一种无血清冻存液在猪胎儿成纤维细胞上的应用研究

在猪胎儿成纤维细胞(porcine fetal fibroblasts, PFF)冻存过程中,血清品质常常制约着细胞的冻存效果。为了解决这个问题,本研究旨在开发一种无血清冻存液应用于猪胎儿成纤维细胞冻存。用3种不同冻存液冻存猪胎儿成纤维细胞,每种冻存10管。冻存30 d后复苏细胞,测定冻存细胞存活率,细胞增殖活力以及电转后细胞活性。结果显示:自制无血清细胞冻存液,冻存猪胎儿成纤维细胞后存活率达95.33%;细胞增殖活力以及电转后细胞活性均显著高于标准胎牛血清冻存液(p<0.05),与特级胎牛血清冻存液效果相当(p>0.05)。因此,自制冻存液冻存猪胎儿成纤维细胞效果稳定,能够替代含血清冻存液,有良好的推广应用前景。     

猪卵母细胞的体外受精及多精受精

对用于猪体外受精(IVF)的研究方法和技术,如传统的液滴IVF、透明带下注射精子受精(SUZI)、卵母细胞质内单精注射受精(ICSI)及细管IVF等进行了简述。与其它动物相比,进行猪卵的体外受精研究,多精受精现象特别明显。大量的研究表明,猪卵的多精受精不但与其品种特性有关,而且与卵母细胞成熟的程度、透明带的异常、受精时获能精子的浓度、输卵管分泌物、受精液蛋白添加成分、NaHCO3浓度、咖啡因、pH值以及温度等因素密切相关。     

猪精原干细胞研究进展

精原干细胞（spermatogonial stem cells,SSCs）是雄性生殖系干细胞,位于睾丸曲细精管基膜上,既具有自我更新潜能,又具有定向分化潜能,是自然状态下出生后动物体内在整个生命过程中进行自我更新并能将基因传递至子代的惟一成体干细胞。根据国内外最新相关进展,系统评述了猪SSCs分离纯化、体外培养及移植等方面的现状及问题。     

猪气管剥离标本的制作

猪气管剥离标本的制作陈景红四川省石柱县中学６４８１００）ｌ选材选择新鲜、完整的猪肺，要带有一段完整的气管、（新鲜的肺有韧性。完整指无划口，无缺失，带一段气管既便于操作也便于修整）。２剥离将材料──猪肺洗净后，平放在解剖盘中，用镊子插入肺组织并顺着支气...     

精参颗粒的主要药效学实验研究

目的：研究精参颗粒的补益（改善气虚和血虚作用）、抑茵、抗炎及镇痛作用,同时探讨并确定其量效和时效关系。方法：采用“气虚”、“血虚”、“气血双虚”和“耳廓肿胀”、“足肿胀”、“子宫炎症”小鼠、大鼠模型及体内、外的抗菌试验,观察精参颗粒灌胃给药不同剂量和不同时间对各模型病证的影响。结果：①3．0g/kg、5．0g/kg和7．0g像g精参颗粒灌胃14天可显著提高“气虚”小鼠游泳耐力（P〈0．01）,对萎缩的胸腺、脾脏、肝脏具有促进恢复的作用（P〈0．05或P〈0．01）;②中、高剂量精参颗粒对小鼠“血虚”和大鼠“气血双虚”证所致的红细胞（RBc）、血红蛋白（Hb）、白细胞（WBC）、血小板（PLT）、体重下降具有显著升高作用（P〈0．05或P〈0．01）,其中对大鼠“气血双虚”证的作用随给药时间延长而增强;③对金黄色葡萄球菌、绿脓杆菌、大肠杆菌,中、高剂量精参颗粒具有明显抑制和抗感染作用（P〈0．01）;④中、高剂量精参颗粒对小鼠和大鼠各炎症有显著的抗炎作用（P〈O．05或P〈0．01）,其中对大鼠子宫炎的作用随给药时间延长而增强;⑤中高剂量精参颗粒能显著提高小鼠热板所致的痛阈（P〈0．01）;明显降低冰醋酸所致的扭体次数（P〈0．05或P〈0．01）;表明精参颗粒具有镇痛作用。结论：①精参颗粒具有改善“气虚和血虚”等病证、抑制和抗细菌感染、抗炎及镇痛作用;且其作用呈现较好的量效及时效关系。②按小鼠（5．0g／kg）和大鼠（3．5眺g）有效剂量计算,并结合临床用药的可操作性,拟推荐精参颗粒临床成人日用剂量为一次12g（1包）,一日3次。⑧根据动物实验的时效关系,结合慢性盆腔炎临床反复发作和治疗周期较长的特点,拟推荐临床用药4周为一疗程。     

一种简单实用的提取植物DNA的方法

ASimpleandPracticalMethodforIsolationofPlantDNAQIUGuo－Hua,XUZeng－Fu．CAOJi－Xiang,LIBao－Jian（BiotechnologyResearchCenter,ZhongshanUniversity,Guangzhou510275）植物DNA的提取是植物分子生物学和基因工程研究的一种基本的技术,至今已有了数种方法[1,2]。本文报道一种新的方法,与以前的方法相比,它最大的不同点是：将SDS同时用作细胞膜的去污剂和蛋白质的变性剂,变性的蛋白质通过离心而木是酚抽除去,使得整个操作过程变得简单快捷,比较温和,1h左右就可以获得纯化的植物DNA。用这种方法得到的DNA能…     

介绍一种快速混合的苏木精染液

苏木精(Hematoxylin)系从豆科植物苏木(Caesa lpinia sappan)中用乙醚浸制提炼而成,是一种常用的优良核染料。在苏木精的结构式中(见图1、2),因无双键的醌式结构,无发色团,所以它本身不是染料而必须经氧化成为氧化苏木精时才能染色。这个过程称为“成熟”。使苏木精“成熟”的方法有二:一是在苏木精溶液中加入氧化剂如氧化汞,高锰酸钾,碘酸钠,重铬酸钾等,使苏木精脱氢成为醌,才呈发色团,这个过程较快;二是配制成苏木精原液并暴露于空气中3-6周,使其氧化,这个过程较慢。另外,由于苏木精对组织的亲合力很小,即使成熟后变为氧化苏     

猪细小病毒vp2基因的表达和类病毒颗粒的构建

利用PCR方法从病毒基因组中获得了vp2完整的编码区并经测序后克隆到原核表达载体pET-32(a)上.在大肠杆菌中诱导表达了VP2与Trx-His-S Tag的融合蛋白,通过免疫家兔获得了高特异性的兔抗血清.另外,将vp2克隆到pFastBacDUAL载体上,利用昆虫表达系统,即AcMNPV的Bac-to-Bac系统对vp2进行了表达,经SDS-PAGE和Western blot分析,表达产物为64kD,并且该蛋白能在昆虫细胞中形成类病毒颗粒.     

一种简单实用的连续半薄切片技术

一种简单实用的连续半薄切片技术常规的半薄切片法有两种：一是干刀法，玻璃刀不装水槽，切一片，用尖头镊子夹取切片边缘，放到滴有蒸馏水的载玻片上。二是湿刀法，玻璃刀上安装水槽，切一片，待其在水中展开，用睫毛针从边缘捞起，放于载玻片的水滴中。在没有专用超薄切...     

小鼠嵌合体制作技术的研究

本研究采用人工聚合技术,将小鼠不同品系和同品系之间的8细胞胚聚合为一体,分别用PBS FCS、BWW和BMOC-Ⅲ培养20—24小时后观察了发育率。其结果在昆明白←→C57中分别有88.1％(59/67)、95.0％(115/121)和94.8％(73/77),在昆明白←→昆明白中分别有93.3％(28/30)、87.7％(121/138)和86.7％(52/60)的嵌合胚发育为桑椹胚或胚泡,各处理组之间无显著差异。将141枚嵌合胚分别移植给12只受体,结果有7只小鼠妊娠共产仔25只。在昆明白←→C57的17只嵌合体小鼠中毛色为黑、白和杂色的各有3、4和10只。     

介入法建立猪急性心肌梗死再灌注模型及其效果评价

目的:用介入法行球囊堵闭猪冠状动脉左前降支(LAD)建立急性心肌梗死再灌注动物模型并评价其效果。方法:选用小型雄性家猪11只。麻醉后经股动脉或颈总动脉置入经皮腔内冠状动脉成形术(PTCA)球囊至冠状动脉LAD远端,堵闭血流60 min。术中持续静脉滴注胺碘酮。60min后负压撤除球囊及鞘管。行超声心动图、磁共振成像(MRI)评价心肌梗死模型建立情况,并行病理分析。结果:2只猪死于心肌梗死模型制备,存活的9只猪经超声心动图、MRI检测显示梗死部位均为左心室前壁、心尖区,部分模型累及室间隔,左室下壁,并有室壁瘤、室速等心肌梗死常见并发症的发生。结论:运用介入法经PTCA球囊LAD可成功建立猪急性心肌梗死模型。这种模型重复性强,可控性好,模型的梗死部位基本一致,易于术后评价。     

A型猪流感病毒山东分离株鉴定及其HA基因序列分析

从山东各地疑似流感发病猪分离到10株流感病毒,经国家流感中心鉴定均为A型流感病毒H9N2亚型.将其中一株Sw/SD/1/2003(H9N2)的血凝素全基因(HA)进行克隆与测序,与GenBank收录的其它猪流感和禽流感H9N2 亚型的HA基因进行比较,发现Sw/SD/1/2003(H9N2)的血凝素基因在核苷酸序列方面同广西1999年分离的禽流感毒株Ck/GX/99(H9N2)和2000年云南分离的禽流感毒株Ck/YN/2000(H9N2)的同源性最高;进化树分析表明Sw/SD/1/2003 (H9N2) 起源于禽源的H9N2亚型流感病毒;Sw/SD/1/2003 的HA氨基酸裂解位点与其他H9N2亚型不同,Sw/SD/1/2003 的HA氨基酸裂解位点是R-S-L-R-G, 而其它猪流感和禽流感H9N2亚型都是R-S-S-R-G.     

A型猪流感病毒山东分离株鉴定及其HA基因序列分析

从山东各地疑似流感发病猪分离到10株流感病毒,经国家流感中心鉴定均为A型流感病毒H9N2亚型。将其中一株Sw／SD／1／2003(H9N2)的血凝素全基因(HA)进行克隆与测序,与GenBank收录的其它猪流感和禽流感H9N2亚型的HA基因进行比较,发现Sw／SD／1／2003(H9N2)的血凝素基因在核苷酸序列方面同广西1999年分离的禽流感毒株Ck／GX／99(H9N2)和2000年云南分离的禽流感毒株Ck／YN／2000(H9N2)的同源性最高;进化树分析表明Sw／SD／1／2003(H9N2)起源于禽源的H9N2亚型流感病毒;Sw／SD／1／2003的HA氨基酸裂解位点与其他H9N2亚型不问,Sw／SD／1／2003的HA氨基酸裂解位点是R-S-L-R-G,而其它猪流感和禽流感H9N2亚型都是R-S-S-R-G。     

A simple and reliable method for clinical assessment of odor thresholds

We investigated whether presenting of dilutions of phenyl ethyl alcohol at random succession according to the method of constant stimuli can replace the standard procedure of presenting a various number of dilutions in a staircase paradigm. Forty-six men and 44 women, aged 19-76 years, participated in this study. Phenyl ethyl alcohol was diluted in a ratio of 1:2, starting from 4%. Presentation of the odorant followed a three-alternative, temporal forced-choice paradigm with two blanks in addition to the odorant. Twenty dilutions were administered in a randomized order. Odor threshold was obtained by logistic regression of the correct and incorrect identifications of the probe containing the odorant. Thresholds were also calculated on the basis of the first 16 dilution steps only. Results from these procedures were compared with 'gold-standard' threshold assessment employing a three-alternative, temporal forced-choice staircase paradigm with seven reversals using 16 dilutions of phenyl ethyl alcohol. The method of constant stimuli took a shorter and less variable testing time than the staircase technique. The use of 20 dilution steps provided no better results than the use of 16 steps. The method of constant stimuli exhibited a good test-retest reliability (r = 0.7; P < 0.001) comparable to that of the staircase method and provided unbiased results highly correlated (r = 0.8; P < 0.001) with those of the staircase technique with similar inter-test variability. Applying 16 dilutions (1:2 steps) of phenyl ethyl alcohol at random succession in a three-alternative, temporal forced-choice paradigm is thus a simple and reliable procedure for the reproducible assessment of odor thresholds that may be contemplated as an alternative to the 'gold-standard' staircase method of clinical odor threshold assessment.     

深圳福田红树林生态造林方法技术研究

为了增加深圳红树林生物多样性而引种海南岛的银叶树、海芒果、海桑、水椰、海莲、地瓣海桑、正红树等,其技术关键是抗低温使引种成功,为了大面积造林成功,选择当地优势种秋茄,桐花树、白骨壤及木榄进行繁殖试验,其关键问题是不同种类适合于不同水深及淤泥深度,大面积造林成功的技术关键是种植时间、插植方法等。     

A simple field validation of daily transpiration derived from sapflow using a porometer and minimal meteorological data

Heat-pulse techniques are routinely used to estimate transpiration from canopies of woody plants typically without any local calibration, mainly because of the difficulty of doing so in the field and, frequently, lack of detailed weather data. This is despite concerns that the techniques may produce erroneous values under certain conditions, such as when evaporative demand is high. In this study, we used a micrometeorological approach to validate transpiration from irrigated olives deduced from heat-pulse technique by ascertaining precise values for the parameters that are critical for converting heat-pulse velocity to sapflow. The micrometeorological approach involved limited data on stomatal conductance (g_s), obtained hourly with a porometer on four contrasting days, and was used to calibrate a simple model for predicting conductance. Predicted stomatal conductance (g_sm) agreed well with that measured, and when both were used to calculate hourly transpiration, they produced values that were within 10% of each other. This was despite brief underestimations of transpiration based on g_sm (T_m) in the early hours of the day that arose from poor determination of incident radiation at this time. We then used T_m to iteratively set the values for the various parameters, including the time-out value that accounts for zero-flow conditions, needed to convert heat-pulse velocity to sapflow, for the four days. The best fit between T_m and transpiration from sapflow (T_s) was obtained with time-out value set to 120 s. All heat-pulse velocity data were therefore analysed with this time-out value to obtain sapflow and, hence, transpiration (T_s). Comparison of T_m and T_s for the whole season showed that the former tended to produce higher values on certain days when vapour pressure deficit (D) was high in summer (December–February). While T_s occasionally produced larger values than T_m under the mild conditions of autumn (March–April). Totals of the daily transpiration during the 190-day period were within 10% of each other. Responsible Editor: Yan Li     

A simple urea leaf-feeding method for the production of 13C and 15N labelled plant material

The use of ¹³C isotope tracer techniques in terrestrial ecology has been restricted by the technical requirements and high costs associated with the production of ¹³C enriched plant material by ¹³CO₂ release in labelling chambers. We describe a novel, simple and relatively inexpensive method for the small-scale production of ¹³C and ¹⁵N labelled plant material. The method is based on foliar feeding of plants with a urea solution (97 atom% ¹³C, 2 atom% ¹⁵N) by daily misting. Maize was grown in a greenhouse in a compost–soil mixture and enclosed in clear polythene bags between urea applications. Final enrichment in 27 d old maize shoots was 211 ¹³C (1.34 atom% ¹³C) and 434 ¹⁵N (0.52 atom% ¹⁵N). Enrichments of hot-water extractable fractions (289 ¹³C, 469 ¹⁵N) were only slightly higher than those observed in plant bulk material, which suggests that daily urea applications ensured fairly uniform labelling of different biochemical fractions and plant tissues. Recovery of applied excess ¹³C and ¹⁵N in plant shoots was 22% and 42%, respectively. Roots were less enriched (21 ¹³C and 277 ¹⁵N), but no attempts were made to recover roots quantitatively.