原代培养大鼠松果体细胞的增殖与分化——免疫组织化学研究

目的：探讨体外培养大鼠松果体细胞的生长、增殖及分化。方法：采用MTT测定法BrdU及5－HT免疫组织化学方法。结果：相差显微镜观察可见培养的松果体细胞呈小圆形或不规则形,初期聚集成巢,以后逐渐散贴壁,有强折光性,细胞体随培养时间的延长而增大,MTT检测证实。培养的松果林体细胞增生较活跃,细胞倍增时间在培养第9天,第11天左右高峰。BrdU免疫组化法对松果体细胞分裂与增殖的观察及统计结果与MTT检验结果相符。5－HT阳性细胞占培养细胞总数的80％以上,结论：体外培养能获得生长增殖旺盛,具有一定生理功能的松果体细胞。     

中脑神经前体细胞的体外分离、培养和特性

为了解中脑神经前体细胞的体外培养特性和建立中脑神经前体细胞的体外分化调控机制提供细胞模型。本实验采用含有丝分裂源表皮生长因子(EGF)的无血清培养基培养来源于大鼠胚胎E14.5天的中脑神经前体细胞,应用免疫细胞化学方法了解其前体细胞特性。结果发现中脑神经前体细胞呈神经前体细胞特征性标记Nestin免疫染色阳性,无分化细胞标记;细胞克隆实验证实中脑神经前体细 胞有自我更新能力;在EGF刺激下增殖迅速;当撤去EGF后置于含胎牛血清的培养基和被覆多聚赖氨酸(PLL)的培养皿内,中脑神经前体细胞可分化成神经元和星形胶质细胞。本试验证明我们培养的中脑神经前体细胞具有增殖、自我更新能力和多向分化潜能特性。     

山羊精原干细胞的鉴定及培养体系优化的研究

该研究优化了山羊精原干细胞(goat spermatogonial stem cells,g SSCs)培养体系,使山羊精原干细胞能在体外长期培养,维持自我更新的能力并保持未分化状态。取3~5月龄山羊睾丸,采用两步酶消法结合差速贴壁方法得到山羊精原干细胞悬液,分别通过形态学观察、碱性磷酸酶(alkaline phosphatase,AKP)染色、特异基因表达及蛋白质水平的分析对培养的细胞进行鉴定;并以山羊睾丸支持细胞(goat sertoli cells,g SCs)、小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)和层黏连蛋白(laminin,L)为饲养层,观察饲养层对山羊精原干细胞体外增殖的影响。结果表明,山羊精原干细胞体外增殖形成克隆簇,AKP染色呈阳性。经RT-PCR检测,Oct-4、C-myc、Cyclin D1、Ngn3和TERT等干细胞特异基因均有表达。细胞免疫组化结果显示,Oct-4、SSEA-1、α6-integrin、Vasa和Thy-1蛋白质呈阳性。克隆簇统计显示,在山羊睾丸支持细胞上形成的山羊精原干细胞(goat spermatogonial stem cells,g SSCs)克隆数与其他两组比较差异显著(P0.05)。山羊睾丸支持细胞饲养层上的精原干细胞可在体外传3~4代,培养时间为2个月。结果证明,通过两步酶消法和差速贴壁法可以分离获得山羊精原干细胞,且山羊睾丸支持细胞能够促进g SSCs的增殖。     

大鼠部分肝切后血清对体外培养肝细胞的刺激作用

目的探讨大鼠部分肝切后血清对体外培养肝细胞生长状况的影响。方法对Sprague-Dawley大鼠进行肝部分切除,分别在术后第12、24、36h于心腔内穿刺取血,制备刺激血清。采用酶消化法分离获取Sprague-Dawley乳鼠的肝细胞,在加有10％上述刺激血清的DMEM培养基中进行肝细胞体外培养。倒置显微镜下观察培养细胞的生长状态,采用免疫细胞化学方法检测肝细胞中白蛋白和纤维蛋白原的表达情况。结果发现在肝切后血清刺激作用下,原代培养的肝细胞生长加快,存活时间增长。细胞传代培养后仍用制备血清加以刺激,可产生胶原样细胞外基质,并且在基质上粘附的肝细胞呈现克隆样生长状态,其胞浆内白蛋白和纤维蛋白原均呈阳性表达。结论研究初步表明,体外培养乳鼠肝细胞时加入大鼠部分肝切后血清,可以有效刺激细胞的生长,促进细胞外基质的产生,从而利于肝细胞在体外较长时间存活、增殖和功能保持,同时此种肝细胞体外培养方式还为肝脏细胞生物学研究增添了新的实验途径。     

定向诱导小鼠ES细胞为肝脏细胞及其凝血因子Ⅷ, Ⅸ的表达

凝血因子Ⅷ, Ⅸ缺陷导致血友病A和B的发生, 肝细胞是产生凝血因子的器官, 利用肝细胞进行替代治疗是纠正凝血因子缺陷的根本办法, 但是其来源及利用存在难以克服的问题. ES细胞具有体外培养时保持未分化状态和无限的增殖能力, 具有在一定条件下可以分化发育成各个胚层细胞的潜能, 利用ES细胞源性肝细胞作为供体细胞, 解决了肝细胞来源困难、增殖能力差的难题. 体外分阶段定向诱导E14小鼠ES细胞分化为肝细胞, ICG摄取及PAS反应结果证实ES源性细胞具备肝细胞功能, 在此基础上, 利用RT-PCR法对诱导细胞初步进行了凝血因子Ⅷ, Ⅸ表达分析, 为进一步开展利用ES细胞纠正凝血因子缺乏奠定了研究基础.     

鲫鱼松果体的显微和超微结构研究

本文作者发现鲫鱼的松果体与一般硬骨鱼不同,它除由背囊和背囊内褶中松果管所组成的松果体外,还有退化的旁突体和副松果体.背囊是单层柱状纤毛上皮,其腔与第3脑室相通,松果管由光感觉细胞、支持细胞、节细胞、丰富的血管和无髓神经纤维构成.松果体既是光感受器又有内分泌的功能.       

藏羚与几种牛科动物成纤维细胞的特性比较

为研究高原低氧环境对哺乳动物细胞生长特性的影响,本文以高原低氧动物藏羚和同科的阿尔巴斯白绒山羊、蒙古绵羊及鲁西黄牛的耳源组织细胞为材料,利用DMEM /F12 培养液进行培养,建立了4 种动物的体外培养成纤维细胞系,并对其进行形态学观察、生长曲线绘制、凋亡检测、核型分析等生物学特性检测。结果表明:4 种动物的细胞经差异贴壁法联合消化排除法,可获得纯化的成纤维细胞,细胞形态一致且活力稳定,4 种动物细胞的形态与生长曲线无明显差异;藏羚、绒山羊、绵羊、牛同代次细胞生长倍增时间分别为26 h、22 h、23 h 和22 h;经流式细胞仪检测,细胞凋亡率分别为5.5% 、2. 7% 、8. 6% 和1.5% ;藏羚、绒山羊、绵羊与牛的染色体数目分别为60、60、54 和60。藏羚与绒山羊、牛染色体数目相同,且除性染色体外,其余均为端部着丝粒类型,而绵羊存在6 条中着丝粒类型染色体。该结果表明,在体外培养条件下,藏羚的体细胞生长特性与同科的其他物种的细胞生长特性存在明显差异。这为从细胞水平上研究藏羚对环境低氧适应的分子机理提供了基础。     

外泌体——癌症的“双刃剑”

正外泌体定义外泌体(exosome)是细胞对外分泌的小囊泡,常为30~120纳米大小的膜包裹结构,可特异性地包裹一些蛋白质、脂质或核酸等物质,具有生物活性,能够被受体细胞吸收,实现细胞间的物质运输和信息传递。20世纪80年代初研究人员在体外培养的正常细胞或者肿瘤细胞内发现细胞外泌现象,细胞向其培养基中分泌带有细胞膜特征的囊泡结构。1983年Rose M.Johnstone等发现体外培养的绵羊网格红细胞在成熟过程中会向外分泌     

星形胶质细胞通过CX47促进少突胶质前体细胞增殖

目的:研究星形胶质细胞(AST)对少突胶质前体细胞(OPC)生长分化的影响及作用机制。方法:用P3SD乳鼠,建立星形胶质细胞与少突胶质前体细胞原代培养体系。实验分两部分,第一部分为常规OPC培养组、AST分泌因子组、AST与OPC直接接触式培养组;第二部分为AST与OPC直接接触式培养组、缝隙连接干扰对照组、缝隙连接干扰组。免疫荧光鉴定OPC与AST细胞纯度,光镜观察细胞生长状态,流式细胞术检测细胞周期,转录组测序分析不同状态下OPC中缝隙连接蛋白CX47差异表达,PCR和Western blot检测鉴定转录组测序CX47差异表达,EDU检测CX47干扰后OPC增殖能力,流式细胞术测干扰CX47后细胞周期变化。结果:光镜与流式检测发现,与AST接触培养的OPC增殖能力最强、新生细胞数目最多。转录组测序分析和PCR验证显示,AST接触调控OPC增殖的主要差异表达蛋白为CX47,干扰CX47后细胞周期阻滞在G1期,OPC增殖能力明显下降。结论;AST可通过缝隙连接蛋白CX47调控细胞周期,促进OPC增殖。     

生物学中的组织培养

一、组织培养(Tissue culture)的含义组织培养,简单地说即把来自机体的细胞、组织或器官放于类似于体内的体外环境中使其存活或/和生长、增殖。严格地区分,它又可分为细胞培养(cell culture):指细胞在体外的生长、增殖,但培养中的细胞不再结合成组织;组织培养:指组织在体外存活生长,并保持其结构和/或功能及进行分化;器官培养(Organ cu     

Day-night changes in plasma melatonin levels,synaptophysin expression and ultrastructural properties of pinealocytes in developing female sheep under natural long and short photoperiods

The aim of the present study was to analyze the 24-h rhythm in plasma melatonin concentration and the day-night differences in synaptophysin expresion and ultrastructural characteristics of the pinealocytes in developing female sheep. Ewes of three different ages were examined: infantile (1-6 months old), pubertal and early fertile age (9-24 months old) and adult (36-60 months old). Experiments were conducted under natural non-stimulatory (long) and stimulatory (short) photoperiods. The obtained results were similar for both analyzed photoperiods. Plasma melatonin concentration, measured in samples obtained every 4 h, showed a similar pattern in the three age groups, with peak values at 02:00 h and troughs at 14:00 h. Mean value of plasma melatonin levels in 9-24 month-old sheep was significantly greater than that in younger or older sheep. The weight of pineal glands obtained at night (02:00 h) was significantly higher than in daylight (14:00 h). Pubertal and early fertile sheep had the largest pineal glands. The pineal volume, and the total number of pinealocytes per gland of 9-24 months-old sheep differed significantly from that of younger or older sheep. The pineal volume, and the mean volume of pinealocytes was significantly greater in animals killed at night. Number of pinealocytes did not vary between animals killed during daylight or at night. The mean volumen of pinealocytes did not show statistical differences between the age groups. In quantitative ultrastructural analysis of pinealocyte cells, the relative volume of mitochondria, rough endoplasmic reticulum and Golgi complexes was significantly greater in 9-24 month-old sheep and in animals killed at night. The relative volume of lipid droplets was highest in older sheep. Collectively, the data support the existence of developmental changes in pinealocyte morphology and quantity, partially in coincidence with a higher melatonin secretion rate.     

多重实时荧光PCR检测牛、山羊和绵羊源性成分

根据牛、山羊和绵羊线粒体细胞色素b基因序列, 设计特异性引物和以不同荧光素标记的Taqman探针。通过对PCR反应体系和反应条件的优化筛选, 建立能同时鉴别牛、山羊和绵羊源性成分的多重实时荧光PCR方法。采用本文方法与国标GB/T 20190-2006方法分别对17种不同源性动物DNA和200份不同来源样品DNA进行牛羊源性成分检测, 数据显示两者检测结果符合率达100%, 特异性相当。与国标方法相比, 本试验方法不需电泳、酶切和测序, 即可在一个PCR反应中同时鉴别检测牛、山羊和绵羊3种源性成分, 检测效率提高近3倍; 灵敏度更高, 比国标方法灵敏10倍; 适用性更广, 除了饲料, 还适用于肉品、奶品、生皮和动物油脂等动物产品的牛羊源性成分检测。     

绒山羊和绵羊KAP6.1基因CDS区克隆与分析

目的:克隆绒山羊、绵羊KAP6.1基因CDS全序列,分析序列特征和蛋白结构。方法:RT-PCR法克隆基因并测序,生物信息学软件分析序列特征和结构。结果:绒山羊、绵羊KAP6.1基因CDS序列全长252 bp,编码83个氨基酸;绒山羊、绵羊KAP6.1蛋白在基本理化特性、二级结构以及空间三维结构上的差异较小。结论:绒山羊与绵羊KAP6.1蛋白结构上的差异将会在一定程度上影响其功能。     

Xenobiotic biotransformation in livers and lungs of adult black-tailed deer: comparison with domestic goat and sheep

1. The capacity of liver and lung tissue of black-tailed dear (Odocoileus hemionus columbianus) to biotransform xenobiotics was compared in vitro to the domestic sheep and goat. Donor animals were all females of varying ages. Tissues from the black-tailed deer were collected in the wild. A variety of biotransformation enzymes were measured in both microsomal and cytosolic fractions. 2. Deer liver was lower in total cytochrome P450 concentration, but mono-oxygenase activities were greater compared to sheep and goat. The opposite was true for the lung. 3. Epoxide hydrolase activities were significantly different in deer vs sheep and goat. 4. In general, both hepatic and pulmonary activities were more similar between sheep and goat than either species compared to the deer, however, the magnitude of the hepatic differences did not exceed 5-fold. 5. Based on these limited results, there is no reason to discredit the sheep or goat as a toxicity testing model for deer.     

Attempted Cross-Transmission of Coccidia Between Sheep and Goats and Description of Eimeria ovinoidalis sp. n.

SYNOPSIS.
Attempted infection of 2 young lambs with oocysts of Eimeria christenseni from a goat was unsuccessful. Negative results were obtained also when young kids were fed oocysts of Eimeria ninakohlyakimovae from sheep. There was no difficulty in infecting lambs with the sheep coccidium resembling E. ninakohlyakimovae nor goats with the goat coccidium E. christenseni. Oocysts from the goat measured 38.4 × 26.7 m, but were easily distinguished from Eimeria ahsata from the sheep by sporocyst size and shape, and from Eimeria ovina by oocyst size. Eimeria ninakohlyakimovae -like oocysts from sheep averaged 23.0 ×18.2 m and were morphologically indistinguishable from previously reported goat coccidia.
Since no cross infections of sheep and goats could be accomplished with oocysts of Eimeria sp. characteristic of one or the other host, I concluded that sheep coccidia previously known as E. ninakohlyakimovae are distinct from morphologically similar goat coccidia and therefore constitute a separate species. Since the name E. ninakohlyakimovae was first used for coccidia from the goat, the sheep coccidium is renamed Eimeria ovinoidalis with oocyst structure and endogenous stages similar to those previously described from the sheep.     

Attempted cross-transmission of coccidia between sheep and goats and description of Eimeria ovinoidalis sp. n.

Attempted infection of 2 young lambs with oocysts of Eimeria christenseni from a goat was unsuccessful. Negative results were obtained also when young kids were fed oocysts of Eimeria ninakohlyakimovae from sheep. There was no difficulty in infecting lambs with the sheep coccidium resembling E. ninakohlyakimovae nor goats with the goat coccidium E. christenseni. Oocysts from the goat measured 38.4 X 26.7 microns, but were easily distinguished from Eimeria ahsata from the sheep by sporocyst size and shape, and from Eimeria ovina by oocyst size. Eimeria ninakohlyakimovae-like oocysts from sheep averaged 23.0 X 18.2 microns and were morphologically indistinguishable from previously reported goat coccidia. Since no cross infections of sheep and goats could be accomplished with oocysts of Eimeria sp. characteristic of one or the other host, I concluded that sheep coccidia previously known as E. ninakohlykimovae are distinct from morphologically similar goat coccidia and therefore constitute a separate species. Since the name E. ninakohlyakimovae was first used for coccidia from the goat, the sheep coccidium is renamed Eimeria ovinoidalis with oocyst structure and endogenous stages similar to those previously described from the sheep.     

Mapping of mitochondrial DNA of individual sheep and goats: Rapid evolution in the D loop region

Mitochondrial DNA (mtDNA) from sheep and goat was compared by restriction endonuclease analysis and heteroduplex mapping in the electron microscope. The fragment patterns produced by endonuclease Hae III from three individual sheep and two goat mtDNAs all differed from each other. The three sheep mtDNAs had identical Eco RI and Hind III fragments, but the two goat mtDNA patterns differed from each other as well as from sheep mtDNA. We estimate that each sheep mtDNA differs from each other by 0.5–1% of its nucleotide sequences, the two goat mtDNAs by 1–2%, and there is a 6–11% sequence difference between sheep and goat mtDNAs. We have mapped the Eco RI and Hind III sites of goat and sheep mtDNA and determined the positions of the D loop, which marks the replication origin, relative to the restriction map. The D loops are at homologous positions on the mtDNAs from both species, but the goat D loop is only 75% as long as the sheep D loop. Regions with a high degree of sequence divergence occur at both ends of the D loop. We suggest that a duplication of about 150 base pairs has occurred in the region where the sheep and goat D loops differ in length. We discuss mtDNA evolution in terms of divergence of isolated “mitochondrial DNA clones.”     

Toxoplasmosis II. Studies of Animal Sera Using Complement-Fixation Tests

Serological studies were performed in guinea pigs, a sheep, calf, goat and two pigs experimentally infected with toxoplasmosis. The direct complement-fixation method was effective in detecting antibodies in guinea-pig, goat and sheep sera. The modified complement-fixation technique supplementing complement with normal bovine serum fraction, was required when testing bovine serum. With swine sera best reactions occurred in the indirect complement-fixation test and definite but low grade reactions were produced in the direct test after pro-complementary activity was removed by pH treatment of the sera. Allergic skin reactions were produced in the experimental animals but improvement in the antigen is necessary before the test could be used generally in the field as a diagnostic method for animal toxoplasmosis.     

Recombinant bovine interferon alpha causes only a temporary prolongation of the lifespan of the corpus luteum

Abstract

A method is described for developing a sheep‐ vs. goat‐specific DNA marker using sequence characterized amplified regions (SCARs) derived from a random amplified polymorphic DNA (RAPD) marker from sheep DNA samples. A sheep 645 bp DNA fragment that was absent in goat DNA was identified by analyzing pools of sheep and goat DNA with RAPD primers. This fragment was cloned and partially sequenced to design extended, strand‐specific 24‐mer oligonucleotide primers. Each primer contained the original 10 bases of the RAPD primer and the following 14 internal bases. The pair of primers resulted in the amplification of a single band of 645 bp when used to amplify sheep DNA, and in no amplification when used to amplify goat DNA. These SCAR primers successfully amplified the equivalent of DNA from one nucleated sheep cell in a sample of 5000 nucleated goat cells. This level of sensitivity is especially desirable for research involving the detection of interspecific chimerism.     

山羊、绵羊MT-Ⅳ分子特性研究

金属硫蛋白(MTs)是一类低分子量、金属和半胱氨酸含量高的细胞质蛋白, 哺乳动物的MTs包括MT-I、MT-II、MT-Ⅲ和MT-IV 4种亚型, 其中MT-IV只在磷状复层扁平上皮细胞中表达, 相关研究报道较少。本研究根据GenBank已公布的动物MT-IV基因序列, 设计出扩增山羊和绵羊MT-IV基因的特异性PCR引物MT-IVSP1和MT-IVSP2, 利用RT-PCR的方法, 分别从山羊和绵羊的瘤胃组织mRNA中, 克隆出山羊和绵羊的MT-IV基因编码区序列(均为189 bp), 序列登录GenBank, 获得序列号EF470251和EF624067。通过序列分析, 表明山羊和绵羊两个物种MT-IV基因编码区全编码均为189 bp、编码62个氨基酸, 其中绵羊的MT-IV含有20个半胱氨酸, 而山羊第61位保守的半胱氨酸被色氨酸所代替。两个物种的MT-IV均不含芳香族氨基酸, 含有MTs特有的C-X-C、C-X-X-C、C-C-X-C-C结构, 无明显的跨膜结构域, 无信号肽, 是一种细胞质蛋白。二级结构分析表明两个物种的MT-IV二级结构大多数为无规则卷曲结构, 分别在第7~9和第49~51氨基酸残基性存在折叠结构, 不存在螺旋结构。三级结构预测结果表明两个物种MT-IV的三级结构由a和b两个结构域组成, 其中β结构域相同, a结构域山羊少一个半胱氨酸残基, 其结构与绵间存在明显差异, 这一差异可能对山羊MT-IV的生理功能产生一定影响, 有必要深入研究。