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1.
影响人类胚胎生殖细胞分离克隆的因素   总被引：7，自引：0，他引：7  
探讨胚龄培养液、细胞因子、饲养层细胞等因素对人类胚胎生殖细胞分离克隆的影响。以4～13周龄流产胎儿生殖嵴或性腺为材料，小鼠胎儿成纤维细胞(MEF)、人胎儿成纤维细胞(hEF)、STO细胞和牛胎儿成纤维细胞(bEF)作饲养层，高糖DMEM(Dulbecco's minimum Eagle’s medium)为基础培养液，添加10^-4mol／L 2Me(2-巯基乙醇)、胎牛血清(FBS)或新生牛血清(NBS)、白血病抑制因子(LIF)、碱性成纤维细胞生长因子(bFGF)、干细胞冈子(SCF)等为培养液，探讨影响人类胚胎生殖细胞分离克隆的因素。结果表明7～12周流产儿适于人类胚胎生殖细胞的分离克隆；人类胚胎生殖细胞体外培养以添加15％FBS为宜：添加4ng／mL LIF 4ng／mL bFGF 20ng／mL SCF能明显改善人类胚胎生殖细胞的分离克隆；MEF、STO、bEF和hEF可提高人原始生殖细胞(PGCs)的贴壁率，能促进PGCs源的人类胚胎干细胞(ES)的分离效率；以MEF作为饲养层效果较好。  相似文献   

2.
从原始生殖细胞分离克隆牛胚胎干细胞的研究   总被引：8，自引：0，他引：8  
从５～１３周龄的牛胎儿生殖嵴及其周围组织采用与其儿成纤维细胞共培养的方式分离得到大量原始生殖细胞（ＰＧＣｓ）集落。这些集落的细胞经多次克隆传代具有胚胎干（ＥＳ）细胞的诸多特征,如有连续传代的能力（有两个胎儿分别传至６代和８代）,细胞集落有典型鸟巢状结构。ＡＫＰ染色阳性,体外分化实验具有形成类胚体力能力。上述表明该细胞具有多能性,类似ＥＳ细胞。  相似文献   

3.
为提高昆明小鼠胚胎生殖细胞(embryonic germ cells, EG cells)建系效率，以怀孕8.5～12.5 d小鼠胎儿为材料，比较了胎儿后1/3部位的组织共培养、生殖嵴共培养、差速贴壁和穿刺生殖嵴4种分离胚胎原始生殖细胞(primordial germ cells，PGCs)的方法以及不同传代方式对EG细胞克隆的影响。结果表明：生殖嵴共培养和差速贴壁方式获得了较好分离EG细胞的效果，与其它两组比较差异显著；手工传代方式与连同成纤维细胞一起消化的传代方式相比获得了较高传代比率。对所分离EG细胞经形态观察、AKP染色和体外分化能力检测，证实其符合小鼠EG细胞的集落状生长、细胞未分化特性及细胞多能性等特征。  相似文献   

4.
本文从第19期（孵化68~72h）鸡（Gallus domesticus）生殖嵴分离到原始生殖细胞（PGC），通过PGC-体细胞共培养进行了原代和传代培养，并对PGC做了c-kit免疫组化鉴定。通过改变PGC的体外培养条件，诱导PGC分化成了神经样细胞、上皮样细胞和骨骼肌样细胞，并分别通过神经元特异性烯醇化酶（NSE）和角蛋白（keratin）免疫组化进行了分化细胞的鉴定，均呈阳性。结果表明培养的鸡胚PGC在体外可分化成神经样细胞、上皮样细胞和骨骼肌样细胞。  相似文献   

5.
鸡胚原始生殖细胞的分离、培养及鉴定   总被引：2，自引：2，他引：2  
采用Ficoll密度梯度离心法分离第19期(孵化68～72 h)艾维茵鸡(Gallus domesticus)生殖嵴原始生殖细胞(PGC)，通过体细胞-PGC共培养进行了原代和传代培养，对PGC进行了碱性磷酸酶(AKP)和过碘酸雪夫氏反应(PAS)鉴定，并用细胞增殖核抗原(PCNA)免疫细胞化学检测了传代于鸡胚饲养层上PGC的增殖活性。培养的PGC经AKP和PAS鉴定均为阳性，AKP阳性为红棕色，PAS阳性为深红色，PCNA染色显示PGC集落为红棕色，表明PGC处于增殖状态。同时比较了不同体外培养条件(5%~20%胎牛血清(FCS), ITS培养液(M199+ 10 μg/mL 胰岛素+5 μg/mL转铁蛋白+3×10-8 mol/L亚硒酸钠), 条件培养液, 15%FCS+ITS, 15%FCS+40% 条件培养液)下PGC增殖情况。发现未经密度梯度离心的PGC生长较离心的PGC好，且5%FCS在原代培养中起较好的促增殖作用；而传代培养时在不同培养条件下，5%FCS组或ITS组形成的PGC集落的直径较大，而仅用单纯的条件培养液效果并不明显。结果表明除鸡胚饲养层可作为PGC增殖的支持体系外，在体细胞-PGC共培养方式下5%FCS或单独的ITS也可作为PGC体外增殖的一种培养体系。  相似文献   

6.
本研究克隆了广西沼泽型水牛γ-干扰素(interferon-γ,IFN-γ)基因,并构建了水牛IFN-γ原核表达质粒。从健康沼泽型水牛静脉无菌采血,分离外周血单个核细胞(Peripheral blood mononuclear cells,PBMCs),用刀豆素A(Concanavalin A,ConA)诱导培养13 h后,提取细胞总RNA,用水牛IFN-γ基因特异性引物通过RT-PCR扩增水牛IFN-γ成熟肽编码区cDNA序列,将其克隆到pMD18-T载体中。RT-PCR产物电泳可见约457 bp大小目的片段。经过限制性酶切分析,测序证实克隆得到的基因序列正确。将IFN-γ成熟肽编码区基因片段切下亚克隆到表达载体pET-32a 中,构建成重组表达质粒pET-mIFN-γ。PCR、双酶切电泳和序列测定结果均证实已插入约457 bp的IFN-γ基因片段。经异丙基硫代-β-D-半乳糖苷(IPTG)诱导,水牛IFN-γ基因在大肠杆菌中获得了高效的表达,融合蛋白的分子量为35 ku,表达量占菌体总蛋白的43.6%。  相似文献   

7.
对水牛和小鼠腔前卵泡的分离方法进行了系统探索,旨在找出一种水牛和小鼠腔前卵泡的有效分离方法。实验一分别用梳刮法、剪碎法和显微分离法分离回收水牛卵巢腔前卵泡;实验二采用梳刮法分离水牛胎牛、青年牛和成年牛腔前卵泡;实验三分别用浓度为0(CK)、0.02%、0.04%、0.08%的胶原酶消化水牛卵巢20 m in,然后采集腔前卵泡;实验四采用梳刮法、剪碎法和显微分离法分离小鼠卵巢的腔前卵泡。结果表明,梳刮法能有效地分离回收水牛和小鼠卵巢的腔前卵泡。该法分离水牛卵巢所得腔前卵泡的原始卵泡最多,次级卵泡最少,而分离小鼠卵巢所得腔前卵泡的次级卵泡最多,原始卵泡最少;水牛胎牛卵巢可分离得到较多的腔前卵泡;酶消化有助于提高剪碎法的腔前卵泡分离效率。  相似文献   

8.
对来自6批次147枚采用去透明带-双半卵融合法(简称去透明带法)克隆的牛体细胞囊胚进行质量分级,然后除了20枚优质胚胎用来移植外,其余的127枚克隆囊胚均采用低渗分离细胞核法进行细胞计数。结果表明:127枚采用去透明带法克隆的7 d囊胚细胞数平均为129.8个,其中优质大囊胚细胞数平均为218.9±45.0个(117~281),中等大小的优质囊胚细胞数为134.9±43.7个(85~233),不同体积大小的囊胚细胞数差异极显著(P<0.01)。结果表明,从优质囊胚比例以及其细胞数来衡量,采用去透明带克隆方法可以获得质量较高的牛囊胚。  相似文献   

9.
研究了水牛胎儿成纤维细胞（buffalo fetal fibroblasts,BFF）转染外源基因后的细胞生物学特性。纯化含有绿色荧光蛋白（green fluorescent protein,GFP）和新霉素抗性基因的线性化载体,用脂质体法转染体外培养3代的BFF细胞,应用抗生素G418筛选2周后挑取转基因抗性细胞集落扩大培养。结果发现：经过G418筛选后细胞增殖活力下降,胰蛋白酶消化法共分离获得162个转基因水牛阳性细胞集落,转移到24孔板扩大培养后,132个（85．2％）细胞集落可以存活,转移到4孔板中后仅有52个（32．1％）细胞集落能继续生长,最终能在35mm培养皿中大量培养的只有15个（〈9．3％）。抗性细胞由梭形、成簇生长变为多角形、不规则形弥漫生长,立体感减弱,细胞生长速度减慢,活力下降。染色体核型异常率增加,10个抗性细胞集落核型正常率平均为61％,显著低于对照组86．7％（P〈0．01）。研究结果为建立和优化BFF细胞转染外源基因平台奠定了工作基础。  相似文献   

10.
以昆明系小鼠(Mus musculus Km)胚胎为材料,以丝裂霉素(10 μg/mL)处理的MEF为饲养层,研究了在胚胎干细胞(embryonic stem cells,ESCs)培养液中分别添加血清替代物(knockout serum replacement,KSR)、胎牛血清(fetal bovine serum,FBS)和FBS PD98059(50 μgol/L)对昆明系小鼠胚胎贴壁、内细胞团(inner cell mass,ICM)集落形成及ESCs分离培养的影响.结果表明,在ESCs培养液中添加KSR,胚胎贴壁率显著低于添加FBS(P<0.05),ICM集落形成率和1代ESCs集落出现率差异不显著(P>0.05),2～5代ESCs集落出现率显著高于添加FBS(P<0.05),2株ESCs传到7代;在ESCs培养液中添加FBS PD98059,胚胎贴壁率、ICM集落形成率和1～5代ESCs集落出现率均显著低于添加KSR或FBS(P<0.05);用0.5 g/L胰酶 0.2 g/L EDTA离散消化ICM细胞和ESCs并结合机械分割,1～5代ESCs集落出现率显著高于用2.5 g/L胰酶 0.2 g/LEDTA(P<0.05).实验结果表明,在ESCs培养液中添加KSR,较添加FBS或FBS PD98059更适合用于分离培养昆明系mESCs,用0.5 g/L胰酶 0.2 g/L EDTA离散消化ICM细胞和ESCs并结合机械分割优于用2.5 g/L胰酶 0.2 g/L EDTA.  相似文献   

11.
【目的】以富士（Fuji）、 秦冠（Qinguan）嫁接在平邑甜茶（Malus hupehensis Rehd.）上的当年生盆栽苗为试验材料,采用砂培方法,研究了缺氮胁迫和干旱对富士和秦冠生长情况、 光合参数、 植株各部位氮磷钾含量及氮素利用效率的影响,分析比较了低氮干旱条件下富士和秦冠生长及氮素利用的差异,以期为果树生产高效肥水利用提供理论指导。【方法】试验共设四个处理: 正常氮正常水（ZZ）、 低氮正常水（DZ）、 正常氮干旱（ZG）、 低氮干旱（DG）。氮素和水分均设置两个水平,分别为正常氮（6 mmol/L NO-3-N）、 低氮（0.3 mmol/LNO-3-N）、 正常供水（保持盆中砂子相对含水量为饱和含水量的80%~85%）、 干旱处理（保持盆中砂子相对含水量为饱和含水量的60%~65%）。【结果】富士和秦冠的生物量（茎和叶）、 株高茎粗等生长指标以及光合速率、 气孔导度、 蒸腾速率均为正常氮正常水（ZZ）＞低氮正常水（DZ）＞正常氮干旱（ZG）＞低氮干旱（DG）,并且相对应处理下秦冠的以上指标均高于富士;正常供水下,缺氮处理使富士、 秦冠的根冠比比正常氮处理均有所增加,富士提高了2.05%,秦冠提高了22.40%。富士和秦冠的氮、 磷、 钾含量均表现出正常氮正常水（ZZ）＞低氮正常水（DZ）＞正常氮干旱（ZG）＞低氮干旱（DG）; 氮、 钾元素含量在植株各部位的分布顺序依次是叶＞根＞茎,磷元素则是根＞叶＞茎;光合氮素利用效率（PNUE）和氮素利用效率表现为秦冠处理之间差异极显著,富士处理之间差异不显著;秦冠的PNUE和NUE明显高于富士,在低氮正常水（DZ）处理下,秦冠氮肥利用率比富士高42.07%,在低氮干旱（DG）处理下高64.14%;低氮胁迫下富士和秦冠的NUE显著提高,并且秦冠提高的幅度高于富士。【结论】施用氮肥能够显著提高富士与秦冠的干物质量,同等水肥条件下,秦冠生长优于富士;水分亏缺会减少叶片对氮的吸收,干旱条件下适度增施氮肥,可提高果树的抗旱能力;低氮干旱胁迫下秦冠的生长指标、 光合指标及氮素利用效率指标均优于富士,表现出较强的抗低氮干旱胁迫的能力。  相似文献   

12.
Laser-induced breakdown spectroscopy (LIBS) is a new technique for the analysis of plant material. This study investigates the application of LIBS to pasture-based plant samples. The LIBS measurements were obtained from pelletized pasture samples (100 samples) that had been also analyzed by inductively coupled plasma–optical emission spectroscopy (ICP-OES) following microwave digestion for calibration and comparison purposes. Comparisons for elements sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), manganese (Mn), iron (Fe), copper (Cu), zinc (Zn), boron (B), phosphorus (P), and sulfur (S) showed that LIBS could be used for almost all the standard profile total elements with concentrations down to low mg/kg levels (observed error of Na: 0.024 percent, K: 0.18 percent, Mg: 0.016 percent, Ca: 0.073 percent, P: 0.017 percent, Mn: 31 mg/kg, Fe: 150 mg/kg, Zn: 6.6 mg/kg, and B: 1.1 mg/kg). Elemental analysis at less than mg/kg levels was not possible using LIBS. The elements S and Cu were particularly difficult to analyze with reliability using LIBS at the concentration levels found in the plant samples. Replacing microwave digestion and subsequent ICP analysis with a direct analysis of dried plant samples using LIBS has the potential to improve the productivity and reduce the cost of testing.  相似文献   

13.
正The Center for Agricultural Resources Research(CARR),the Institute of Genetics and Developmental Biology(IGDB),Chinese Academy of Sciences,invites applicants for several research group leader positions.CARR is one of the research organizations in Chinese Academy of Sciences(CAS).We seek nominations and applications from individuals who have expertise and a record of accomplishment in research areas related to ecology,agro-hydrology,  相似文献   

14.
The responses of tallgrass prairie plant communities and ecosystem processes to fire and grazing are well characterized. However, responses of invertebrate consumer groups, and particularly soil-dwelling organisms, to these disturbances are not well known. At Konza Prairie Biological Station, we sampled soil macroinvertebrates in 1994 and 1999 as part of a long-term experiment designed to examine the effects and interactions of annual fire, mowing, and fertilization (N and P) on prairie soil communities and processes. For nearly all taxa, in both years, responses were characterized by significant treatment interactions, but some general patterns were evident. Introduced European earthworms (Aporrectodea spp. and Octolasion spp.) were most abundant in plots where fire was excluded, and the proportion of the total earthworm community consisting of introduced earthworms was greater in unburned, unmowed, and fertilized plots. Nymphs of two Cicada genera were collected (Cicadetta spp. and Tibicen spp.). Cicadetta nymphs were more abundant in burned plots, but mowing reduced their abundance. Tibicen nymphs were collected almost exclusively from unburned plots. Treatment effects on herbivorous beetle larvae (Scarabaeidae, Elateridae, and Curculionidae) were variable, but nutrient additions (N or P) usually resulted in greater densities, whereas mowing usually resulted in lower densities. Our results suggest that departures from historical disturbance regimes (i.e. frequent fire and grazing) may render soils more susceptible to increased numbers of European earthworms, and that interactions between fire, aboveground biomass removal, and vegetation responses affect the structure and composition of invertebrate communities in tallgrass prairie soils.  相似文献   

15.
Abstract

A 3-year study was carried out to investigate quality parameters in 14 tree fruit and berry species grown in southern Norway. The species were blueberry, apple, aronia, sour cherry, sweet cherry, red raspberry, strawberry, blackcurrant, gooseberry, red currant and elderberry, harvested along with wild bilberry, cloudberry and lingonberry. Significant differences between species were identified for all quality parameters. The coefficient of variation between species was lowest for pH (12.5%), dry matter (18.9%) and soluble solids (25.3%), followed by titratable acids (59.3%), total phenolics (83.8%), antioxidant capacity FRAP (85.7%) and antiradical power by the DPPH-assay (97.8%), total monomeric anthocyanins (132%) and ascorbic acid (137%). Average coefficient of variation within species were lower and ranged from 4 (pH) to 62% (ascorbic acid). Only the FRAP values were significantly affected by harvesting year with lower levels in 2004 than in 2005 and 2006. There were significant interactions between species and harvesting year for dry matter, soluble solids, pH, ascorbic acid and FRAP. The results indicate generic ranges in composition within species independent upon growing location and climate, and the composition of the tree fruits and berries is not likely to deviate from these ranges. It is concluded that desirable composition of tree fruits and berries and their products should primarily be achieved by selection among species rather than searching fors broadened variation within individual species.  相似文献   

16.
Potassium (K) fixation and release in soil are important factors in the long-term sustainability of a cropping system. Changes in K concentration and characteristics of K fixation and release in rhizosphere and nonrhizosphere soils in the rapeseed (Brassica napus L.)–rice (Oryza sativa L.) rotation were investigated using a rhizobox system. The concentrations of different forms of K in both rhizosphere and nonrhizosphere soils decreased with plants compared to without plants, regardless of K fertilizer application. Potassium uptake by crops mainly came from the rhizosphere soil. In the treatment without K fertilizer (–K), the main form of K supplied by the soil to the crops was 1.0 mol L?1 nitric acid (HNO3) nonextractable K, followed by nonexchangeable K, and then exchangeable K. In the treatment with K fertilizer (+K), the main K forms supplied by the soil to the crops were exchangeable K and nonexchangeable K. The amount and rate of K fixation after one cycle of the rapeseed–rice rotation was greater in rhizosphere soil than in nonrhizosphere soil. The amount and rate of K fixation of soil in the +K treatment were significantly less than in the –K treatment. The cumulative amounts of K released with 1.0 mol L?1 ammonium acetate (NH4OAc) and 1.0 mol L?1 HNO3 extraction increased with the increasing numbers of extractions, but the K-releasing power of soil by successive extraction decreased gradually and finally became almost constant. The release of K was less in rhizosphere soil than in nonrhizosphere soil. The release of K in the +K treatment was similar to that in the –K treatment in rhizosphere soil, but the K release in nonrhizosphere soil was greater with the +K than the –K treatment. Overall, the information obtained in this study will be helpful in formulating more precise K fertilizer recommendations for certain soils.  相似文献   

17.
Biologically enhanced dissolution offers a method to speed removal of chlorinated solvent dense non-aqueous-phase liquid (DNAPL) sources such as tetrachloroethene (PCE) and trichoroethene (TCE) from aquifers. Bioremediation is accomplished by adding an electron donor to the source zone where fermentation to intermediates leading to acetic acid and hydrogen results. The hydrogen and possibly acetic acid are used by dehalogenating bacteria to convert PCE and TCE to ethene and hydrochloric acid. Reductive dehalogenation is thus an acid forming process, and sufficient alkalinity must be present to maintain a near neutral pH. The bicarbonate alkalinity required to maintain pH above 6.5 is a function of the electron donor: 800 mg/L of bicarbonate alkalinity is sufficient to achieve about 1.2 mM TCE dechlorination with glucose, 1.7 mM with lactate, and a much higher 3.3 mM with formate. Laboratory studies indicate that in mixed culture, formate can be used as an electron donor for complete conversion to ethene, contrary to pure cultures studies indicating it cannot. Various strategies can be used to add electron donor to an aquifer for DNAPL dehalogenation while minimizing pH problems and excessive electron donor usage, including use of injection-extraction wells, dual recirculation wells, and nested injection-extraction wells.  相似文献   

18.
Staff members of the Department of Botany of Palacký University in Olomouc and Gene Bank Department – Workplace Olomouc, Research Institute of Crop Production in Prague, Czech Republic, conducted an expedition in seven European countries (Austria, Czech Republic, France, Germany, Italy, the Netherlands, Switzerland) in August/September 1999 to collect wild Lactuca spp. germplasm and study its geographic distribution, ecology and biodiversity. During the mission, more than 600 locations were visited resulting in the collection of 602 seed samples (accessions) of wild Lactuca species and 13 seed samples of related genera (Chondrilla and Mycelis). Lactuca serriola f. serriola, L. serriola f. integrifolia, L. saligna and L. viminea subsp. chondrilliflora were prevalent in southern Europe (Italy, France), however, only L. serriola was common in central and western Europe (Austria, Czech Republic, Germany, Netherlands, Switzerland). The greatest diversity of Lactuca species was found in France, where also the most seed samples (165) were collected. The most characteristic habitats with a high density of Lactuca spp. populations were observed along roads and highways, grassy ditches, ruderal communities, and dust-heaps. Natural infections by powdery mildew (Erysiphe cichoracearum) and downy mildew (Bremia lactucae) on some wild Lactuca spp. were observed. Recent observations concerning the geographic distribution, population structure, habitats, and natural occurrence of diseases of Lactuca spp. are discussed. This assemblage of genetic resources of Lactuca spp. can serve as the basis of future studies of species diversification, spatial population structure, plant microevolution, domestication processes, and genetic variability of host-parasite interactions.  相似文献   

19.
Inoculation of wheat seedlings with the plant growth-promoting bacterium Azospirillum brasilense Cd was immobilized in alginate microbeads and, without applying any stress, significantly increased the quantity of several photosynthetic pigments, such as chlorophyll a, chlorophyll b, and the auxiliary photoprotective pigments violaxanthin, zeaxanthin, antheroxanthin, lutein, neoxanthin, and β-carotene. This resulted in greener plants with no apparent visible stress. After monitoring the quantity of photosynthetic pigments for 4 weeks, we observed that inoculated plants had higher quantities of pigments in shoot and stem. The greatest difference in the quantity of all pigments between inoculated and noninoculated plants occurred in the first week of growth. Regardless of treatment, the quantity of pigments in stems was three to four times less than the quantity of these pigments in shoots. Application of Azospirillum, either as liquid inoculant or as alginate microbeads, did not alter the positive effect of the bacteria on pigment production or the positive response of the plants towards A. brasilense Cd inoculation.  相似文献   

20.
Three slow-growing legume trees used for desert reforestation and urban gardening in the Sonoran Desert of Northwestern Mexico and the Southwestern USA were evaluated whether their growth can be promoted by inoculation with plant growth-promoting bacteria (Azospirillum brasilense and Bacillus pumilus), unidentified arbuscular mycorrhizal (AM) fungi (mainly Glomus sp.), and supplementation with common compost under regular screenhouse cultivation common to these trees in nurseries. Mesquite amargo (Prosopis articulata) and yellow palo verde (Parkinsonia microphylla) had different positive responses to several of the parameters tested while blue palo verde (Parkinsonia florida) did not respond. Survival of all tree species was over 80% and survival of mesquite was almost 100% after 10 months of cultivation. Inoculation with growth-promoting microorganisms induced significant effects on the leaf gas exchange of these trees, measured as transpiration and diffusive resistance, when these trees were cultivated without water restrictions.  相似文献   

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