酒酒球菌液氮超低温保存

【目地】为安全、长期的保藏酒酒球菌,本文研究了菌体生长时间、冷冻方法、解冻温度、菌密度以及保护剂等对酒酒球菌细胞冷冻存活率的影响,找到最优液氮超低温保存方法。【方法】采用平板计数法测定冷冻存活率。【结果】实验结果表明酒酒球菌的最佳保存方法为:首先在稳定期前期离心收集菌体;其次加入保护剂(20 g/L酵母浸提物,40V/V甘油,20 g/L蔗糖,30 g/L谷氨酸钠)稀释菌体,使菌密度为109CFU/mL;然后直接投入液氮冷冻;最后在37℃温水浴中迅速解冻。保存6个月后,其中21株酒酒球菌的冷冻存活率达到99%以上。【结论】初步研究表明酵母浸提物,甘油,蔗糖,谷氨酸钠复合保护剂对酒酒球菌的保护效果较好,液氮超低温保存可用于酒酒球菌的长期保存。     

仔猪副伤寒活疫苗中耐热保护剂应用相关参数的研究

【目的】研究并确定耐热保护剂在仔猪副伤寒活疫苗中应用的各参数,为制备细菌耐热保护剂活疫苗提供参考。【方法】将耐热保护剂与制苗菌液等体积混合,分别采用3种常用冻干曲线(1、2、3)进行冻干,抽样检测确定耐热保护剂配套冻干曲线。在确定合适冻干曲线的基础上,进一步研究耐热保护剂与不同菌龄菌液(发酵培养12、15、18和21 h)、不同浓度菌液(菌液终浓度分别为3×1010、5×1010和7×1010 CFU/m L)、不同感作时间(分别作用0、24和48 h)、耐热保护剂的不同保存时间(2-8°C分别保存0、10、20、30和40 d),以及不同分装量(2、3和4 m L)等多种不同参数对疫苗质量的影响。【结果】配套冻干曲线研究表明,曲线2冻干的产品性状、活菌存活率与耐老化指标效果最好;菌龄研究表明,37°C发酵培养18 h(位于对数生长末期或稳定前期),其冻干菌存活率达78%-81%,优于其它时间;配苗试验表明,菌液适宜终浓度为3×1010-5×1010 CFU/m L;最适感作时间为2-8°C感作24 h,冻干菌存活率可达85.3%-90.5%;耐热保护剂保存期试验表明,2-8°C保存40 d与0 d(配制当日)的保护剂冻干效果基本一致;配苗分装量试验表明,7 m L西林瓶中分装3 m L或4 m L,其冻干菌存活率与2 m L基本一致,但耐老化试验中活菌存活率比2 m L略高。【结论】收获发酵培养18 h的菌液、配苗终浓度采用3×1010-5×1010 CFU/m L,使用2-8°C保存40 d内的耐热保护剂,让保护剂与制苗用菌液2-8°C感作24 h,采用3-4 m L进行定量分装,按曲线2冻干为制备仔猪副伤寒耐热保护剂活疫苗的适合参数。     

微流控法制备载卵母细胞水凝胶微球及其玻璃化保存

目的 通过微流控法制备载卵母细胞海藻酸钠微球,在低浓度保护剂下实现卵母细胞玻璃化保存。方法 采用流动聚焦型微流控芯片,通过调整芯片结构、海藻酸钠溶液浓度和流速比,制备大小均匀、空包率低、低温耐受的载卵母细胞海藻酸钠水凝胶微球。在低浓度低温保护剂下将微球玻璃化保存,复温后检测存活率,采用细胞松弛素B和氯化锶孤雌激活卵母细胞,与Cryotop玻璃化法对比卵母细胞存活率和卵裂率、囊胚率。结果 制备的海藻酸钠微球在冷冻复温前后的体积稳定且结构完整,在将卵母细胞包封在海藻酸钠水凝胶中后,空包率低,存活率、卵裂率和囊胚率与新鲜组相比无显著差异。在低浓度低温保护剂10% DMSO+10%乙二醇（EG）+0.5 mol/L海藻糖中玻璃化冻存后卵母细胞的存活率达到92.48%,卵裂率70.80%,囊胚率20.42%,与高浓度保护剂15% DMSO+15% EG+0.5 mol/L海藻糖中Cryotop玻璃化法相比无显著性差异。结论 本文设计制作了三通道内部交联芯片并用于卵母细胞玻璃化保存的微流控系统,可生成大小均匀、空包率低、低温耐受的载卵母细胞海藻酸钠水凝胶微球,在低浓度保护剂下实现玻璃化保存,为卵母细胞玻璃化保存方法提供新思路。     

L-干燥法和冷冻真空干燥法保藏放线菌效果的比较

L-干燥法和冷冻真空干燥法保藏不同放线菌效果是不一致的。在低温5℃多数放线菌用L-干燥法保藏,无论是干燥后及保存11个月以后其成活率均比冷冻真空干燥法高,但L-干燥法对温度较敏感,在温度37℃保藏成活率有下降的趋势。而冷冻真空干燥法在短期内保藏受温度影响较小。两种方法使用三种保护剂:10%脱脂牛奶粉,3%谷氨酸钠,1%蛋白胨+10%蔗糖,其中以10%脱脂牛奶粉为最理想。因此,采用L-干燥法以10%脱脂牛奶粉为保护剂,在低温5℃长期保存放线菌是较理想的方法之一。两种方法三种保护剂保存11个月后对葡萄糖异构酶     

L-干燥法和冷冻真空干燥法保藏放线菌效果的比较

L-干燥法和冷冻真空干燥法保藏不同放线菌效果是不一致的。在低温5℃多数放线菌用L-干燥法保藏,无论是干燥后及保存11个月以后其成活率均比冷冻真空干燥法高,但L-干燥法对温度较敏感,在温度37℃保藏成活率有下降的趋势。而冷冻真空干燥法在短期内保藏受温度影响较小。两种方法使用三种保护剂:10%脱脂牛奶粉,3%谷氨酸钠,1%蛋白胨+10%蔗糖,其中以10%脱脂牛奶粉为最理想。因此,采用L-干燥法以10%脱脂牛奶粉为保护剂,在低温5℃长期保存放线菌是较理想的方法之一。两种方法三种保护剂保存11个月后对葡萄糖异构酶     

正交法优化嗜酸氧化亚铁硫杆菌冷冻干燥保护剂

利用正交实验方法,以甘油、海藻糖、蔗糖和牛血清蛋白为因素,对嗜酸氧化亚铁硫杆菌（Acididfiobacillus ferrooxidans,A．ferrooxidans）冷冻干燥保护剂的最优化配比进行了研究。直观分析、因素指标分析和方差分析的结果表明：由甘油、海藻糖、蔗糖和牛血清蛋白组成的冷冻干燥保护剂中,对存活率影响的主次顺序依次为：甘油〉海藻糖〉牛血清蛋白〉蔗糖。保护剂的最优化组合为甘油5％、海藻糖15％、蔗糖18％、牛血清蛋白10％。经过验证,该组合的保护剂可使冷冻干燥嗜酸氧化亚铁硫杆菌的存活率达到94％。     

粪微生态制品移植治疗孤独症病例报道

目的孤独症(autism spectrum disorders,ASD)发病机制不明,患病率逐渐增多,缺乏客观诊断指标,常规治疗主要依靠教育和行为训练,但效果十分有限。本研究通过胃肠镜粪微生态制品移植(fecal microbiota transplantation,FMT)治疗儿童孤独症1例,探索其疗效和可行性。方法参照本课题组报道的方法,收集通过严格筛选的供体粪便,体外制备粪微生态制品。孤独症患儿,男性,8岁,确诊孤独症5年余,合并功能性消化不良症状。静脉麻醉下,分别经胃镜、肠镜行粪微生态制品大肠和小肠注入。以临床评价、患儿的生活行为观察(来自患儿主要照顾者),孤独症行为评定检查表(autism behaviorchecklist,ABC),儿童期孤独症评定量表(childhood autism rating scale,CARS)进行治疗前后评估和对比。结果治疗前患儿ABC量表评分为112分,CARS量表积分为38分;治疗后8周,ABC量表评分降低至76分,CARS量表积分降低至32分,积分改善非常显著。语言、沟通、感觉、运动、自我照顾的各项能力整体提高,同时患儿在睡眠障碍、胃肠道症状、情绪方面有明显改善,尤其是胃肠道症状(呃逆)显著缓解。结论粪微生态制品移植对该例孤独症患儿的治疗,疗效明显,未见明显副反应,提示粪微生态制品移植对孤独症患儿可能是一种潜在的新疗法,但其长期疗效、不良反应有待多样本的深入研究。     

木犀草素脂质体冻干工艺优化及其对LX-2细胞的影响

[目的]优化木犀草素脂质体的冻干工艺,考察其对LX-2细胞增殖的影响。[方法]采用冷冻干燥法制备木犀草素脂质体冻干粉,以外观、再分散性、粒径及包封率为指标,通过单因素考察和Box-Behnken响应面法优选最佳冻干工艺,MTT法检测其对LX-2细胞增殖的影响。[结果]最佳冻干工艺为预冻温度-80℃,预冻时间12 h,干燥时间24 h,冻干保护剂为总量8%的蔗糖-乳糖-甘露醇(1.0∶1.0∶2.0)联合使用;与冻干前比较,冻干粉在4℃下30 d内较稳定;脂质体对LX-2细胞的抑制作用强于原药(P<0.01),并呈浓度和时间依赖性。[结论]按最优工艺制备的脂质体冻干粉包封率为89.03%,与预测值90.12%吻合度高达98.79%;且脂质体对LX-2细胞增殖的抑制率显著高于原药(P<0.01)。     

《儿童粪菌移植技术规范的共识》解读

粪便微生物群移植,即粪菌移植(fecal microbiota transplantation,FMT)是通过各种方式将健康捐赠者的粪便菌群移植入患者的消化道内,改善其肠道微生态,以达到治疗疾病的目的。中华预防医学会微生态学分会儿科微生态学组于2016年组织制定了《儿童粪菌移植技术规范的共识》。本文就该共识涉及的儿童粪菌移植适应证、必备条件、方法学(包括捐赠者筛查、排除及剔除的标准、粪菌液制备、粪菌移植途径和剂量的选择、粪菌移植后的观察及处理以及粪菌移植后的随访)等方面的关键问题进行解读。     

仙客来愈伤组织的超低温保存

为了避免连续继代造成仙客来愈伤组织的变异, 对仙客来愈伤组织进行了超低温冷冻保存研究。以继代后处于对数生长期的愈伤组织为实验材料, 首先在含有不同蔗糖浓度的培养基上预培养不同时间, 转至不同的冰冻保护剂中直接液氮冷冻或-20oC预冷冻2 h, 然后液氮超冷冻保存, 37oC水浴迅速解冻, 并用相应蔗糖浓度的液体培养基洗涤, 以中性红染色测定细胞的存活率, SPSS13.0软件进行统计学分析。结果表明: 预培养基中蔗糖浓度、预培养时间、降温方式、冷冻保护剂等对解冻后材料相对存活率存在不同程度的影响, 筛选出4%蔗糖浓度预培养3 d、9号保护剂、0oC停留30 min后直接冷冻为超低温保存的最佳方案, 通过简单的方法获得了较好的愈伤组织保存效果。     

Effects of Vendor and Genetic Background on the Composition of the Fecal Microbiota of Inbred Mice

The commensal gut microbiota has been implicated as a determinant in several human diseases and conditions. There is mounting evidence that the gut microbiota of laboratory mice (Mus musculus) similarly modulates the phenotype of mouse models used to study human disease and development. While differing model phenotypes have been reported using mice purchased from different vendors, the composition and uniformity of the fecal microbiota in mice of various genetic backgrounds from different vendors is unclear. Using culture-independent methods and robust statistical analysis, we demonstrate significant differences in the richness and diversity of fecal microbial populations in mice purchased from two large commercial vendors. Moreover, the abundance of many operational taxonomic units, often identified to the species level, as well as several higher taxa, differed in vendor- and strain-dependent manners. Such differences were evident in the fecal microbiota of weanling mice and persisted throughout the study, to twenty-four weeks of age. These data provide the first in-depth analysis of the developmental trajectory of the fecal microbiota in mice from different vendors, and a starting point from which researchers may be able to refine animal models affected by differences in the gut microbiota and thus possibly reduce the number of animals required to perform studies with sufficient statistical power.     

Fecal Microbiota in Healthy Subjects Following Omnivore,Vegetarian and Vegan Diets: Culturable Populations and rRNA DGGE Profiling

In this study, the fecal microbiota of 153 healthy volunteers, recruited from four different locations in Italy, has been studied by coupling viable counts, on different microbiological media, with ribosomal RNA Denaturing Gradient Gel Electrophoresis (rRNA-DGGE). The volunteers followed three different diets, namely omnivore, ovo-lacto-vegetarian and vegan. The results obtained from culture-dependent and -independent methods have underlined a high level of similarity of the viable fecal microbiota for the three investigated diets. The rRNA DGGE profiles were very complex and comprised a total number of bands that varied from 67 to 64 for the V3 and V9 regions of the 16S rRNA gene, respectively. Only a few bands were specific in/of all three diets, and the presence of common taxa associated with the dietary habits was found. As far as the viable counts are concerned, the high similarity of the fecal microbiota was once again confirmed, with only a few of the investigated groups showing significant differences. Interestingly, the samples grouped differently, according to the recruitment site, thus highlighting a higher impact of the food consumed by the volunteers in the specific geographical locations than that of the type of diet. Lastly, it should be mentioned that the fecal microbiota DGGE profiles obtained from the DNA were clearly separated from those produced using RNA, thus underlining a difference between the total and viable populations in the fecal samples.     

两种粪便保存方法对肠道菌群结构研究的影响

目的比较2种粪便保存方法（室温法和Invitek公司的粪便稳定剂保存法）对菌群结构研究的影响。方法应用PCR-变性梯度凝胶电泳技术（PCR-DGGE）方法,对用2种方法保存的3位志愿者粪便样品进行菌群结构的分析。结果室温法保存粪便样品24h后,S1个体菌群结构与原始样品的菌群结构相似度为83％,S2和S3的菌群结构与其原始样品的相似度仅为77％。而使用粪便稳定剂保存1d,期间各时间点样品菌群结构与原始样品相比变化较小,相似度在80％-90％。结论粪便稳定剂具有一定的稳定样品菌群结构的作用,在新鲜粪佰样品不能寺刻讲行深冻的情况下,使用粪便稳定剂是一种较好的样品保存方法。     

Modifying effects of fermented brown rice on fecal microbiota in rats

Brown rice fermented by Aspergillus oryzae (FBRA) is a fiber-rich food. Effects of dietary administration of FBRA on rat fecal microbiota composition were examined. Male Wistar rats were fed a basal diet or a 5% FBRA- or 10% FBRA-containing diet, and fecal microbiota was analyzed by culture and terminal-restriction fragment length polymorphism (T-RFLP) analysis. The viable cell number of lactobacilli significantly increased after feeding 10% FBRA diet for 3 weeks compared with that in the basal diet group and that in the same group at the beginning of the experiment (day 0). An increase in the viable cell number of lactobacilli was also observed after feeding 10% FBRA for 12 weeks compared with the effect of a basal diet. T-RFLP analysis showed an increase in the percentage of lactobacilli cells in feces of rats fed 10% FBRA for 14 weeks. Lactobacilli strains isolated from rat feces were divided into six types based on their randomly amplified polymorphic DNA (RAPD) patterns, and they were identified as Lactobacillus reuteri, L. intestinalis and lactobacilli species based on homology of the partial sequence of 16S rDNA. FBRA contains lactic acid bacteria, but their RAPD patterns and identified species were different from those in rat feces. These results indicated that dietary FBRA increases the number of lactobacilli species already resident in the rat intestine.     

不同保存方法对川金丝猴粪便DNA提取效果的影响

目的:川金丝猴(Rhinopithecus roxellana)是我国特有珍稀物种,其粪便作为一种非损伤性样品,为珍稀濒危动物的种群数量调查、遗传多样性评价、亲缘关系、系统进化等研究带来了很大便利,本研究试图建立高效、简便的粪便样品保存方法。方法:在现有珍稀濒危动物粪便样品保存方法的基础上,分别使用干燥法、冷冻法和干燥-冷冻法保存川金丝猴的粪便样品,比较了不同保存方法的DNA提取效果,以及对mtDNA控制区片段的PCR扩增成功率和微卫星基因的PCR扩增效率。结果:干燥法、冷冻法和干燥-冷冻法三种不同保存方法保存粪便1周时间后,提取的粪便DNA样本扩增mtDNA片段的成功率均为92%,微卫星基因的扩增成功率分别为79%、78%、80%;保存2个月后,mtDNA片段扩增成功率分别为80%、76%和80%,微卫星基因扩增成功率分别为65%、61%、67%;保存6个月后,mtDNA片段扩增成功率分别为56%、52%和64%,微卫星基因扩增成功率分别40%、34%、46%。因此,随着保存时间的增长,三种方法的保存效率都将明显降低,但干燥-冷冻法得到的DNA样本扩增成功率相对较高。结论:粪便样品能够为川金丝猴的遗传多样性评价等相关研究提供有效信息,干燥-冷冻法保存能够更为有效的保证DNA的提取和基因扩增效率。     

Comparison of fecal preservation and extraction methods for steroid hormone metabolite analysis in wild crested macaques

Since the non-invasive field endocrinology techniques were developed, several fecal preservation and extraction methods have been established for a variety of species. However, direct adaptation of methods from previous studies for use in crested macaques should be taken with caution. We conducted an experiment to assess the accuracy and stability of fecal estrogen metabolite (E1C) and glucocorticoid metabolite (GCM) concentrations in response to several preservation parameters: (1) time lag between sample collection and fecal preservation; (2) long-term storage of fecal samples in 80% methanol (MeOH) at ambient temperature; (3) different degrees of feces drying temperature using a conventional oven; and (4) different fecal preservation techniques (i.e., freeze-drying, oven-drying, and field-friendly extraction method) and extraction solvents (methanol, ethanol, and commercial alcohol). The study used fecal samples collected from crested macaques (Macaca nigra) living in the Tangkoko Reserve, North Sulawesi, Indonesia. Samples were assayed using validated E1C and GCM enzyme immunoassays. Concentrations of E1C and GCM in unprocessed feces stored at ambient temperature remained stable for up to 8 h of storage after which concentrations of both E1C and GCM changed significantly compared to controls extracted at time 0. Long-term storage in 80% MeOH at ambient temperature affected hormone concentrations significantly with concentrations of both E1C and GCM increasing after 6 and 4 months of storage, respectively. Drying fecal samples using a conventional oven at 50, 70, and 90 °C did not affect the E1C concentrations, but led to a significant decline for GCM concentrations in samples dried at 90 °C. Different fecal preservation techniques and extraction solvents provided similar results for both E1C and GCM concentrations. Our results confirm previous studies that prior to application of fecal hormone analysis in a new species, several preservation parameters should be evaluated for their effects on hormone metabolite stability. The results also provide several options for fecal preservation, extraction, and storage methods that can be selected depending on the condition of the field site and laboratory.     

Dysbiosis of gut microbiota was closely associated with psoriasis

Psoriasis is an autoimmune disease and gut microbiota participate in the establishment of intestinal immunity. This study was performed to identify the fecal microbial composition of psoriasis patients, and investigated the influence of subgroup(type and severity) on the fecal microbial composition, and to define the key microbiota in the pathogenesis of psoriasis. Fecal samples from 35 psoriasis patients and 27 healthy controls were sequenced by 16 S rRNA and then analyzed by informatics methods. We found that the microbiota of the psoriasis group differed from that of the heathy group. The relative abundances of Firmicutes and Bacteroidetes were inverted at the phylum level, and 16 kinds of phylotype at the genus level were found with significant difference. No microbial diversity and composition alteration were observed among the four types of psoriasis. The microbiota of psoriasis patients in the severe state differs from those of psoriasis patients with more mild conditions and also the healthy controls. The veillonella in fecal microbiota showed a positive relationship with h-CRP in blood. This research proved that psoriasis patients have a significant disturbed microbiota profiles. Further study of psoriasis based on microbiota may provide novel insights into the pathogenesis of psoriasis and more evidence for the prevention and treatment of psoriasis.     

Terminal restriction fragment length polymorphism analysis for human fecal microbiota and its application for analysis of complex bifidobacterial communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to characterize and compare human fecal microbiota among individuals. T-RFLP patterns of fecal 16S ribosomal DNA (rDNA) PCR products from three adults revealed host-specific bacterial communities and were in good agreement with those reported in our previous study. In addition, we applied T-RFLP analysis for the analysis of complex bifidobacterial communities in human fecal samples. The developed method based on Bifidobacterium genus-specific PCR and T-RFLP could identify more than one bifidobacterial species. T-RFLP patterns of Bifidobacterium genus-specific PCR products from the fecal samples were host-specific as well as those of fecal 16S rDNA PCR products. These results were confirmed by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the genus Bifidobacterium and Bifidobacterium species- and group-specific PCR. Our study demonstrates that T-RFLP analysis is useful for assessment of the diversity of the human fecal microbiota and rapid comparison of the community structure among individuals, and that the applied method is useful for rapid and sensitive analysis of bifidobacterial community.     

The impact of perinatal probiotic intervention on gut microbiota: double-blind placebo-controlled trials in Finland and Germany

Specific probiotic combinations during early feeding, via the mother or incorporated in early formula-feeding, mold the intestinal microbiota composition in infants. The objective was to analyze the impact of probiotic administration to mother or infant on gut microbiota composition in 6-month-old Finnish and German infants. In Finland probiotics were given to mothers (n = 79) for 2 months prior to and 2 months after delivery. In Germany probiotics were started in infants (n = 81) at weaning, at the latest at 1 month of age, and continued for 4 months. A breast-fed group of 6-month-old infants (22 from Finland, 8 from Germany) were compared. Gut microbiota were analyzed by FCM-FISH and qPCR methods. In breast-fed infants a trend toward higher counts of bifidobacteria was detected in Finland (p = 0.097) as against Germany, where a more diverse microbiota was reflected in higher Akkermansia (p = 0.003), Clostridium histolyticum (p = 0.035) and Bacteroides-Prevotella (p = 0.027) levels and a higher percentage of Akkermansia (p = 0.004). Finnish LPR + BL999 intervention group (Lactobacillus rhamnosus LPR and Bifidobacterium longum BL999) had higher percentages of fecal Lactobacillus-Enterococcus (9.0% vs. 6.1% placebo, p = 0.003) and lower bifidobacteria levels (10.03 log cells/g vs. 10.68 log cells/g placebo, p = 0.018). Probiotic treatment had different impacts on gut microbiota composition in Finnish and German infants due to differences in mode of feeding and the early commensal microbiota. Probiotic treatment had different impacts on gut microbiota composition in Finnish and German infants due to differences in mode of feeding and the basic commensal microbiota.     

Epithelial-mesenchymal interactions allow for epidermal cells to display an in vivo-like phenotype in vitro

We here report that preservation of the basic epithelial-mesenchymal interactions allows for highly complex ex vivo function of epidermal cells. The approach taken is based on the preparation of organ fragments that preserve the basic epithelial/mesenchymal interactions but also ensure appropriate diffusion of nutrients and gases to all cells. Human and mice keratinocytes in such organ fragments, remain viable, proliferate and express epidermal-specific gene products when cultured in serum-free medium without added growth factors, for several weeks in vitro. When implanted into syngeneic animals they remain viable, become vascularized and continue to function and transcribe tissue-specific gene products for several months. Such fragments allow primary cells ex vivo to preserve most of the functional attributes of the in vivo system. Clearly, the effect of the extracellular matrix is critical in this system in order for the cells to proliferate and differentiate ex vivo. We are not aware of any other system which allows for localized expression of epidermal-specific genes ex vivo for significant periods in culture in defined serum-free medium.