小鼠胚胎成纤维细胞饲养层制备条件的研究

为了建立活力稳定的小鼠胚胎成纤维细胞饲养层,本实验以13．5～14．5d的ICR小鼠胚胎为实验材料,以MTT法测定细胞活力,探讨了不同浓度的丝裂霉素C、丝裂霉素C不同处理时间及不同的成纤维细胞接种密度对MEF活力的影响。结果表明：10μg／ml的丝裂霉素C处理后的MEF活力比其他浓度（1μg／ml、5μg／ml、20μg／ml、30μg／ml）丝裂霉素C处理后的MEF活力稳定;用10μg／ml的丝裂霉素C处理2．5h,MEF活力比处理1．5h、3．5h、4．5h稳定;以2．5～35×10^5／ml密度接种于96孔板上,每孔200μl,MEF活力较接种密度为1．5×10^5／ml、4．5×10^5／ml、7．5×10^5／ml稳定。结论：用10μg／ml丝裂霉素C处理MEF 2．5h,2．5～3．5×10^5／m;密度接种于96孔板上,每孔200μl,以10％的胎牛血清培养液培养能得到活力稳定高效的小鼠胚胎成纤维细胞饲养层。     

丝裂霉素C对ICR小鼠胚胎成纤维细胞饲养层制备的影响

目的：探索不同浓度的丝裂霉素c（MMC）处理对ICR小鼠胚胎成纤维细胞饲养层制备的影响,用于分离和培养干细胞。方法：取13．5d胎龄ICR鼠胚分离原代成纤维细胞,利用不同浓度MMC处理胚胎成纤维细胞制备饲养层,比较观察MMC对细胞增殖的抑制作用,制作细胞生长曲线;取妊娠3．5d的ICR小鼠胚胎在不同浓度MMC处理的饲养层上培养,观察胚胎贴壁和增殖情况。结果与结论：胎儿成纤维细胞贴壁生长,增殖迅速,6代后细胞逐渐衰老;MMC能有效抑制胎儿成纤维细胞的增殖,最佳处理方法为20μg／ml、2h。妊娠3．5d的ICR小鼠胚胎在10、20μg／mlMMC处理的饲养层上孵出率和贴壁率显著高于5／Lg／mlMMC处理的饲养层,在5、10和20μg／m[MMC处理的饲养层上,内细胞团增殖率无显著差异。因此,ICR小鼠胚胎成纤维细胞最佳处理方法为20μg／mlMMC作用2h。     

饲养层细胞的接种密度对分离培养胚胎干细胞的影响

建立有效的昆明白小鼠胎儿成纤维细胞(Mouse embryonic fibroblast,MEF)饲养层培养体系,用于分离和培养小鼠胚胎干细胞(Embryonic stem cell,ES细胞)的研究.(1)取怀孕14.5 d昆明白小鼠胎儿分离成纤维细胞,利用体外培养体系分离传代,选取生长旺盛并且已纯化的P3代的MEF,经丝裂霉素处理后.用细胞计数板计算活细胞数,分别按1×104、1×106、1×108/mL密度接种,制备饲养层,观察不同密度饲养层的生长状况.(2)取怀孕4 d的昆明白小鼠囊胚,接种在不同密度饲养层上.观察不同密度饲养层上囊胚、ICM及ES细胞的克隆生长情况.结果显示:囊胚在密度为1 × 106/mL的饲养层上,贴壁率和ICM孵出率分别为(97.0±3.606)%和(96.3±2.887)%,显著高于其他2组;密度为1×104/mL饲养层上的ES细胞克隆形成率高于密度为1×104/mL(差异极显著,P<0.01)和1×108/mL饲养层上的ES细胞克隆形成率(差异显著,P<0.05);而1×104/mL饲养层和1×108/mL饲养层上的ES细胞克隆形成率差异不显著(P>0.05).结果表明:以1×104/mL密度接种的MEF作为饲养层,最适合用于分离培养昆明白小鼠ES细胞,有利于囊胚的发育,ICM的增殖,促进ES细胞的增殖,并起到抑制其分化的作用.     

丝裂霉素C对ICR小鼠胚胎成纤维细胞饲养层制备的影响

<正>目的:探索不同浓度的丝裂霉素C(MMC)处理对ICR小鼠胚胎成纤维细胞饲养层制备的影响,用于分离和培养干细胞。方法:取13.5d胎龄ICR鼠胚分离原代成纤维细胞,利用不同浓度MMC处理胚胎成纤维细胞制备饲养层,比较观察MMC对细胞增殖的抑制作用,制作细胞生长曲线;取妊娠3.5d的ICR小鼠胚胎在不同浓度MMC处理的饲养层上培养,观察胚胎贴壁和增殖情况。结果与结论:胎儿成纤维细胞贴壁生长,增殖迅速,6代后细胞逐渐衰老;     

小鼠胚胎干细胞成纤维细胞液养层的制备

采用受孕12.5 d、13.5 d、14.5 d的ICR品系小鼠的胚胎制备原代成纤维细胞,在相同条件下观察不同胚龄胚胎成纤维细胞的增殖情况。结果表明受孕13.5 d胚胎为分离小鼠胚胎原代成纤维细胞的最适胚龄。分离出的原代成纤维细胞在37 ℃时,用0.25%胰蛋白酶0.02%EDTA消化组织的时间为3 min,可在培养3 d后传代。F1代和F2代较F3代成纤维细胞分泌的抑制干细胞分化因子LIF的量要多。小鼠胚胎成纤维细胞冻存于-135 ℃~-150 ℃10 个月,复苏后能正常传代。丝裂霉素C抑制胚胎成纤维细胞增殖的最适浓度为10μg/105细胞,作用时间为2.5 h,丝裂霉素C处理过的胚胎成纤维细胞可以在12 d内即不增殖也不死亡,但在6 d内使用效果最佳。     

丝裂霉素C对ICR小鼠胚胎成纤维细胞饲养层制备的影响

<正>目的:探索不同浓度的丝裂霉素C(MMC)处理对ICR小鼠胚胎成纤维细胞饲养层制备的影响,用于分离和培养干细胞。方法:取13.5d胎龄ICR鼠胚分离原代成纤维细胞,利用不同浓度MMC处理胚胎成纤维细胞制备饲养层,比较观察MMC对细胞增殖的抑制作用,制作细胞生长曲线;取妊娠3.5d的ICR小鼠胚胎在不同浓度     

水牛胚胎生殖干细胞饲养层体系的建立

本试验对水牛胚胎生殖干细胞饲养层体系建立进行了研究。首先比较了组织块法和酶消化法培养水牛胎儿成纤维细胞的差异;其次探讨丝裂霉素处理水牛胎儿成纤维细胞的有效时间;最后以建立的饲养层体系培养水牛胚胎生殖干细胞。结果表明：（1）组织块法培养的细胞状态优于酶消化法,组织块培养法更适于水牛胎儿成纤维细胞的分离培养;（2）第三代的水牛胎儿成纤维细胞核型正常,状态良好,可用于饲养层的制备;（3）通过MTT和Brdu法检测,确定10mg／L的丝裂霉素C处理水牛胎儿成纤维细胞3．5h能有效抑制成纤维细胞增殖且细胞形态良好;（4）在本试验条件下制备的饲养层能有效维持水牛胚胎生殖千细胞至8代。     

影响制备小鼠胎儿成纤维细胞饲养层的因素

为了建立活力稳定高效的小鼠胎儿成纤维细胞饲养层,实验以13.5~14.5 d的ICR小鼠胚胎为实验材料,以MTT法测定细胞活力,探讨了用丝裂霉素C处理MEF后,含有不同浓度胎牛血清的培养基对MEF活力的影响,以及不同传代次数的MEF对其活力的影响。结果表明:用含10%的胎牛血清培养液培养丝裂霉素C处理后的MEF,其活力比含2%、5%、20%的FBS培养液稳定。对不同传代次数的MEF进行处理后,第1、3代的MEF活力比第5、7代的MEF活力稳定。结论:用丝裂霉素C处理第1到第3代的MEF,以10%的胎牛血清培养液培养能得到活力稳定高效的小鼠胎儿成纤维细胞饲养层。     

昆明小鼠胚胎干细胞培养体系的优化

为了更高效地分离昆明小鼠胚胎干细胞,本研究从饲养层、胚胎发育阶段和培养液方面进行优化。将3代以内的小鼠胎儿成纤维细胞(MEF)用丝裂霉素C处理后,分别按1×104、1×105、1×106·mL-1密度接种,以H-DMEM+15%KSR+LIF为培养液,观察不同密度饲养层对昆明小鼠胚胎干细胞(ES细胞)生长的影响,并研究胚胎发育阶段和培养液中分别添加干细胞生长因子(SCF)、SCF+胰岛素对昆明小鼠ES细胞分离克隆的影响。结果显示,胚胎在密度为1×105·mL-1的饲养层上,F1代和F2代ES细胞克隆形成率均显著高于其他2组(P<0.05)。囊胚的F2代ES细胞克隆形成率显著高于桑椹胚(P<0.05),培养液中添加SCF显著提高昆明小鼠胚胎贴壁率(P<0.05),同时添加SCF和胰岛素得到昆明小鼠最高胚胎贴壁率及F1、F2代ES细胞克隆形成率。所分离的ES细胞显示AKP染色强阳性,Oct-4、SSEA-1的免疫荧光检测阳性,具有ES细胞的特点。由此认为,发育至囊胚的胚胎在MEF密度为1×105·mL-1上,培养液中同时添加SCF和胰岛素更适合昆明小鼠ES细胞的分离培养。     

人胚胎原始生殖细胞的原代培养

从5－9周人胚胎生殖嵴中消化分离原生殖细胞（PGC），将其置于经过丝裂霉素C处理过的单层小鼠胚胎成纤维细胞（PMEF）饲养层上培养。结果，经过培养形成的克隆以及克隆的生长方式基本符合小鼠胚胎干细胞（ES）的特性，表现为细胞核大，细胞排列紧密，界限不清，集落边缘可见分化的成纤维细胞等；将其接种于裸鼠皮下，经过29d生长，得到具有3个胚层组织结构的畸胎瘤。     

Phase I evaluation of CCNU (lomustine) in tumor-bearing cats

1-(2-Chloroethyl)3-cyclohexyl-1-nitrosourea (CCNU) is an alkylating agent in the nitrosourea subclass. A prospective evaluation of CCNU was done to determine the maximally tolerated dosage of CCNU in tumor-bearing cats. Response data were obtained when available. Twenty-five cats were treated with CCNU at a dosage of 50-60 mg/m3 body surface area. Complete hematologic data were available for 13 cats. Neutropenia was the acute dose-limiting toxicity. The median neutrophil count at the nadir was 1,000 cells/microL (mean, 2,433 cells/microL; range, 0-9,694 cells/microL). The time of neutrophil nadir was variable, occurring 7-28 days after treatment, and counts sometimes did not return to normal for up to 14 days after the nadir. Based on these findings, a 6-week dosing interval and weekly hematologic monitoring after the 1st treatment with CCNU are recommended. The nadir of the platelet count may occur 14-21 days after treatment. The median platelet count at the nadir was 43,500 cells/microL. No gastrointestinal, renal, or hepatic toxicities were observed after a single CCNU treatment, and additional studies to evaluate the potential for cumulative toxicity should be performed. Five cats with lymphoma and 1 cat with mast cell tumor had measurable responses to CCNU. Phase II studies to evaluate antitumor activity should be completed with a dosing regimen of 50-60 mg/m3 every 6 weeks.     

Furosemide continuous rate infusion in the horse: evaluation of enhanced efficacy and reduced side effects

Continuous rate infusion (CRI) of furosemide in humans is considered superior to intermittent administration (IA). This study examined whether furosemide CRI, compared with IA, would increase diuretic efficacy with decreased fluid and electrolyte fluctuations and activation of the renin-angiotensin-aldosterone system (RAAS) in the horse. Five mares were used in a crossover-design study. During a 24-hour period, each horse received a total of 3 mg/kg furosemide by either CRI (0.12 mg/kg/h preceded by a loading dose of 0.12 mg/kg IV) or IA (1 mg/kg IV q8h). There was not a statistically significant difference in urine volume over 24 hours between methods; however, urine volume was significantly greater after CRI compared with IA during the first 8 hours ([median 25th percentile, 75th percentile]: 9.6 L [8.9, 14.4] for CRI versus 5.9 L [5.3, 6.0] for IA). CRI produced a more uniform urine flow, decreased fluctuations in plasma volume, and suppressed renal concentrating ability throughout the infusion period. Potassium, Ca, and Cl excretion was greater during CRI than IA (1,133 mmol [1.110, 1,229] versus 764 mmol [709, 904], 102.7 mmol [96.0, 117.2] versus 73.3 mmol [65.0, 73.5], and 1,776 mmol [1,657, 2.378] versus 1,596 mmol [1,457, 1,767], respectively). Elimination half-lives of furosemide were 1.35 and 0.47 hours for CRI and IA, respectively. The area under the excretion rate curve was 1,285.7 and 184.2 mL x mg/mL for CRI and IA, respectively. Furosemide CRI (0.12 mg/kg/h) for 8 hours, preceded by a loading dose (0.12 mg/kg), is recommended when profound diuresis is needed acutely in horses.     

Prevalence of gastric lesions in racing Alaskan sled dogs

Human and equine athletes are reported to have a high prevalence of gastric disease, and anecdotal evidence suggests a similar phenomenon applies to racing sled dogs. To investigate the prevalence of gastric disease in racing sled dogs, we conducted 2 gastroscopy studies on dogs competing in the annual Iditarod Sled Dog Race. A pilot study of dogs that were either dropped from the 2000 Iditarod Sled Dog Race because of illness or that finished the race indicated that, approximately 5 days after competing, 10 of 28 dogs (35%) had endoscopic evidence of gastric ulceration, erosion, or hemorrhage. The next year, an endoscopic study of 73 dogs participating in the 2001 Iditarod race was performed in order to evaluate a larger population of dogs. Data from 70 of these dogs could be used; 34 (48.5%) had ulceration, erosion, gastric hemorrhage, or some combination of these findings. When this group of 70 dogs was compared retrospectively to a control group of 87 dogs presented to the Texas A&M University (TAMU) Veterinary Medical Teaching Hospital, the Iditarod sled dogs had a significantly higher prevalence (P = .049) of gastric lesions. These findings suggest that, similar to athletes of other species, elite canine athletes have an increased prevalence of gastric disease compared to the canine population at large.     

Internal Parasites and Haematological Values in Cattle Slaughtered in Buea Subdivision of Cameroon

Tropical Animal Health and Production -     

The Epidemiology of Gastrointestinal Nematodes of Dairy Cattle in Central Kenya

The epidemiology of H. placei and of other gastrointestinal nematodes in yearling dairy cattle was examined on two farms in Kiambu District, central Kenya during each of 13 one-month periods from April 1993 to April 1994. On each farm, 32 newly weaned dairy calves were given a single dose of albendazole and then placed on experimental pastures. Twelve of the animals were designated for bi-monthly slaughter (n = 2) and analysis of worm population characteristics and 20 were designated for blood and faecal collection and for weighing. Two parasite-free tracer calves were grazed alongside the weaner calves each month throughout the study period and were also slaughtered for analysis of worm populations. Faecal egg counts, haematological and serum pepsinogen determinations, herbage larval counts, and animal live weight changes were recorded monthly. The study revealed that Haemonchus placei, Trichostrongylus axei, Cooperia spp. and Oesophagostomum radiatum were responsible for parasitic gastroenteritis and that H. placei was the predominant nematode present in the young cattle on both farms. Faecal egg counts from resident cattle and necropsy worm counts revealed that pasture larval levels were directly related to the amount of rainfall. The total worm burdens in the animals were highest during the rainy season (March–June and October–December) and lowest during the dry seasons (July–September and January–February). The very low recovery of immature larvae of H. placei from the tracer calves indicated that arrested development is not a feature of the life cycle of this parasite in central Kenya. The maintenance of the parasite population depended on continuous cycling of infection between the host and the pasture. The agroclimatic conditions of the study area were such that, in general, favourable weather conditions for the development and survival of the free-living stages of gastrointestinal nematodes existed all year round.     

Diversity of Ectoparasites in Sheep Flocks in Sa~o Paulo,Brazil

The occurrence of ectoparasites in sheep flocks is frequently reported but seldom quantified. Sheep production used to be a predominantly family activity in the state of Sa~o Paulo (Brazil), but it began to become a commercial activity in the past decade. Thus, information about the ectoparasites existing in sheep flocks has become necessary. The present data were obtained by means of questionnaires sent to all sheep breeders belonging to the `Associaça~o Paulista de Criadores de Ovinos' (ASPACO; Sa~o Paulo State Association of Sheep Breeders). Response reliability was tested by means of random visits paid to 10.6% of the respondents. Most of the properties (89.5%) reported the presence of one or more ectoparasites. Screw-worm (Cochliomyia hominivorax) was the most frequent ectoparasite (72.5%), followed by bot fly larvae (Dermatobia hominis, 45.0%), ticks (Amblyomma cajennense) and Boophilus microplus, 31.3%) and finally lice (Damalinia ovis, 13.8%). Combined infestations also occurred, the most common one being screw-worm with bot fly larvae (36.0%) followed by bot fly larvae with ticks (13.9%), screw-worm with ticks (9.3%), bot fly larvae with lice (6.9%), and ticks with lice (5.0%). The most common triple combination was screw-worm, bot fly larvae and ticks (12.8%). Breeds raised for meat or wool were attacked by bot fly larvae and ticks more often than other breeds. Lice were only absent from animals of indigenous breeds. The relationships among these ectoparasites are discussed in terms of sheep breeds, flock size, seasonality and the ectoparasitic combinations on the host.     

Chemotaxis of macrophage in inflammation

Our particular attention in this article was given to natural mediators for macrophages isolated from the sites of tissue injury. A number of chemotactic factors, which may satisfy many criteria making them acceptable as inflammatory leucocyte chemotactic factors, has been separated. Among them, our laboratory has isolated three macrophage (monocyte) chemotactic factors (MCF-a, -b and -c). Their purification, characterization and functional specificity are discussed.     

Evaluation of biohydrogenation rate of canola vs. soya bean seeds as unsaturated fatty acids sources for ruminants in situ

An experiment was conducted to study disappearance of C14 to C18 fatty acids, lag times and biohydrogenation (BH) rates of C18 fatty acids of ground soya bean and canola seeds in situ. Three ruminally fistulated Dallagh sheep were used to determine ruminal BH of unsaturated fatty acids (UFAs). Differences in the disappearance of fatty acids through the bags and lag times were observed between the oilseeds. We saw that the longer the incubation time of the oilseeds in the rumen, the lower the content of C18:2 and C18:3. Significantly higher lag times for both C18:2 and C18:3 were observed in ground canola compared to ground soya bean. BH rates of C18:2 and C18:3 fatty acids in soya bean were three times higher than those of canola. These results suggest that the fatty acid profile of fat source can affect the BH of UFAs by rumen micro‐organisms. So that UFAs of canola had higher ability to escape from ruminal BH. It seems that fatty acid profile of ruminant products is more affected by canola seed compared to soya bean seed.     

Serum biochemical values and mineral element contents of tissues in yaks

Serum biochemical values and the wet weight to dry weight ratios of tissues were determined in yaks in Shandan county of Gansu province. The liver, kidney, heart and muscle contents of seven elements in yaks were also analysed. Most serum biochemical values were similar to those of cattle, camels and sheep, but the calcium concentration was considerably above the normal range for other ruminants. The liver contained the highest concentrations of copper, manganese and cobalt and the kidney of selenium, iron and calcium.Abbreviations Alb albumin - ALT alanine aminotransferase - AST aspartate aminotransferase - BUN blood urea nitrogen - Chol cholesterol - Glob globulin - -GT -glutamyl transpeptidase - IP inorganic phosphorus - TP total protein