香菇多糖鸡肉鸡脾淋巴细胞功能及信息传导的影响

本实验研究了香菇多糖对肉鸡脾淋巴细胞转化率及细胞内信息分子一氧化氮（NO）、Ca^2 及cAMP、cGMP的影响。发现香菇多糖能剂量依赖性促进脾淋巴细胞转化率及细胞内NO、Ca^2 浓度，当作用时间为20min时，能增加cAMP和cGMP浓度，而当作用时间为60min，对cAMP和cGMP浓度无显著影响。以上结果提示，香菇多糖能显著提高脾淋巴细胞免疫功能，其作用是通过改变细胞内信息传导实现的。     

欧洲鳗鲡淋巴细胞转化试验条件的建立

通过对培养时间、培养温度、刀豆蛋白A(ConA)质量浓度和鳗鲡血清质量分数4个参数的测定,确定欧洲鳗鲡(Anguilla anguilla)外周血淋巴细胞转化试验四甲基偶氮唑(MTT)法的实验条件。结果表明,在培养时间为42-90 h,培养温度分别为15.0℃、20.0℃、25.0℃和30.0℃时,细胞培养液中ConA质量浓度分别为0、10μg/mL、20μg/mL和30μg/mL,鳗鲡血清质量分数分别为0、5%、10%和15%时,淋巴细胞于20.0℃、含10μg/mL ConA,10%鳗鲡血清的RPMI1640细胞培养液中培养66 h后可获得最好的淋巴细胞转化效果。方差分析结果显示,4个因素中仅鳗鲡血清水平对淋巴细胞转化试验OD值具有极显著影响(P<0.01),而温度、时间和ConA对其影响均不显著(P>0.05)。本研究建立的鳗鲡外周血淋巴细胞转化试验条件为鳗鲡的细胞免疫建立了研究方法。     

香菇多糖对肉鸡脾淋巴细胞功能及信息传导的影响

本实验研究了香菇多糖对肉鸡脾淋巴细胞转化率及细胞内信息分子一氧化氮(NO)、Ca2+及cAMP、cGMP的影响。发现香菇多糖能剂量依赖性促进脾淋巴细胞转化率及细胞内NO、Ca2+浓度,当作用时间为20min时,能增加cAMP和cGMP浓度,而当作用时间为60min,对cAMP和cGMP浓度无显著影响。以上结果提示,香菇多糖能显著提高脾淋巴细胞免疫功能,其作用是通过改变细胞内信息传导实现的。     

草鱼胸腺细胞培养方法的初步研究

草鱼胸腺细胞培养方法主要有机械分散法和消化分离法。胸腺细胞绝大部分是由大、中、小淋巴细胞组成．而淋巴细胞为悬浮细胞．培养悬浮细胞时由于细胞中期分裂相与培养的细胞不易区分，其传代时阃只能凭知胞计数推测其密度来确定是否可以进行细胞传代。在培养草鱼胸腺细胞时．在细胞培养液中加入PHA．观察培养结果可发现能使草鱼胸腺细胞成团生长．增殖旺盛。     

鲫、草鱼外周血中淋巴细胞和单核细胞对鲤血管内皮细胞的粘附

借鉴哺乳动物内皮细胞体外培养技术，结合鱼类细胞自身特点，纯化培养鲤血管内皮细胞并传至3代，以此作为粘附材料。应用淋巴细胞分离液离心技术并结合玻璃粘附法分离纯化鲫、草鱼外周血中淋巴细胞、单核细胞，并与上述3代内皮细胞进行免疫粘附试验，同时进行免疫粘附动力学观察。结果显示，鲫、草鱼两类细胞对内皮细胞的粘附率分别为0.09±0.013，0.20±0.018；0.11±0.015，0.21±0.023。表明鲤科鱼类不同种的同类细胞粘附率差异不大，而同种不同类细胞则差异显著。动力学观察分析表明，淋巴细胞粘附较快，60min进入平台期；单核细胞粘附慢，120min进入平台期。初步证明鱼类内皮细胞具有免疫介导作用。     

动物采血技术

<正>针对动物疫病诊断、检测过程中,经常需要采血做实验室检测,针对常采的猪、牛、羊、鸡、犬等5种动物的采血技术我们通过长期的实践和体会,总结选择出不同的采血部位和采血方法及应注意的事项,以供读者参考。1猪的采血我们在实践中把猪的采血方法分为3种。一是体重50kg以上的肥猪和大型种猪,采用耳静脉采血;二是体重     

日本蟳血淋巴细胞的初步研究

对日本蟳血淋巴细胞有形成分进行显微观察分析，根据细胞大小、有无颗粒及颗粒的大小进行分类。结果表明：日本蟳血淋巴细胞可区分为3种，即大颗粒细胞、小颗粒细胞和无颗粒细胞。在此基础上比较了不同性别、不同发育阶段及饥饿状态下，日本蟳血淋巴细胞的形态、密度及3种类型细胞所占比例等指标的数据变化，并与其他甲壳动物的血细胞进行了比较。     

断奶应激影响仔猪血清中某些激素水平

郑州牧业工程高等专科学校王老七等，选用24头21日龄仔猪分为对照组和试验组．对照组14头，分别于21、27、30、33、36、42、49日龄由颈静脉采血，每次2头，公母各一；试验组10头，分别于30、33、36、42、49日龄从颈静脉进行采血，每次2头，公母各一．结果表明：试验组与对照组相比，在基础及EWS状态下。不同性别仔猪皮质醇及ACTH水平不同．断奶后第2、5、8d，     

壳聚糖和益生菌对暗纹东方鲀抗病力和免疫功能的影响

为了解壳聚糖和益生菌刺激和增强免疫应答的可能作用,本试验在基础饲料中添加0.2%、0.5%、1.0%的壳聚糖,0.1%、0.2%、0.4%的益生菌,甘露聚糖与益生菌混合物,壳聚糖与益生菌的混合物,在室内水泥池中喂养暗纹东方鲀(3.15±0.05)g 2个月,根据抗菌活力筛选出免疫增强饲料组(0.2%壳聚糖,0.1%、益生菌,甘露聚糖与益生菌混合物,壳聚糖与益生菌混合物)测定免疫功能。结果表明,甘露聚糖与益生菌混合物仅提高头肾T淋巴细胞转化,壳聚糖与益生菌混合物显著增强头肾T、B淋巴细胞转化和脾脏B淋巴细胞体外培养时分泌的IgM。筛选出的四种免疫增强饲料均能使头肾B淋巴细胞体外培养时分泌的IgM显著增加。无论是脾脏B淋巴细胞还是头肾B淋巴细胞,体外培养时所分泌的IFN-α都不同程度地受抑制。     

重金属混合物对鲫淋巴细胞DNA损伤的研究

采用单细胞凝胶电泳技术，研究了重金属混合物对鲫淋巴细胞DNA的损伤。结果表明：重金属混合物对鲫淋巴细胞DNA有损伤作用。随着剂量增加，损伤加重，在实验浓度范围内。存在剂量-效应关系。但与作用时间无明显的相关性。DNA损伤可望成为水环境基因毒剂指标。     

鳗鲡血液细胞的研究

本文通过鳗鲡血液有形成份Ｗｒｉｇｈｔ染色并进行显微分析，辩识其红血细胞、嗜中性粒细胞、嗜酸性粒细胞、嗜碱生粒细胞、小淋巴细胞、单核细胞和血栓细胞并加以分析。     

Functional partition of cells in the gill epithelium of the Japanese eel, Anguilla japonica, and the role of hormones

Viable chloride and pavement cells were isolated from the gill epithelium of Japanese eel, Anguilla japonica, by a 3-step percoll gradient centrifugation at low speed. Viability of the isolated cells were tested by the trypan blue exclusion test, rhodamine-123, or pre-labelling the cells with fluo-3 Ca²⁺ dye and examined by laser confocal microscopy. Isolated chloride cells responded to ionomycin with a rapid increase in Ca²⁺ fluorescence, which was abolished by chelating external Ca²⁺ with EGTA. Peptide hormones, including arginine vasotocin, isotocin, insulin-like growth factor I and II, and urotensin I increased Ca²⁺ entry, urotensin II had no effect, and eel corpuscles of Stannius extract reduced residual Ca²⁺ fluorescence. Isolated chloride cells and pavement cells from eels were analyzed for their enzymatic activities involved in intermediary and nitrogen metabolism. Chloride cells had high levels of glutaminase I, glutamate dehydrogenase, glutamate oxalacetate transaminase, HCO₃-ATPase and carbamoyl phosphate synthetase II. Pavement cells had highly active glucose-6-phosphate dehydrogenase and AMP deaminase. Both had high levels of lactate dehydrogenase compared with other tissues.     

哺乳动物干细胞的研究进展

介绍了干细胞的概念和分类,综述了哺乳动物胚胎干细胞和胚胎生殖细胞的分离培养、生物学特性、定向诱导分化以及几种重要的成体干细胞。讨论了干细胞的应用和存在的问题。     

Characterization of the pattern of cytokeratin proteins in the epidermal cells of loach,Misgurnus anguillicaudatus (Cyprinformes)

The pattern of cytokeratin proteins in the epidermal cells of loach was studied by immunotechniques and partial separation of the epidermal cells. Two monoclonal antibodies, namely 8F7 and 1C45, against the cytokeratin proteins of the loach epidermis were prepared. these two monoclonal antibodies exhibit distinctive results in immunohistochemical staining. The 8F7 monoclonal antibody stains mainly with the epithelial cells, while the 1C45 monoclonal antibody stains specifically with the club cells. The pattern of cytokeratin proteins in the club cells and the epithelial cells of various epidermal layers was further determined by partial separation of these cells. Immunoblotting analysis of the cell fractions confirms the cytokeratin proteins to be differentially expressed in the club cells and the epithelial cells. However, the cytokeratin proteins expressed in the epithelial cells of the basal, middle and outer layers are same. The results indicate that differentiation of the epithelial cells seems limited during their translocation from basal to upper layers, but in those cells that do differentiate into club cells, the cytokeratin pattern changes.     

Cytotoxicity of surfactants to the FHM-sp cell line

Cytotoxicity of eight surfactants was determined by the neutral red assay with the fathead minnow (FHM)-sp cell line, a cell line in suspension culture from fish. The toxicity ranking of the surfactants was benzalkonium chloride > benzethonium chloride > sodium linear - dodecylbenzene–sulfonate (LAS) > potassium laurate > sodium dodecylsulfate > polyoxyethylene(20)sorbitan monolaurate > polyoxyethylene(20)sorbitan monooleate > betaine. The toxicity ranking of the surfactants classified into four groups based on the ion of the hydrophilic group was cationic surfactants > anionic surfactants > non-ionic surfactants > amphipathic surfactants. The FHM-sp cells, as well as the chinook salmon embryo (CHSE)-sp cells, could be inoculated directly to the microplate wells without dispersion by trypsin treatment of cell sheets at room temperature. Therefore, the cytotoxicity assay of the surfactants could be carried out quickly by using the FHM-sp cell line. The FHM-sp cell line had similar or higher sensitivity to sodium dodecylsulfate compared with several cell lines from mammals. The cytotoxicity assay could be shortened by the procedure exposing the surfactants to the FHM-sp cells before the cell monolayer formation in the microplate wells. To use the FHM-sp cell line as a screening tool prior to in vivo testing, studies on the correlation between in vivo data and in vitro data on the toxicity of surfactants are necessary.     

莱茵衣藻type I metacaspase基因克隆及其参与调控程序性细胞死亡研究

Metacaspase是在原生动物、真菌和植物中发现的一种具有底物精氨酸/赖氨酸特异性的半胱氨酸蛋白酶。根据蛋白质结构特征可将metcaspase分为typeⅠ与typeⅡ两种类型,均参与调控多种植物与原生动物程序性细胞死亡。本研究根据GenBank数据库莱茵衣藻(Chlamydomonas reinhardtii) type I metacaspase基因(GenBank No. XM_001696904)序列,采用巢式PCR克隆获取type I metcaspase基因开放阅读框(ORF)序列并命名为CrMC1。CrMC1 ORF全长987 bp,推测编码1个包含405个氨基酸的蛋白质。通过与已知物种type I metacaspase氨基酸序列进行同源序列比对发现,CrMC1具有高度保守的p20、p10、连接区结构域以及精氨酸、半胱氨酸活性中心位点。研究显示,在H2O2诱导的莱茵衣藻程序性细胞死亡过程中,CrMC1表达量显著提高(P<0.05),2 h后达到峰值,3 h时下降至对照组同一水平。结果表明,莱茵衣藻type I metacaspase基因CrMC1参与H2O2诱导的莱茵衣藻程序性细胞死亡调控。     

Differentiation and development of Leydig cell, and induction of spermatogenesis during testicular differentiation in the Japanese eel, Anguilla japonica

The initial appearance and the development of Leydig cells (LCs), the sites of steroid hormone production in the testis, were investigated ultrastructurally during testicular differentiation in the Japanese eel, Anguilla japonica. In addition, the effects of a single injection of human chorionic gonadotropin (HCG; 5 IU g body weight^-1) on histological changes of the testes and serum 11-ketotestosterone (11-KT) were examined at various stages (15–18, 20–23, 26–29, 32–35, 38–41 and 46–50 cm body length (BL)) of testicular differentiation. Testicular differentiation was morphologically characterized by the development of loose connective tissue on the medial side in animals 18–29 cm in BL. Ultrastructurally, LCs were first identified in the loose connective tissue of the testis of the 23 cm fish. In the testes of fish over 32 cm, clusters of LCs were distributed throughout the interstitial region accompanying the increase in number of spermatogonia. In fish larger than 32 cm, spermatogenesis was induced by administration of HCG; serum 11-KT levels were also raised. On the other hand, there was no effect on spermatogenesis or serum 11-KT levels in fish less than 29 cm, or in the controls. These result suggests that morphological differentiation of LCs occurs in testis of the 23 cm eel, and subsequently, the testes of eels of BL more than 32 cm acquire the capability to produce steroid hormones.     

文蛤消化道粘液细胞研究

运用组织学和组织化学染色方法,在光镜水平上研究了文蛤消化道粘液细胞的类型,分布与组化特性。H.E染色法显示,粘液细胞主要存在于消化道上皮组织中,仅唇瓣结缔组织中发现有少量的粘液细胞,ABPAS染色法显示;文蛤消化道粘液细胞主要分为4种类型：Ⅰ型,呈红色,AB阴性,PAS阳性,含有中性粘液物质,Ⅱ型：呈蓝色,AB阳性,PAS阴性,含有酸性粘液物质;Ⅲ型：AB和PAS均呈阳性,但PAS阳性较强,两种粘液物质均含有,但中性粘液物质含量多;Ⅳ型：AB和PAS均呈阳性,但AB阳性较强,两种粘液物质均含有,但酸性粘液物质含量多,不同部位粘液细胞的类型,数量不同,唇瓣粘液细胞的数量较少,食道含有大量的粘液细胞,胃,肠,直肠中粘液细胞的数量以胃中最少,肠次之,直肠最多,AB-PAS（AB pH1.0）染色法显示：粘液细胞所含酸性粘液物质包括硫酸性粘液物质和涎酸性粘液物质,且唇瓣和直肠中粘液细胞所含的涎液酸性粘液物质比硫酸性粘液物质多,酶组化研究结果显示,肠粘液细胞具有弱酸性磷酸酶,弱碱性磷酸酶活性。     

穿膜肽引导的体外表达转录因子蛋白Sox2进入红鳍东方鲀精巢细胞系

为了探索适用于体外培养的鱼细胞外源基因转入方法, 本研究通过构建红鳍东方鲀(Takifugu rubripes)转录因子Sox2的重组表达载体pET32a(+)-Sox2-11R-6His, 诱导表达并纯化得到了C末端连接多聚精氨酸(11R)的重组蛋白Sox2-11R-6His, 以其与红鳍东方鲀精巢细胞系细胞共孵育12 h后, 光镜观察结合Western Blot检测发现重组蛋白进入细胞的效率与浓度呈剂量依赖关系且最佳孵育浓度为8 μg/mL, 当重组蛋白质量浓度达到10 μg/mL时, 表现出明显的细胞毒性。对外源蛋白进行免疫荧光标记定位, 发现重组蛋白分布于细胞质, 部分进入到细胞核中。证明了穿膜肽11R可以有效运载转录因子重组蛋白至红鳍东方鲀的细胞系细胞中。本研究旨在将广泛应用于哺乳动物的细胞基因递送载体穿膜肽应用于鱼类细胞系细胞。

     

不同冻存液对长江鲟鳍条细胞冷冻效果的影响

为了探究不同浓度配比冻存液对长江鲟(Acipenser dabryanus)鳍条细胞(Dabry′s sturgeon fin-derived cells,DSFCs)冷冻效果的影响,选择传代培养处于对数生长期的DSFCs,将第8代DSFCs置于液氮(-196℃)中冻存。根据冻存液中培养基(MEM)、胎牛血清(Fetal bovine serum,FBS)及二甲基亚砜(Dimethyl sulfoxide,DMSO)的配比不同,分为8个实验组:①含不同浓度DMSO配比实验组:5%DMSO+45%MEM+50%FBS,10%DMSO+40%MEM+50%FBS,20%DMSO+30%MEM+50%FBS;②含不同浓度FBS配比实验组:10%DMSO+90%FBS+0%MEM,10%DMSO+70%FBS+20%MEM,10%DMSO+50%FBS+40%MEM,10%DMSO+30%FBS+60%MEM,10%DMSO+10%FBS+80%MEM,对各组分采用形态学观察、CCK-8法、流式细胞仪检测细胞凋亡和细胞周期,综合分析不同冻存液组分对DSFCs冻存后活力的影响。结果显示,不同浓度DMSO配比实验组中,DMSO的浓度为20%时,细胞复苏后难以贴壁,存活率下降非常明显,仅为38.3%;而5%和10%DMSO细胞生长状态良好,存活率分别为80.2%和78.7%。不同浓度FBS配比实验组中,90%、30%和10%实验组细胞活力显著高于70%和50%实验组。FBS浓度为30%和10%的冻存组细胞凋亡率显著高于90%的冻存组(8.24%,15.35%vs 3.54%),3组之间相互比较有明显差异(P<0.05);细胞周期结果显示,与FBS浓度为90%的冻存组比较,FBS浓度为30%和10%冻存组G 0/G 1期细胞比例均明显增加(P<0.05),S期和G 2+M期细胞比例均显著降低(P<0.05),FBS浓度30%与10%冻存组比较差异不明显(P>0.05)。研究表明,长江鲟细胞的长期冷冻保存宜采用稍低浓度的DMSO(5%~10%)和高浓度的FBS。