硝基苯染毒致小鼠脑的毒性作用

为探讨硝基苯染毒致小鼠脑的毒性作用,用灌胃的方法对试验小鼠进行了硝基苯染毒,染毒剂量分别为26、52、105mg／kg,每日染毒1次,共30d。于末次染毒后第2d将小鼠脱颈椎处死,立即取出脑,检测SOD和GSH—Px活性和．MDA含量,并对其显微及超微结构进行了观察。结果,硝基苯对小鼠脑有明显的毒性作用,主要表现为大脑血管间隙扩大,周围神经纤维疏松;小脑、海马区部分浦肯野氏细胞核固缩、核溶解,锥体细胞核固缩;神经细胞肿胀,线粒体明显肿胀,粗面内质网形态扩张;大部分胶质细胞电子密度增加,胞核变小、核固缩,胞质内细胞器明显减少;有些神经纤维脱髓鞘,神经微丝溶解;随着染毒剂量的加大,MDA含量逐渐升高,CAT和SOD活性不断显著降低。结果表明,硝基苯可以通过氧化应激和诱发细胞凋亡对小鼠脑组织造成损伤。     

氧化应激在硝基苯致小鼠肾损伤中的作用

为探讨硝基苯对小鼠肾的毒性作用，采用灌胃法对试验小鼠用硝基苯进行染毒，染毒剂量分别为26、52、105mg／kg，每天染毒1次，共30d。于末次染毒后第2d将小鼠脱颈处死，立即取出肾通过相应处理用于抗氧化指标检测及显微和超微结构的观察。结果，染毒小鼠主要表现为近曲小管上皮细胞不同程度水样变性，线粒体肿胀，脊断裂；内质网脱核糖颗粒；细胞核发生形变；肾小球血管内皮细胞肿胀，血管扩张；随着染毒剂量的加大，肾和血清中MDA含量逐渐升高，SOD和GSH—Px酶活性不断降低，与对照组比较，均差异显著（P〈0．05）或极显著（P〈0．01）。结果表明，硝基苯能够诱发小鼠肾细胞发生氧化胁迫，并诱导细胞凋亡的发生。     

小檗碱透皮制剂对小鼠骨髓细胞的微核效应

本采取小鼠骨髓细胞微核试验方法。研究小檗碱透皮制剂对体细胞的诱变性。结果表明：阴性对照组微核率为1．125±0．9‰,环磷酰胺阳性对照组的微核率为5．125±0．976‰,与阴性对照组比较,差异极显（P＜0．01）;小檗碱透皮制剂低,中,高剂量组微核率分别是1．125±0．69‰,1．5±0．9‰,1．875±0．787‰,与阴性对照比较,差异均不显（P＞0．05）,试验结果为阴性。小檗碱透皮制剂对小鼠骨髓细胞不具诱变性。     

沙咪珠利对小鼠精子畸形和骨髓细胞微核的影响

为评价沙咪珠利的安全性,对其进行了小鼠精子畸形和骨髓细胞微核试验研究。选择健康ICR小鼠随机分为受试物高、中、低剂量组,阳性对照组和阴性对照组,进行精子畸形试验和骨髓细胞微核试验,计算精子畸形率和微核率。结果显示,受试物各剂量组精子畸形率与骨髓细胞微核率与阴性对照组比较,差异不显著(P0.05),与阳性对照组比较,差异有统计学意义(P0.01)。表明沙咪珠利对小鼠精子和骨髓细胞微核无影响,不具有遗传毒性。     

抗氧化酶在硝基苯中毒小鼠睾丸细胞凋亡中的作用

将10周龄雄性昆明小鼠随机分为硝基苯染毒组（26mg／kg、52mg／kg、105mg／kg）、花生油溶剂对照组、生理盐水对照组。对各组试验小鼠进行处理后，于第30d检测小鼠血清和睾丸中SOD、GSH-Px、CAT的活性及MDA含量，并通过DNA Ladder条带和透射电镜检测睾丸细胞的凋亡。结果，染毒组血清及睾丸中SOD、GSH—Px、CAT的活性显著或极显著（P〈0.01或P〈0．05）低于溶剂对照组，MDA含量显著或极显著（P〈0．01或P〈0，05）高于溶剂对照组，染毒组均显示DNA Ladder条带；电镜下细胞核皱缩，异染色质边聚，呈现典型凋亡现象。结果表明，硝基苯中毒能够诱使睾丸发生氧化应激，进而导致睾丸细胞发生凋亡，抗氧化酶在睾丸细胞凋亡中具有一定的作用。     

复方硝基酚钠诱发小鼠骨髓细胞和睾丸精原细胞染色体畸变的观察

研究了鱼虾促生长药物添加剂复方硝基酚钠对体细胞和雄性生殖细胞的致突变性。骨髓细胞染色体畸变分析以每公斤体重用２５０、８３．３、２５ｍｇ的复方硝基酚钠分别给小鼠经口染毒，每日１次，连续５ｄ，同时设空白对照组和阳性对照组。睾丸精原细胞染色体畸变分析以每公斤体重用２５０、１２５、６２．５ｍｇ的复方硝基酚钠给雄性小鼠经口染毒，每日１次，连续５ｄ，同时设空白对照组和阳性对照组。上述两种致突变试验结果均为阴性。因此认为复方硝基酚钠对哺乳动物体细胞和雄性生殖细胞均未显示致突变作用     

隆朋对小鼠骨髓嗜多染红细胞微核率的影响

隆朋(Rompun)即二甲苯胺噻嗪,它作为镇静、镇痛和肌松剂广泛用于兽医临床,特别对反刍动物有较好的镇麻效果,且具有用量小、作用迅速等特点。农牧大学闫章年等以隆朋为主要成分研制的鹿化学保定剂眼乃宁应用于鹿业生产受到广泛好评。隆朋属高毒物质,但对动物细胞有无遗传损伤尚未见报道。本试验通过小鼠骨髓嗜多染红细咆(PCE)微核率的变化,对隆朋的遗传毒理学效应进行初步评价。 1 材料和方法     

羊胎盘复方制剂对小鼠睾丸染色体与骨髓细胞微核的致畸变作用

研究了不同羊胎盘制剂对小鼠睾丸染色体与骨髓细胞微核的致畸作用,实验显示,羊胎盘提取物（GPE）及其制剂（GPP）的各剂量组小鼠睾丸染色体的断片数、易位数、染色体（包括常染色体和性染色体）单价数、畸变细胞数及其畸变率与阴性对照组相比差异不显著（P〉0．05）,但与环磷酰胺（CTX）处理的阳性对照组相比差异则极显著（P〈0．01）;GPE、GPPI及GPPII3个剂量组小鼠骨髓嗜多染红细胞微核率与阴性对照组相比,无统计学意义（P〉0．05）。而阳性对照组的小鼠骨髓嗜多染红细胞微核率则显著高于其他实验组（P〈0．01）。综上所述,羊胎盘提取物及其复方制剂对小鼠无致畸作用。     

乙酰胆碱酯酶在硝基苯致小鼠神经毒性中的作用

为探讨硝基苯中毒所致乙酰胆碱酯酶的变化规律，本试验用不同剂量的硝基苯对小鼠进行灌胃染毒，1次/d，共30 d，采用碱性羟胺比色法测定小鼠脑乙酰胆碱酯酶活性和肝乙酰胆碱含量，并在试验期间记录小鼠的临床表现。结果表明：染毒期间小鼠表现为行动迟缓、反应迟钝、抽搐及情绪异常如兴奋、敏感、不停跳跃等症状，随着染毒剂量的增加，时间的延长，中毒症状越明显；乙酰胆碱酯酶活性表现为明显的抑制作用，肝乙酰胆碱含量增加。结论：硝基苯具有神经毒性，能够抑制肝和脑乙酰胆碱酯酶活性，且表现为明显的剂量效应。     

体外诱导牛BMSC向上皮样细胞分化的研究

为培育转基因肉牛提供种子细胞以及进一步丰富牛骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)的多向分化潜能,利用细胞免疫荧光染色和分子生物学方法,初步探讨表皮生长因子和胰岛素体外诱导牛BMSC向上皮样细胞分化的可能性。利用含细胞因子的诱导液对纯化稳定的P4代牛BMSC进行体外诱导,并对诱导后的细胞进行细胞角蛋白18的细胞免疫荧光观察和细胞角蛋白19的RT-PCR鉴定。结果表明,诱导后细胞经细胞角蛋白18免疫荧光染色后出现明显的荧光。RT-PCR结果显示诱导分化后细胞角蛋白19基因在细胞中表达。因此,在体外,表皮生长因子和胰岛素可诱导牛BMSC初步分化为上皮样细胞。     

The morphological effects of vincristine sulfate on canine bone marrow cells

This study documents the morphologic changes observed in the bone marrow aspirate biopsies from dogs 6 and 24 hours after receiving a single therapeutic dose (0.025 mg/kg) of vincristine sulfate (Oncovin: Eli Lilly & Co., Indianapolis, Ind.) intravenously. The most striking cytologic changes were observed in the erythroid cell line. Abnormalities included increased numbers of mitotic figures, abnormal nuclear configurations, and fragmented nuclei. Erythroid cells in metaphase were prominent in marrow samples collected 6 hours post-vincristine, accounting for a mean of 27% of all erythroid precursors. Fragmented nuclei and atypical nuclear configurations were seen in low numbers (mean = 7%) of erythroid cells from these animals. In contrast, marrow collected from dogs 24 hours post-vincristine exhibited low numbers (mean = 1%) of erythroid cells in metaphase, but erythroid cells with atypical nuclear configurations and fragmented nuctei accounted for a mean of 41% of the erythroid cells present. Less dramatic increases in the number of mitotic non-erythroid cells were seen 6 hours post-vincristine (mean = 5% of non-erythroid cells) and 24 hours post-vincristine (mean = 1% of non-erythroid cells). Only rare nuclear fragmentation was observed in these cell lines. Significant alterations in megakaryocytes and myeloid to erythroid (M:E) ratios were not observed in samples taken 6 hours post-vincristine; however, M:E ratios were considerably higher in three of the four samples taken from dogs 24 hours post-vincristine. Similar time-related changes were observed in four clinical cases in which bone marrow aspirates were performed after vincristine administration.     

Differentiation of canine bone marrow stromal cells into voltage- and glutamate-responsive neuron-like cells by basic fibroblast growth factor

We investigated the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into voltage- and glutamate-responsive neuron-like cells. BMSCs were obtained from the bone marrow of healthy beagle dogs. Canine BMSCs were incubated with the basal medium for neurons containing recombinant human basic fibroblast growth factor (bFGF; 100 ng/ml). The viability of the bFGF-treated cells was assessed by a trypan blue exclusion assay, and the morphology was monitored. Real-time RT-PCR was performed to evaluate mRNA expression of neuronal, neural stem cell and glial markers. Western blotting and immunocytochemical analysis for the neuronal markers were performed to evaluate the protein expression and localization. The Ca²⁺ mobilization of the cells was evaluated using the Ca²⁺ indicator Fluo3 to monitor Ca²⁺ influx. To investigate the mechanism of bFGF-induced neuronal differentiation, the fibroblast growth factor receptor inhibitor, the phosphoinositide 3-kinase inhibitor or the Akt inhibitor was tested. The bFGF treatment resulted in the maintenance of the viability of canine BMSCs for 10 days, in the expression of neuronal marker mRNAs and proteins and in the manifestation of neuron-like morphology. Furthermore, in the bFGF-treated BMSCs, a high concentration of KCl and L-glutamate induced an increase in intracellular Ca²⁺ levels. Each inhibitor significantly attenuated the bFGF-induced increase in neuronal marker mRNA expression. These results suggest that bFGF contributes to the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells and may lead to the development of new cell-based treatments for neuronal diseases.     

Optimisation of bone marrow aspiration from the equine sternum for the safe recovery of mesenchymal stem cells

Reasons for performing study: Mesenchymal stem cell (MSC) therapy for orthopaedic disease is being used with increasing frequency; there is a need to define a safe, reliable and effective technique for the recovery of MSCs from the sternum of the horse. Objectives: To describe an optimised safe technique for obtaining bone marrow‐derived MSCs from the sternum of the Thoroughbred horse. Methods: The anatomical relationship of the sternum with the heart and internal anatomy was demonstrated in cadavers. Sternal anatomy was evaluated ultrasonographically and after midline sectioning. Sternebrae were examined histologically after aspiration to determine the effect of needle insertion. The quality of the aspirate was evaluated as the number of colony‐forming units from sequential and separately aspirated 5 ml aliquots and assessed for their multipotency using trilineage differentiation. Results: The optimal safe location for the needle was the 5th sternebra because it had a safe dorsoventral thickness and was cranial to the apex of the heart. This sternebra could be reliably identified ultrasonographically. Aspirates could also be obtained from the 4th and 6th sternebrae, although the former is between the front limbs and the latter closer to the heart. Minimal disruption of the internal bony architecture was seen after needle insertion through the thin outer cortex and the first 5 ml aliquot contained the greatest number of colony‐forming units of mesenchymal stem cells with trilineage capabilities. Conclusions: Accurate placement of a Jamshidi needle into the medullary cavity of the 4th–6th individual sternebrae is facilitated by the use of ultrasonography and enables aspiration of bone marrow reliably with minimal damage to the sternum and risk to the horse. Potential clinical relevance: Sternal marrow aspiration as described is a safe and reliable technique to obtain MSCs for orthopaedic cell‐based therapies.     

Epidemiology: Culture of bovine bone marrow progenitor cells in vitro

Summary

In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400g). The use of 3% bovine leucocyte‐conditioned medium, produced by stimulation of blood lymphocytes with 4 pg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte‐conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose‐based medium requires different culture conditions, which are different from the culture conditions for human cells.     

Radioprotective effects of fucoidan on bone marrow cells: improvement of the cell survival and immunoreactivity

Fucoidan is a sulfated polysaccharide purified from brown algae including Fucus vesiculosus and has a variety of biological effects including mobilization of hematopoietic progenitor cells. Recently, we demonstrated that fucoidan stimulates the antigen-presenting functions of dendritic cells. In this study, we investigated the radioprotective effects of fucoidan on bone marrow cells (BMCs), which are the main cellular reservoir for the hematopoietic and immune system. To evaluate the effects of fucoidan, we assayed cell viability and immune responses. In a viability assay, fucoidan significantly increased the viability of BMCs. Based on the results of flow cytometric analysis, the increased viability of fucoidan-treated BMCs was attributed to the inhibition of radiation-induced apoptosis. Furthermore, fucoidan altered the production of immune-related cytokines from BMCs and increased the capability of BMCs to induce proliferation of allogeneic splenocytes. Taken together, our study demonstrated that fucoidan has radioprotective effects on BMCs with respect to cell viability and immunoreactivity. These results may provide valuable information, useful in the field of radiotherapy.