传代培养骺板软骨细胞作为组织工程种子细胞的生物学研究

目的:定量观测体外培养骺板软骨细胞合成蛋白多糖聚集体的变化,评价其不同代次生物学功能活性。方法:酶消化分离原代骺板软骨细胞,传代培养至第6代。改进的二甲基亚甲蓝(DMB)比色法测定各代细胞胞外基质的硫酸糖胺多糖(Glycosaminoglycans,GAG)含量,细胞-玻片甲苯胺蓝染色光镜观察。结果:原代软骨细胞具有最高的硫酸GAG分泌活性,(70±8)μg/cm2,至第3代则出现了非常明显的下降,(53±10)μg/cm2,第4代以后很低下;甲苯胺蓝染色显示原代细胞对甲苯胺蓝呈明显的红色异染反应,第3代异染反应明显减弱,第4代以后异染反应微弱,其结果与硫酸GAG含量变化相一致。结论:骺板软骨细胞在体外培养过程中,随传代次数的增加而功能活性逐渐降低,第3代以后的细胞不适于作为骺板组织工程种子细胞和用于细胞生物学等方面研究。     

Anaerobic 4-hydroxyproline utilization: Discovery of a new glycyl radical enzyme in the human gut microbiome uncovers a widespread microbial metabolic activity

ABSTRACT

The discovery of enzymes responsible for previously unappreciated microbial metabolic pathways furthers our understanding of host-microbe and microbe-microbe interactions. We recently identified and characterized a new gut microbial glycyl radical enzyme (GRE) responsible for anaerobic metabolism of trans-4-hydroxy-l-proline (Hyp). Hyp dehydratase (HypD) catalyzes the removal of water from Hyp to generate Δ¹-pyrroline-5-carboxylate (P5C). This enzyme is encoded in the genomes of a diverse set of gut anaerobes and is prevalent and abundant in healthy human stool metagenomes. Here, we discuss the roles HypD may play in different microbial metabolic pathways as well as the potential implications of this activity for colonization resistance and pathogenesis within the human gut. Finally, we present evidence of anaerobic Hyp metabolism in sediments through enrichment culturing of Hyp-degrading bacteria, highlighting the wide distribution of this pathway in anoxic environments beyond the human gut.     

聚合酶链反应法和分离培养法筛检小肠结肠炎耶尔森菌方法的比较研究

目的 比较针对小肠结肠炎耶尔森菌的两种不同检测方法,即ail基因联合foxA基因的聚合酶链反应(polymerase chain reaction,PCR)检测和分离培养法的差异。 方法 从生猪咽拭子和回盲部肠内容物标本中提取细菌基因组DNA并进行小肠结肠炎耶尔森菌保守基因foxA和粘附侵袭位点基因ail的PCR检测;菌株分离培养按常规方法进行。将两种方法的检出结果进行统计分析,以判定哪种更有优势。 结果 700份标本中,小肠结肠炎耶尔森菌特异性基因PCR检测阳性402份,阳性率57.43%;小肠结肠炎耶尔森菌分离培养阳性278份,阳性率39.71%。在PCR检测阳性的402份标本中,分离小肠结肠炎耶尔森菌271株,分离阳性率为67.41%;PCR检测阴性的298份标本中,分离小肠结肠炎耶尔森菌7株,分离阳性率仅为2.35%。 结论 本次实验证实,检测来源于生猪标本中的小肠结肠炎耶尔森菌,采用foxA和ail基因联合的PCR检测法,检出率明显高于小肠结肠炎耶尔森菌分离培养法(P0.05)。     

台式压力蒸汽灭菌器的应用及其灭菌效果观察

目的观察台式压力蒸汽灭菌器在特殊物品灭菌中的应用及其灭菌效果。方法通过化学指示剂监测法,观察了小型台式压力蒸汽灭菌器对细胞培养用品和配液的灭菌效果。结果用内源蒸汽台式压力蒸汽灭菌器,在121℃条件下对细胞培养配液灭菌25 m in,对细胞培养配液达到灭菌通过;在132℃条件下灭菌12 m in,对手术器械、玻璃培养器皿、胶塞等物品包达到灭菌通过。灭菌处理的细胞培养器皿和细胞配液都不影响细胞生长。结论用台式压力蒸汽灭菌器对眼科实验室的细胞培养器皿和配液进行灭菌具有良好的适用性和有效性。     

新安医家“固本培元”法临床应用的数据分析

目的分析新安医家运用"固本培元"法防治疾病的相关证治规律。方法选取12位新安医家的12本医籍,运用"固本培元"法共678条医案,建立数据库,运用SAS 8.0软件进行统计分析。结果医案中出现的症状主要为:脾气虚(腹胀、腹痛、便溏),肾气虚(腰膝酸软、眩晕、小便清长),肾阳虚(畏寒、下肢肿、腰痛);常用药物为人参、黄芪、白芍、茯苓、甘草、当归、陈皮、附子等。由数据挖掘出的基本方(KDD)为:人参、黄芪、白术、茯苓、甘草、白芍、当归、陈皮、半夏、柴胡、附子、肉桂、鹿角、紫河车、黄芩、知母。结论 "固本培元"法的治疗重点在于脾、肾并治,以补脾气、温补脾阳,补肾气、温补肾阳和督脉等为其基本治法。经过数理分析和数据挖掘,得到了一些有效药物,常见症状,常用药物和症状的组合及基本方。为今后临床治疗及实验研究提供了依据和研究线索,为新安医学的进一步研究提供了数据基础。     

人非小细胞肺癌血管内皮细胞的分离培养及鉴定

目的:建立人非小细胞肺癌血管内皮细胞体外分离培养体系,研究其体外生长特性。方法:利用酶消化法分离出肺癌组织微血管片段,采用植块培养法培养原代内皮细胞,先后以局部消化法和差速黏附法进行纯化;应用光镜、免疫细胞化学及免疫荧光技术对所获得的人肺癌血管内皮细胞进行鉴定。结果:所获人非小细胞肺癌血管内皮细胞呈FⅧ-RAg、CD34阳性;电镜下生长状态良好、可传代培养。结论:本研究摸索并建立了人非小细胞肺癌血管内皮细胞的分离培养方法,所获人非小细胞肺癌血管内皮细胞对肿瘤血管生成及抗肿瘤药物的研究具有重要价值。     

Laboratory-induced changes in the mate recognition system ofDrosophila pseudoobscura

Drosophila pseudoobscura stocks maintained in laboratory culture for some time had altered mate recognition systems compared with recently collected control stocks. Three showed a significant deviation from random mating; in one, a tendency toward homogametic mating was recorded. Such deviations are in marked contrast to results from populations which have been recently collected from the field. Asymmetrical mating was observed in two of the crosses in the direction predicted by the Kaneshiro model.This research is supported by New Zealand University Grants Committee Grant 141Z164/D. M. Lambert. The authors with to thank L. Barr, D. F. Poulson, A. Beckenach, R. A. Norman, and B. C. Moore for their kind assistance in sending us cultures.This contribution represents publication No. 12 from the Evolutionary Genetics Laboratory, University of Auckland.     

Impact of organ culturing on metabolic profile of human corneas: preliminary results

Purpose: It is suggested that the quality of corneal graft may depend on modifications that appear in the tissue during culturing. The aim of this study was to investigate the differences in the metabolic profile between cultured and noncultured human corneas. Methods: Corneas from 12 donors were obtained post‐mortem and cultured for 6–20 days. Control corneas were obtained from four patients with malignant melanoma of the chorioidea and were kept frozen at ?80 °C until analysed. The metabolic profiles of the samples were investigated using high‐resolution, magic angle spinning ¹H nuclear magnetic resonance spectroscopy and special software for: (i) analysis of complex mixtures, (ii) principal component analysis and (iii) specialized statistical analysis. Results: Twenty metabolites were detected and assigned in the corneas. Significant differences in metabolic profiles between cultured and noncultured corneas were revealed. It was also shown in samples kept in culture for 9–14 days that the levels of (i) alanine, formate, lactate and (ii) acetate, alanine, arginine, lactate were elevated in comparison with the samples kept for <9 and more than 14 days, respectively. Conclusions: Corneal culturing affects the metabolic profile of the tissue. The increases in the levels of some metabolites within the second week of culturing likely result from variations in tissue metabolic or enzymatic activity caused by changed (organ culture) environment. As the mechanisms responsible for these changes are not clear, further research is indicated.     

细菌芽孢形成机制在微生态制剂生产中的应用

益生菌的生存能力和稳定性是生产者在技术和市场方面临的两大挑战。由于芽孢对多种不利环境有极大的抗性而成为近年来益生菌剂中一个研究热点。芽孢的抗性与多种因素有关,并通过芽孢这种独特结构来实现。Spo0A和σ是芽孢形成过程中基因表达的关键调节因子。杆菌和梭菌在芽孢形成中的主要差别是梭菌形成感受态细胞和淀粉粒的积累。为了得到最高的菌体浓度和芽孢形成率,需要优化培养条件。其中,遗传性、接种量、培养温度、pH值、无机盐、淀粉、菌体浓度、营养因子的限量添加都与芽孢的形成有重要关系。     

Transplantation of human bone marrow‐derived clonal mesenchymal stem cells reduces fibrotic scar formation in a rat spinal cord injury model

This study aimed to evaluate the therapeutic effect on tissue repair and scar formation of human bone marrow‐derived clonal mesenchymal stem cells (hcMSCs) homogeneously isolated by using a subfractionation culturing method, in comparison with the non‐clonal MSCs (hMSCs), in a rat spinal cord injury (SCI) model. The SCI was made using a vascular clip at the T9 level. Cells were transplanted into the lesion site 3 days after injury. A functional test was performed over 4 weeks employing a BBB score. Rats were killed for histological analysis at 3 days, 1 week and 4 weeks after injury. The transplantation of hMSCs and hcMSCs significantly reduced lesion size and the fluid‐filled cavity at 4 weeks in comparison with the control group injected with phosphate buffered saline (PBS) (p < 0.01). Transplantation of hcMSCs showed more axons reserved than that of hMSCs in the lesion epicentre filled with non‐neuronal tissues. In addition, hMSCs and hcMSCs clearly reduced the inflammatory reaction and intraparenchymal hemorrhaging, compared with the PBS group. Interestingly, hcMSCs largely decreased Col IV expression, one of the markers of fibrotic scars. hcMSCs yielded therapeutic effects more than equal to those of hMSCs on the SCI. Both hMSCs and hcMSCs created an increase in axon regeneration and reduced scar formation around the SCI lesion. Copyright © 2017 John Wiley & Sons, Ltd.