布氏鲳鲹GHRH基因克隆、组织和胚胎表达模式

促生长激素释放激素(growth hormone releasing hormone, GHRH)主要生物学功能是刺激垂体细胞分泌生长激素,已被证实是动物体生长轴的重要调控因子之一,布氏鲳鲹是一种生长快速的海洋鱼类,为了揭示其代谢旺盛的调节机制,本研究从GHRH入手,利用RACE技术和qPCR方法对布氏鲳鲹GHRH基因进行了克隆、组织和胚胎表达模式研究。实验结果显示,布氏鲳鲹GHRH基因cDNA序列全长1019bp,5’UTR、3’UTR长度分别为327 bp和164 bp,开放阅读框528 bp,共编码175个氨基酸;同源性分析结果表明,布氏鲳鲹GHRH基因与其它鲈形目鱼类的同源性在91%以上。布氏鲳鲹GHRH基因的表达区域大多都集中在中枢系统,其中下丘脑表达量最高;GHRH在受精卵期到后续发育过程中均检测到表达,其表达水平在仔鱼期达到最高。序列分析、组织及胚胎表达的结果表明,布氏鲳鲹GHRH的调节模式仍然可能通过下丘脑调节垂体释放GH,GHRH在个体发育的较早阶段即开始发挥作用。本研究掌握了布氏鲳鲹GHRH基因的基本规律,为进一步研究生长轴的调控提供了理论参考。     

组织工程支架制备中超临界CO2技术的应用*

作为组织工程研究中三大要素之一,组织工程支架可为细胞的附着、迁移和增殖提供理想的环境。传统的组织工程支架制备方法,如粒子沥滤法、相分离法及静电纺丝法等在理论和技术上已较为成熟,但由于大多需要有机溶剂的参与,在制备过程中仍存在有机溶剂难以去除,以及支架孔洞难以控制、连通性较差等问题。超临界二氧化碳(supercritical carbon dioxide,SC-CO₂)密度近似液体,黏度和扩散系数近似气体,具有流动性强、溶解能力大、传热效率高等特殊的理化性质,与传统工艺相结合,可在绿色温和的反应体系中有效规避上述问题,在组织工程支架制备及药物负载方面具有广阔前景。     

Prediction of matrix-to-cell stress transfer in heart valve tissues

Non-linear and anisotropic heart valve leaflet tissue mechanics manifest principally from the stratification, orientation, and inhomogeneity of their collagenous microstructures. Disturbance of the native collagen fiber network has clear consequences for valve and leaflet tissue mechanics and presumably, by virtue of their intimate embedment, on the valvular interstitial cell stress–strain state and concomitant phenotype. In the current study, a set of virtual biaxial stretch experiments were conducted on porcine pulmonary valve leaflet tissue photomicrographs via an image-based finite element approach. Stress distribution evolution during diastolic valve closure was predicted at both the tissue and cellular levels. Orthotropic material properties consistent with distinct stages of diastolic loading were applied. Virtual experiments predicted tissue- and cellular-level stress fields, providing insight into how matrix-to-cell stress transfer may be influenced by the inhomogeneous collagen fiber architecture, tissue anisotropic material properties, and the cellular distribution within the leaflet tissue. To the best of the authors’ knowledge, this is the first study reporting on the evolution of stress fields at both the tissue and cellular levels in valvular tissue and thus contributes toward refining our collective understanding of valvular tissue micromechanics while providing a computational tool enabling the further study of valvular cell–matrix interactions.     

Tissue proteomics of the low‐molecular weight proteome using an integrated cLC‐ESI‐QTOFMS approach

Analysis of the protein/peptide composition of tissue has provided meaningful insights into tissue biology and even disease mechanisms. However, little has been published regarding top down methods to investigate lower molecular weight (MW) (500–5000 Da) species in tissue. Here, we evaluate a tissue proteomics approach involving tissue homogenization followed by depletion of large proteins and then cLC‐MS (where c stands for capillary) analysis to interrogate the low MW/low abundance tissue proteome. In the development of this method, sheep heart, lung, liver, kidney, and spleen were surveyed to test our ability to observe tissue differences. After categorical tissue differences were demonstrated, a detailed study of this method's reproducibility was undertaken to determine whether or not it is suitable for analyzing more subtle differences in the abundance of small proteins and peptides. Our results suggest that this method should be useful in exploring the low MW proteome of tissues.     

Tissue damage in organic rainbow trout muscle investigated by proteomics and bioinformatics

The response to tissue damage is a complex process, which involves the coordinated regulation of multiple proteins to ensure tissue repair. In order to investigate the effect of tissue damage in a lower vertebrate, samples were taken from rainbow trout (Oncorhynchus mykiss) at day 7 after damage and proteins were separated using 2DE. The experimental design included two groups of rainbow trout, which were fed organic feed either with or without astaxanthin. In total, 96 proteins were found to be affected by tissue damage, clearly demonstrating in this lower vertebrate the complexity and magnitude of the cellular response, in the context of a regenerative process. Using a bioinformatics approach, the main biological function of these proteins were assigned, showing the regulation of proteins involved in processes such as apoptosis, iron homeostasis, and regulation of muscular structure. Interestingly, it was established that exclusively within the astaxanthin feed group, three members of the annexin protein family (annexin IV, V, and VI) were regulated in response to tissue damage.     

On‐target ultrasonic digestion of proteins

In the present work, we report a novel on‐target protein cleavage method. The method utilizes ultrasonic energy and allows up to 20 samples to be cleaved in 5 min for protein identification and one sample in 30 s for on‐tissue digestion. The standard proteins were spotted on a conductive glass slide in a volume of 0.5 μL followed by 5 min of ultrasonication after trypsin addition. Controls (5 min, 37°C no ultrasonication) were also assayed. After trypsin addition, digestion of the tissues was enhanced by 30 s of ultrasonication. The samples were analyzed and compared to those obtained by using conventional 3 h heating proteolysis. The low sample volume needed for the digestion and reduction in sample‐handling steps and time are the features that make this method appealing to the many laboratories working with high‐throughput sample treatment.     

骨软骨复合支架的研究进展

软骨的修复是当前医学界十分棘手的难题,人们采取若干手段均收效甚微。由于软骨缺损时,其下的软骨下骨常出现硬化、退变,而新生软骨是无法与病变的软骨下骨进行整合的,所以在修复软骨的同时,必须重视软骨下骨的修复。近十几年来,人们开始发明和利用各种骨软骨复合支架,进行同时修复软骨与软骨下骨的动物实验研究。在正常骨软骨组织中,软骨与软骨下骨被钙化层所相连,此外钙化层也将软骨与软骨下骨分隔在不同的生存环境中。根据仿生学原理,人们又设计出一种带有隔离层的新型骨软骨复合支架,并取得了较为理想的实验结果。本文就国内外骨软骨复合支架的研完进展作一综述。     

大黄鱼白细胞介素8基因克隆及特征分析

白细胞介素8是一种CXC型趋化性细胞因子,在免疫反应中起着非常重要的作用。本文在构建大黄鱼肌肉组织cD-NA文库的基础上,克隆了白细胞介素8基因。克隆到的白细胞介素8全长为2582bp,基因组包含106bp的5’端非编码区,52bp的外显子Ⅰ,168bp的内含子Ⅰ,133bp的外显子Ⅱ,149bp的内含子Ⅱ,87bp的外显子Ⅲ,682bp的内含子Ⅲ,13bp的外显子Ⅳ和1192bp的3’端非编码区,编码序列285bp,编码94个氨基酸。氨基酸序列具有趋化性因子CXC家族的结构特征,在进化上高度保守,与鲈鱼的同源性在90％以上。在检测的大黄鱼的10种组织中,表达量较高的为肾、肝、肠和脾,脑、心和肌肉中表达量较低。     

Effect of Dietary Mono- and Polyunsaturated Fatty Acids on the Fatty Acid Composition of Pigs' Adipose Tissues

In two experiments with growing-finishing pigs six different dietary fats were added to a conventional diet (control - C) to study the effects of dietary monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) on the fatty acid composition of backfat and kidney fat at similar amounts of double bonds in feed (Exp. 1:7% pork fat - PF, 4.95% olive oil - OO, 3.17% soybean oil - SO) or a constant amount of 5% of processed fats (Exp. 2: partially hydrogenated fat - SAT, fractionated pork fats: olein - OLE, stearin - STE). Compared with the control, PUFA were only slightly increased in backfat of pigs fed PF, OLE, STE or OO, although dietary PUFA intake was up to 70% higher. With SO PUFA were significantly increased in adipose tissues, predominantly at the expense of MUFA. Consequently, a non-linear relationship was found between PUFA intake and proportion in backfat. MUFA were incorporated at the expense of SFA, therefore, adipose tissues of OO fed animals were lowest in SFA. Despite comparable amounts of double bonds in feed (Exp. 1), the degree of unsaturation measured as fat score (sum of double bonds) was in the order SO > OO > PF > C. In contrast, the proportion of SFA was C > PF = SO > OO. Regarding the decisive role of SFA for fat consistency it may be concluded that MUFA should also be considered in feeding recommendations for pigs. Furthermore, in case of a high dietary supply of MUFA, a simple index of double bonds might not be sufficiently conclusive to judge pig fat quality.