CRISPR系统促进脂肪干细胞成骨分化与抑制成脂分化的研究

本研究旨在优化组织培养法分离小鼠脂肪间充质干细胞（adipose-derived stem cells,ASCs）,为研究成骨分化和成脂分化在间充质干细胞分化过程中的相互影响奠定基础。通过细胞形态学观察、细胞生长曲线和流式仪器检测所分离获得的间充质干细胞的特性,利用CRISPR-dCas9系统在快速促进间充质干细胞成骨分化的前提下观察其对成脂分化的影响,并通过生化染色、实时荧光定量PCR和免疫细胞学等手段进行分析。结果显示,接种3~5 d后可见细胞从组织块周围爬出,光镜下可见细胞形态多为成纤维细胞样的梭形细胞,且形态单一均匀,具有较高的爬出率,可以大大提高脂肪间充质干细胞的分离效率;通过CRISPR-dCas9系统激活Runx2和Osterix基因后可以促进间充质干细胞的成骨分化,实时荧光定量PCR及油红O染色结果显示,CRISPR-dCas9系统可以同时抑制间充质干细胞的成脂分化;通过CRISPR-dCas9-KRAB系统同时抑制成骨相关基因Runx2和Osterix后可以促进成脂分化。本研究利用组织贴壁法成功获得了高纯度的脂肪间充质干细胞,具有间充质干细胞的特性和分化能力;利用CRISPR系统可以同时过表达Runx2和Osterix两个基因,可以在进成骨分化的同时抑制成脂分化,表明成脂分化和成骨分化的相关性,为基因编辑在间充质干细胞诱导分化和临床应用方面提供了新的思路和方法。     

Improving the specificity of immunodiagnosis for porcine brucellosis

This report describes the use of cell mediated immunity to improve specificity of current diagnosis for Brucella suis. Diagnosis is problematic due to cross reactions that lead to false positive serological reactions (FPSR) in the standard diagnostic tests. A common cause of this cross reactivity is infection with the organism Yersinia enterocolitica O:9. Gottingen™ mini-pigs were experimentally infected with B. suis biovar I field strain or Y. enterocolitica serotype O:9 biotype 3. Infection was followed for 70 days. During this time whole blood stimulation assays were set up using Brucella specific antigen. IFNγ was measured in the supernatants (SN) from these assays by ELISA. Concurrent standard serological tests were carried out. The results indicate that the IFNγ assay is specifically able to distinguish Y. enterocolitica O:9 infection from a B. suis infection in experimentally infected mini-pigs. These results represent an improvement in diagnostic specificity compared to currently used serological tests. Thus suggesting that in a surveillance setting this test could be applied as a confirmatory test in the face of FPSR. The authors are British Civil Servants and as such their work is subject to British Crown Copyright. This means the exclusive copyright for the article cannot be transferred.     

Bird flu (H7N9) outbreak and its implications on the supply chain of poultry meat in China

During the past two decades, food safety issues in China not only posed serious threats to Chinese consumers but also damaged the image of Chinese products internationally. In China, food safety is not only about scientific discoveries, advanced laboratories, and sanitation equipment; it is more about the role of different players in the food supply chain. The poultry meat supply chain is instrumental in the spread of the avian influenza A virus (H7N9), raising questions about how policymakers respond to such threats and whether industries need to be restructured to manage and control this epidemic so that it does not recur. As a short-term measure, to prevent the spread of this disease, government authorities enforced the closure of live bird markets (LBM) in disease-affected areas of China. However, in the long term, the poultry meat supply chain needs to be restructured. The aim of the current study was to analyze distribution channels for chicken meat in China and then describe arrangements in poultry meat sectors that incorporate small- and medium-scale producers into the supply chain while responding to shifts in LBMs. We also assessed the role of LBMs in spreading H7N9 and how these interventions affect the poultry meat supply chain in the Chinese market.     

草鸡源H9亚型禽流感病毒的分离与鉴定

2010年4月,安徽省某草鸡养殖场发生一起以严重呼吸困难、急性死亡为特征的病例。取发病典型的鸡内脏,按常规方法处理后,接种10日龄SPF鸡胚,经有限稀释法纯化,获得一株病毒,通过HA、HI、分子生物学以及动物回归等试验,最终确诊该病为H9亚型禽流感。     

沙门菌CRISPR的结构与功能研究进展

近年发现沙门菌中恒定地存在两个CRISPR位点,沙门菌利用它们不仅能抵御外源质粒和噬菌体的入侵,还能介导自身短期的表型变化及长期亚类的进化。同时由于其结构的多样性,使之广泛应用于沙门菌分型和进化的研究。论文简要综述了沙门菌CRISPR系统的基本结构、作用机制和功能及其在沙门菌分型和进化应用方面的研究进展。     

Nsp9基因实时荧光定量PCR检测方法的建立及其在感染细胞过程中表达量的变化

试验旨在建立猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus,PRRSV）Nsp9基因的实时荧光定量PCR检测方法,并探究其在感染细胞过程中表达量的变化。根据PPRSVNsp9基因序列设计引物,建立基于SYBR Green Ⅰ检测模式的实时荧光定量PCR。结果显示,PRRSV Nsp9基因在1×10⁴~1×10⁸拷贝/μL范围内有很好的线性关系,扩增产物的熔解曲线只有1个单特异峰,无引物二聚体。该方法与猪戊肝病毒（HEV）、猪流感病毒（SIV）、伪狂犬病病毒（PRV）基因组均无交叉反应,可重复性好,组内变异系数小,可用于临床PRRSV检测。在病毒感染细胞过程中,Nsp9基因表达量逐步升高,36 h达到最高。本研究为探究病毒复制规律与临床疫苗生产奠定了理论基础。     

PRRSVNsp9蛋白基因的RT—LAMP检测方法的建立

根据GenBank中登录的PRRSVNsp9基因序列,应用PrimerExplorerV4软件,在该基因序列中选取保守区设计了4条RT—LAMP引物,旨在建立1种针对Nsp9蛋白基因的逆转录环介导等温扩增（RT-LAMP）的快速检测方法。采用引物分组扩增的方法对引物进行了检测,以确保引物的可靠性。对反应体系、温度以及时间进行了优化,检测了该方法的特异性和灵敏度。结果表明,该方法在等温条件下只需50min就能检测出结果。与RT-PCR方法相比,在判定检测结果时不需要借助昂贵仪器设备,具有高特异性、高灵敏度、操作简便快速等特点,适合于临床检测的应用。     

Interleukin 1, a mediator of immunoadjuvant peptidoglycans

Bacterial peptidoglycans and the synthetic analog muramyl dipeptide possess various immunomodulating properties (adjuvant effect, increase of resistance to infectious agents and to tumor growth). They are able to induce B cell activation and to stimulate macrophages to produce monokines such as Interleukin 1 (IL 1). IL 1 plays an essential role in immune response. It promotes thymocytes maturation and Interleukin 2 secretion by antigen sensitive T cells, which in turn triggers regulatory T cells. Moreover, it is involved in the proliferation and differentiation of B cells.

There is a correlation between the immunoenhancing effect of PG of a definite structure and their ability to induce IL 1 secretion. Non-adjuvant PG were inactive. This suggests that one of the major mechanisms of action of adjuvant PG could be the stimulation of IL 1 synthesis.     

Serotypes of Escherichia coli strains isolated from piglets with diarrhea in swine industrial farms

Serotypes of E. coli strains isolated from piglets, which died with symptoms of diarrhea in 9 swine industrial farms, were determined. Large numbers of serotypes (from 16 to 27) in individual farms were detected. The sets of serotypes from 9 investigated farms differed among each other significantly, depending on the farm and time of examination. It was found that more than one serotype of E. coli may exist in the pig body and contribute to the development of disease. The predominant serotypes, i.e. those comprising more than 10% of serologically determined strains, were found to exist in 6 of the investigated farms and not in the remaining ones. Among the predominant serotypes, particularly important seem to be strains with K88 antigen. For prophylaxis of piglet colibacteriosis in industrial farms in Poland two vaccines for sows are recommended: one containing the K88 antigen only and the other the following serotypes: 0149:K91,K88; 020:K57; 020:K83; 0157:K88; 01:K1; 0136:K78; 024:K?; 078:K80 and 0118:K? Strains belonging to these serotypes were the most prevalent in our strain collection.     

Intramammary infusion of an Enterococcus faecium SF68 preparation promoted the involution of drying off Holstein cows partly related to neutrophil‐associated matrix metalloproteinase 9

A problem for dairy cows following milk stasis is to cope with a high risk of intramammary infection and there is a need to initiate an extensive renewal of secretory modules in mammary glands so that milk production in next lactation may be optimized. We recently reported that ultrasonicated Enterococcus faecium SF68 (SF68) is compatible with cow mammary glands and an enhancer of innate immunity during the immediate post‐milk stasis period. The current study further examines the concomitant effect of ultrasonicated SF68 on mammary tissue remodeling. Four Holstein cows each received intramammary infusions of regular antibiotic dry‐cow formula (positive control) and two different doses of SF68 in different quarters. Analyses of individual quarter secretion samples showed faster neutrophil infiltration, earlier modifications in protein composition, including caseins and lactoferrins, as well as more prompt elevation of the specific unit of 92‐kDa matrix metalloproteinase 9 (MMP9) in SF68‐infused quarters compared to the positive controls. Intramammary infusion of ultrasonicated SF68 seems able to accelerate the regression of mammary synthetic capacity and potentiate the breakdown of glandular extracellular matrix, indicating a more efficient mammary gland involution. Correlation analyses imply that the ability of ultrasonicated SF68 to induce faster neutrophil chemotaxis and the associated MMP9 release is partly responsible.