钙化胎盘中纳米细菌的分离培养与鉴定

目的分离、培养与鉴定钙化胎盘中的纳米细菌,为进一步探讨纳米细菌致胎盘钙化的机制奠定基础。方法剖腹产手术收集25份钙化胎盘组织标本,通过脱矿、过滤、离心处理,用细胞培养的方法进行纳米细菌培养,观察其生长情况。运用透射电镜、扫描电镜观察培养物形态。结果 (1)培养3~4周后,对钙化组织培养标本进行观察,发现部分培养管底部出现紧贴管壁生长的白色沉淀物。(2)扫描电镜见纳米细菌为大颗粒成簇分布。(3)透射电镜可见纳米细菌为针状物的聚集体,大小不一。结论首次从钙化胎盘组织中分离培养鉴定出纳米细菌,表明其感染与胎盘钙化有关,需进一步研究其矿化机制以及所致钙化对后代的影响。     

先天性佝偻病与纳米细菌关系研究的现状与展望

先天性佝偻病是胎儿出生后就发生的特殊阶段的佝偻病,病因并不十分清楚,迁延不愈会影响小儿骨骼发育和免疫功能。纳米细菌导致的胎盘钙化与小儿佝偻病有关,推测纳米细菌与先天性佝偻病有关。因此诊断和防治纳米细菌感染可能对于先天性佝偻病的预防有一定临床意义。     

瘤胃细菌的分离培养研究进展

瘤胃微生物的发酵使反刍动物能够有效利用粗纤维等营养物质,瘤胃作为自然界的天然饲料发酵罐,是微生态菌剂开发的重要来源,包括纤维素、木聚糖、脂肪、毒素降解菌、非蛋白氮利用菌、甲烷氧化菌等.然而,瘤胃中大部分的微生物仍不可被培养,人工培养方法和流程的参差不齐限制了瘤胃菌种资源的开发利用.因此,本文基于国内外已有的瘤胃细菌分离...     

光合细菌的分离、保存及其同化磷能力试验研究

为了研究光合细菌的保藏方法及其同化磷能力,采用液体种室内自然放置、液体种蜡封和穿刺物蜡封等方法对光合细菌进行保藏方法研究,结果表明,液体种蜡封和穿刺物蜡封可作为该菌种的长期保存方法;对两株菌的磷同化能力进行了比较,降解率分别为22%和15%。本研究为获得光合细菌的长期保存方法及其在污水净化中的应用提供参考。     

磁分离仪分离磁性细菌的新方法

磁性细菌胞内可以产生磁性颗粒,因此具有趋磁性,基于这种特性,利用磁分离的原理,本研究开发了一种磁性细菌分离仪,提供了一种分离磁性细菌的新方法。以氧化亚铁硫杆菌为例,使用磁性细菌分离仪进行分离,可以得到强磁菌和弱磁菌。利用透射电镜观察,强磁菌胞内磁性颗粒明显多于弱磁菌;半固体平板磁泳实验也表明强磁菌趋磁性明显强于弱磁菌。各项实验结果表明磁性细菌分离仪可以有效地分离磁性细菌,这是一种分离磁性细菌的新方法,将促进磁性细菌分离培养的研究。     

基于培养组学的健康人口腔细菌分离鉴定初探CSCD

目的 通过对口腔的不同部位进行细菌分离,扩展对口腔微生物多样性的认识,以期为后续的口腔菌群研究提供参考。方法 通过培养组学方法分离口腔不同部位的细菌,采集2个健康人扁桃体、唾液、牙菌斑3个部位的样本,使用13种培养基并于需氧和厌氧条件下进行培养,采用16S rRNA基因全长测序分析。结果 总计获得144株菌,分属36个种,10个科,3个门。其中,链球菌属细菌种类最多。基于16S rRNA基因全长相似度98.65%的阈值判定,获得5种潜在全新细菌。3个部位中,扁桃体分离的细菌种类多样性最丰富。结论 13种培养基中,BHI培养基获得的口腔细菌多样性最好;BAB培养基与TSA培养基更适合口腔链球菌生长,链球菌的比例更高;LBB培养基上只有较单一的口腔细菌生长,不适于广谱分离口腔细菌。     

亚硝酸氧化细菌分离方法的比较

试验对亚硝酸氧化细菌的三种分离方法一稀释法,硅胶平板分离法和富集培养-微口吸管滴分法进行了比较,结果表明稀释法最简便易行,分离效果最好。     

对纳米细菌在牙结石形成过程中的作用的初步研究

目的探讨牙结石中是否含有纳米细菌及其在牙结石形成过程中的作用。方法ELISA法、免疫荧光染色、透射电镜方法、化学成分分析。结果ELISA检测结果显示20例牙周病患者的龈沟液和牙结石分离培养物中阳性结果分别为2例、16例。通过免疫荧光染色、透射电镜方法检测并观察到牙结石及牙结石分离培养物中均存在纳米细菌,化学成分分析证明了纳米细菌分泌晶体成分同牙结石主要化学成分相同。结论本研究证实了牙结石中存在纳米细菌,并且纳米细菌作为矿化中心参与牙结石形成。     

菌种保存方法对冰核细菌冰核活性的影响

本文测定了5种保存方法及3种保存温度,对4株代表3个属4个种(变种)的冰核细菌冰棱活性的影响。结果表明,不同保存方法对冰核细菌的存活力和冰核活性存在着不同程度的影响,保存温度越高,对冰核活性的影响越大,不同菌种对保存的敏感性不同。所适合的保存方法也不同。真空冷冻干燥和灭菌水于-20℃冷冻保存对各菌株的存活力和冰核活性的影响都较小,适用于一般冰棱细菌的长期保存。而10%甘油冷冻保存则不适用于冰核细菌的保存。     

未培养微生物分离培养技术研究进展

自然环境中的微生物具有多样性高的特点,而99%的微生物使用传统的方法仍然是"不可培养的",并且能被培养的小部分微生物不能够代表微生态的多样性。因此,确定新的微生物物种和功能仍然是目前的重要任务。本文将综述基于调整培养基、考虑微生物间相互作用、捕获分离培养、高通量分离培养来分离培养微生物群体中的未培养微生物的方法,及功能性研究的进展情况。     

正常人和肝病患者血中“致密体”及其与纳米细菌的关系

目的研究正常和肝病患者血液中圆球体样微生物的生物学特性,初步确定纳米细菌与该种可滤过物的关系。方法将正常人和肝病患者培养阴性的血培养物进行细菌L型、穿菌和厌氧菌培养。同时取沉淀用透射电镜观察。L型培养液用0．45μm和0．22μm滤器过滤,接种RPMI 1640培养基,细胞培养条件下培养45d,用鼠抗纳米细菌单克隆抗体8D10免疫组化和钙盐染色进行纳米细菌鉴定。血浆纳米细菌培养阳性者12000×g离心后,进行普通和L型细菌培养。结果36／39患者和60／60健康对照血培养液中呈现类似于L型的巨型体、圆球体、原生小体的不明微生物。电镜观察圆球体内为电子致密样物质,周围未见细胞壁结构。电镜和光镜下可见其粘附在红细胞上或存在于红细胞内。滤过后培养物钙盐染色有5／39阳性,但纳米细菌免疫组化染色均阴性。纳米细菌阳性的培养物转种,未见一般细菌、细菌L型和上述血中“致密体”。结论血培养中致密体样微生物与纳米细菌无关,可能在维持机体正常免疫功能上具有一定意义。     

Method for isolation of pancreatic blood vessels,their culture and coculture with islets of langerhans

The only cure available for Type 1 diabetes involves the transplantation of islets of Langerhans isolated from donor organs. However, success rates are relatively low. Disconnection from vasculature upon isolation and insufficient rate of revascularization upon transplantation are thought to be a major cause, as islet survival and function depend on extensive vascularization. Research has thus turned toward the development of pretransplantation culture techniques to enhance revascularization of islets, so far with limited success. With the aim to develop a technique to enhance islet revascularization, this work proposes a method to isolate and culture pancreas-derived blood vessels. Using a mild multistep digestion method, pancreatic blood vessels were retrieved from whole murine pancreata and cultured in collagen Type 1. After 8 days, 50% of tissue explants had formed anastomosed microvessels which extended up to 300 μm from the explant tissue and expressed endothelial cell marker CD31 but not ductal marker CK19. Cocultures with islets of Langerhans revealed survival of both tissues and insulin expression by islets up to 8 days post-embedding. Microvessels were frequently found to encapsulate islets, however no islet penetration could be detected. This study reports for the first time the isolation and culture of pancreatic blood vessels. The methods and results presented in this work provide a novel explant culture model for angiogenesis and tissue engineering research with relevance to islet biology. It opens the door for in vivo validation of the potential of these pancreatic blood vessel explants to improve islet transplantation therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2745, 2019.     

兔肝细胞体外分离及培养方法的实验研究

目的：研究体外兔肝细胞分离及培养方法,比较不同培养基条件下兔肝细胞培养过程。方法：采用非灌注胶原酶消化法分离兔肝细胞,分别采用RPIM1640培养液（含10％新生牛血清）,DMEM培养液（含10％新生牛血清）,DMEM培养液（含10％胎牛血清）培养,计数法观察原代细胞增殖变化,MTF法观察传代细胞增殖情况。培养细膨采用PAS染色法鉴定,电镜观察细胞超微结构。结果：分离的肝细胞细胞活率大于85％;DMEM培养液（含10％胎牛血清）培养肝细胞生长状态较另两种培养液中的肝细胞强,DMEM培养液（含10％新生牛血清）中的细胞增殖能力较RPIM1640培养液（含10％新生牛血清）高,具有统计学意义。PAS染色和透射电镜观察培养细胞胞质中有大量糖原颗粒。结论：本实验采用的分离方法可获得较纯的肝细胞,而且操作简便实用。DMEM培养液较RPIM1640培养液更加适宜原代培养肝细胞生长,胎牛血清对培养肝细胞的生长促进作用明显高于新生牛血清。     

Protoplast isolation, culture, and fusion protocols for kenaf (Hibiscus cannabinus)

Protoplast isolation and culture protocols were developed for ten cultivars of Hibiscus cannabinus L. (kenaf). Leaves from seedling lines maintained in vitro were used as donor tissues. Optimal cell wall digestions were achieved with a combination of cellulysin (1.0%) and macerase (0.5%). Average yields ranged from 0.9×10⁵ to 5.9×10⁶ protoplasts g fw^-1 leaf tissue with viability estimates ranging from 53% to 87%. This protocol was ineffective for leaf tissue taken from plants grown in vivo. Protoplasts harvested from plantlets maintained in vitro produced rapidly growing calluses when plated in semi-solid medium after an initial culture in liquid medium. First cell divisions were observed within four to six days after initial culture in medium containing plant growth regulators 2,4-dichlorophenoxyacetic acid (1.4 M) and kinetin (13.8 M). An electrofusion protocol which did not significantly reduce protoplast viabilities was developed for kenaf protoplasts. The maximum fusion frequency (4.6%) was obtained with an electrofusion voltage of 2.0 kV cm^-1.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - FDA fluorescein diacetate - MS Murashige and Skoog - NAA 1-naphthaleneacetic acid - PGRs plant growth regulators - SCL seed clonal line     

Effects of proteoglycan modification on mineral formation in a differentiating chick limb-bud mesenchymal cell culture system

In the presence of 4 mM inorganic phosphate, differentiating chick limb-bud mesenchymal cells plated in micromass cultures form a mineralized matrix resembling that of chick calcified cartilage. To test the hypothesis that cartilage proteoglycans are inhibitors of cell mediated mineralization, the synthesis, content, and turnover of proteoglycans were altered in this system, and the extent of mineralization and properties of the mineral crystals examined. In all cases where the proteoglycan synthesis or proteoglycans present were modified to provide fewer or smaller molecules, mineralization was enhanced. Specifically, when proteoglycan synthesis was blocked by treatment with 10⁻¹⁰ M retinoic acid, extensive mineral deposition occurred on a matrix devoid of both proteoglycans and cartilage nodules. The crystals, which formed rapidly, were relatively large in size based on analysis by X-ray diffraction or FT-IR microspectroscopy, and were more abundant than in controls. When 2.5 or 5 mM xylosides were used to cause the synthesis of smaller proteoglycans, the extent of mineral accretion was also increased relative to controls; however, the matrix was less affected, and the extent of mineral deposition and the size of the crystals were not as markedly altered as in the case of retinoic acid. Modification of existing proteoglycans by either chondroinase ABC or hyaluronidase treatment similarly resulted in increased mineral accretion (based on ⁴⁵Ca uptake or total Ca uptake) relative to cultures in which the proteoglycan content was not manipulated. Crystals were more abundant and larger than in control mineralizing cultures. In contrast, when proteoglycan degradation by metalloproteases was inhibited by metal chelation with o-phenanthroline, the Ca accretion at early time points was increased, but as mineralization progressed, Ca accumulation decreased. These data provide evidence that in this culture system, proteoglycans are inhibitors of mineralization. J. Cell. Biochem. 64:632–643. © 1997 Wiley-Liss, Inc.     

一种简单的人脐带间充质干细胞分离培养方法

目的 建立一种简单的人脐带间充质干细胞分离培养方法.方法 取新鲜脐带,剪成5 cm长的小段,直接剪碎为糊状,加入含10％胎牛血清的DMEM/F12在培养瓶中培养,光学显微镜下观察细胞的生长特征,运用流式细胞仪检测分析细胞的抗原标志表达,并检测其体外多向分化潜能.结果 运用不剥离血管组织、不用酶消化的组织贴块培养法可以从...     

An improved culture system for virus isolation and detection

Cell culture plays an important role in virology. It provides a platform for the detection and isolation of viruses as well as for the biochemistry and molecular biology based studies of viruses. In the present work, a new system that could permits multiple (different) cell lines to be simultaneously cultured in one dish was developed. In the system, each cell line was cultured in an isolated zone in the same dish or well and the system is therefore called an isolated co-culture system. The usefulness of this novel approach for virus isolation was demonstrated using a model system based on adenovirus.