酶消化、Percoll不连续密度梯度法分离的精原细胞能满足体外培养的需要,可以存活并发生增殖。     

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。     

牛体外受精胚胎培养技术研究

在COCs体外培养过程中添加共培养物质和采用不同体积的液滴进行培养,以探讨牛卵母细胞的体外培养效果。结果表明,卵丘细胞、卵丘细胞+bFF共培养系统对胚胎发育均有促进作用,其中以卵丘细胞+bFF共培养系统对桑椹胚和囊胚的发育最好,其囊胚率为10.63%,显著高于对照组的7.23%(P<0.05),高于卵丘细胞组的8.50%(P>0.05)。用培养皿微滴培养(50μL)和用4孔板大体积培养(500μL)的试验表明,大体积培养比微滴培养有优势,桑椹胚和囊胚率均较高,其中囊胚率为10.23%,显著高于培养皿微滴培养的9.07%(P<0.01)。     

甘氨酸对延边黄牛体细胞克隆重组胚发育的影响

为了提高延边黄牛体细胞克隆重组胚的发育率,研究了在体外培养液中添加不同浓度的甘氨酸对重组胚发育的影响。分别在体外培养液中添加0、7、14、21mmol/L的甘氨酸,结果表明,在体外培养液中添加甘氨酸能够显著提高延边黄牛体细胞克隆重组胚的发育率,且以7mmol/L浓度的甘氨酸囊胚的发育率最高。     

血清和饲养层对水牛精原干细胞体外培养的影响

本研究首先将水牛睾丸经二步酶消化法制成单细胞悬液,依次采用差速贴壁法和Percoll不连续密度梯度离心法分离和纯化精原细胞。在制备好的小鼠胎儿成纤维细胞饲养层上,采用含2．5％血清的培养液对精原细胞进行体外培养,观察血清和成纤维细胞对精原细胞生长的影响,并在体外成功培养了2周通过提取体外培养精原干细胞总FZNA,设计引物并对其进行基因鉴定和免疫细胞化学鉴定,证实体外培养所得的细胞仍保持有精原干细胞的特性,并且该克隆是处在未分化状态的精原干细胞形成的。上述研究可为体外建立水牛精原干细胞长期培养体系提供技术支撑。     

奶牛体外受精胚胎的不同培养方法对比试验

以屠宰场奶牛卵巢为试验材料，研究体外、体内培养法对体外受精胚胎发育的影响。①体外受精胚胎分别在加输卵管上皮细胞单层和颗粒细胞单层（2×106个/ml）的体外培养体系的培养液中培养与临时受体绵羊体内培养对比试验。结果显示：在培养的第8 d囊胚发育率无显著差异，体外培养体系囊胚形成主要集中在第8 d（受精日为第0 d）。体内培养体系囊胚的形成多数在第7 d。加体细胞的体外培养体系与体内培养法囊胚生成率无显著差异，但却显著好于不加体细胞的简单体外培养法。②进行2种方法生产的胚胎的程序冷冻、解冻敏感性试验。结果显示：体内培养法和普通体外法生产的胚胎解冻后存活率（90%；60%）和囊胚孵化率(85.7%；44.4%)存在极显著差异（P<0.01）。     

MDCC—MSB1培养基上清液中DNA活性的定性研究

将 Ｍ Ｄ Ｃ Ｃ Ｍ Ｓ Ｂ１ ４８ 小时培养物 １０００ｒ／ｍ ｉｎ 离心的上清液分别用 Ｒ Ｎ Ａ 酶、 Ｄ Ｎ Ａ 酶和蛋白酶 Ｋ 处理后，进行体外试验。结果表明，只有 Ｄ Ｎ Ａ 酶处理后的上清液失去了体外抑制 Ｍ Ｄ Ｖ“８１４”增殖的作用。将该上清液 １００００ｒ／ｍ ｉｎ 离心所得的沉淀分别用上述酶处理后进行体内试验。结果表明，只有 Ｄ Ｎ Ａ 酶处理的样品失去了体内促进 Ｍ Ｄ Ｖ 京１ 株致瘤的作用。同时，电泳分析结果证明，该上清液中确实存在 Ｄ Ｎ Ａ。     

Development of a cell culture assay for the quantitative determination of vaccination-induced antibodies in rabbit sera against Clostridium perfringens epsilon toxin and Clostridium novyi alpha toxin

Cell culture assays are possible alternatives to replace in vivo neutralization tests currently required for potency testing of clostridial vaccines. Cell culture assays based on the MDCK cell line and the Vero cell line which are sensitive to the Clostridium (C.) perfringens type D epsilon toxin and Clostridium novyi type B alpha toxin, respectively, were developed, and the test conditions were standardized. The antibody titres of vaccinated rabbits measured in vitro were compared with the results of current test procedures recommended by European Pharmacopoeia. The correlation coefficients calculated were significant for all sera tested. The cell culture assays proved to be sensitive, specific, reproducible and reliable. Therefore, these cell culture assays could be suitable in vitro alternatives to the in vivo mouse neutralization experiments required for potency tests of clostridial vaccines, but further validation studies are necessary.     

牛乳腺上皮细胞β-酪蛋白的检测及核型分析

本试验用Western-blotting检测奶牛乳腺上皮细胞培养液中的β 酪蛋白，鉴定该细胞的生理功能是否正常；通过分析染色体数目及核型以鉴定体外培养的奶牛乳腺上皮细胞是否发生转化。结果显示：培养液中含有β 酪蛋白，说明该乳腺上皮细胞生理状况良好；染色体数目正常，染色体数目为60，核型与哺乳动物染色体图谱一致，说明该细胞系在体外培养条件下未发生转化。     

Effects of ghrelin on in vitro development of porcine in vitro fertilized and parthenogenetic embryos

The objective of the present study was to investigate the effect of addition of ghrelin to in vitro culture medium on preimplantation development of porcine in vitro fertilized and parthenogenetic embryos. In Experiment 1, we sought to compare the in vitro developmental competence of IVF and parthenogenetic embryos. No significant (P<0.05) differences were detected for cleavage rate or blastocyst rate between the in vitro fertilization (IVF)- and parthenogenetic activation-derived embryos. In Experiment 2, parthenogenetic embryos were cultured in Porcine Zygote Medium-3 containing various concentrations of ghrelin. The blastocyst rate was remarkably (P<0.05) increased when 5 ng/ml (PA-5) and 500 ng/ml (PA-500) of ghrelin was added to in vitro culture medium compared with the other groups. Total cell number per blastocyst was slightly promoted in the ghrelin treatment groups compared with the controls. However, the ratio of inner cell mass (ICM) cell number/total cell number was significantly reduced in the PA-50 group compared with the controls (P<0.05). In Experiment 3, we cultured in vitro fertilized embryos in Porcine Zygote Medium-3 supplemented with ghrelin at different dosages. The rate of blastocyst formation was markedly (P<0.05) elevated when 500 ng/ml ghrelin was added to culture medium (IVF-500) compared with the controls. Increased total cell numbers (P<0.05) were observed when in vitro fertilized embryos were cultured in IVF-50 and IVF-500 compared with the controls. However, the ratio of ICM cell number/total cell number was decreased in the ghrelin treatment groups compared with the controls (P<0.05). Taken together, the results suggest that ghrelin can enhance blastocyst formation of porcine in vitro fertilized and parthenogenetic embryos while exerting a negative effect on the structural integrity of the blastocysts.     

细胞培养技术在鸡球虫研究中的应用

球虫的细胞培养是球虫在体外合适的细胞体系中进行发育的过程,能让人们在"无控"和"透明"环境下研究抗球虫药物的作用机制和虫体发育机制等.目前,该技术已在球虫学研究中广泛应用,主要集中于细胞培养体系优化、抗球虫制剂的疗效、虫体入侵相关蛋白功能研究以及检测手段的优化等方面.论文从以上几个方面综述细胞培养技术在鸡球虫学研究中的...     

The effect of co-culture on the development of in vitro matured equine oocytes after intracytoplastic sperm injection

It is clear that, in the horse, there are many weak links in the process of in vitro embryo production; an optimal culture system for equine oocytes does not exist, and related data are conflicting. Therefore, the ability of 3 different culture systems to support embryonic development of ICSI horse oocytes was examined. Oocytes (n = 261) suitable for culture were collected from 55 ovaries and divided, according to cumulus morphology, into 2 categories: expanded cumulus and compacted cumulus. Oocytes with expanded and compacted cumulus were cultured for in vitro maturation in TCM 199 + 10% FCS + 0.1 iu/ml FSH/LH at 38.5 degrees C under 5% CO2 in air for 24 and 40 h, respectively. Oocytes (n = 149) reached metaphase II and were subjected to ICSI with frozen semen and then incubated in 3 different culture systems: A) TCM 199 + 10% FCS alone or B) on granulosa cell monolayer, C) SOF + MEM amino acids + 0.8% BSA. Cultural conditions were 39 degrees C and 5% CO2 in air for A and B, while a gas mixture (5% CO2, 5% O2, 90% N2) was used for C. The fertilisation rate was 32%. The cleavage rate in Group A was 74.4% (32/43); 18 embryos reached 2-6 cell stage, eight 8-16 cell, four 16-32 cell and two >32 cell. In Group B, the cleavage rate was 73.5% (36/49) with better results in embryonic development; 14 reached 2-6 cell stage, eighteen 8-16 cell, twelve 16-32 cell and five >32 cell. In Group C, the cleavage rate was significantly lower then in A and B; only 15 of 47 ICSI oocytes (39.1%) cleaved with maximum development to 2-6 cell stage. The remaining oocytes (68.1%) degenerated during culture. In conclusion, IVM horse oocytes can be fertilised in vitro with high efficiency with ICSI and co-culture systems showed to be superior in supporting in vitro embryo culture compared to simple ones. The identification of the factors beneficial to in vitro embryo development provided by the somatic cells could be important to optimise the embryo culture systems for equine embryos.