原位酶谱技术在土壤酶活性研究中的应用进展

原位酶谱可方便、直观地展示土壤中酶活性的二维空间变异,近些年来通过与其他成像技术和分析方法相结合,土壤原位酶谱已发展成为分析土壤微观生态过程及土壤-根系-土壤动物相互作用界面过程的有效方法。然而,土壤原位酶谱技术尚未成熟,亟待深入分析其技术不足、完善技术方法,梳理其在不同领域中的应用现状,拓展并深化其在更多领域研究中的应用前景。基于此,综述并分析了该技术在土壤酶相关研究领域的应用现状,结合原位酶谱技术在底物载体选择与酶活定量方法的优化历程,分析了其存在的问题,发现：(1)可供研究的酶种类较少;(2)结果准确性仍有质疑;(3)在除根际研究外其他领域的研究较少、缺乏系统性,难以直接借鉴。这三者仍是阻碍原位酶谱技术在多领域中推广的最大障碍。综上所述,土壤原位酶谱技术虽仍存在缺陷,但其具有操作简便、测量结果的时空分辨率高等优势,在未来土壤酶学领域研究中仍潜力巨大,研究旨在为推进原位酶谱技术在土壤学和生态学等学科研究中的应用和发展提供理论指导。     

Balanced electrostatic blending approach--an alternative to chemical crosslinking of Thai silk fibroin/gelatin scaffold

In tissue engineering, chemical crosslinking is widely used for conjugating two or more biomaterials to mainly control biodegradability and strength. For example, Thai silk fibroin/gelatin scaffold will offer mechanical strength from Thai silk fibroin and cell attraction from gelatin. However, chemical crosslinking requires crosslinking agent which could potentially pose negative impact from remaining trace amount of chemicals especially in medical application. Here we present an alternative approach to chemical crosslinking—a balance electrostatic blending approach. In this approach, two opposite charge biomaterials were selected for blending, with different ratios. Both materials were bound together with electrostatic force. The maximum binding was achieved when mixture electric potential approaches zero. In this work, we compared this approach with traditionally chemical crosslinking in terms of physical appearance, binding effectiveness, mechanical strength (in dry/wet conditions), in vitro biodegradation, and cell proliferation. We found that 50/50 weight ratio of Thai silk fibroin/gelatin scaffold had almost comparable properties to chemical crosslinked scaffold. It has similar appearance, binding effectiveness, and affinity for cell proliferation. For mechanical properties, even this approach yields lower dry compressive modulus compared with chemical crosslinking. But in wet condition, the compressive modulus from both methods is similar. However, the biodegradation time of non-crosslinked scaffolds is slightly faster than that of chemical crosslinked ones. These results demonstrate that a balance electrostatic approach is an alternative approach to chemical crosslinking when there is a concern of remaining trace amount of crosslinking agent in medical application.     

明胶中细菌内毒素的检测

作者通过鲎试验法检测多种来源、多个批号的药用明胶中细菌内毒素含量并进行合格批号的干扰试验,证明明胶对鲎试剂有干扰作用,其作用表现为浓度越高干扰越大。抗增液无助于消除明胶对鲎试剂的干扰,而有效的稀释可克服这种干扰。并对明胶及含明胶成份的生物制品在细菌内毒素检测中的限值确定及其试验影响进行了讨论。     

明胶碳料的研制及其在明胶液化试验中的应用

自行研制明胶碳粒用于细菌鉴定中的明胶液化试验。将明胶碳粒加入基质中经３７℃ １６￣２０ｈ培养后,如细菌具有明胶酶,其质即变黑色,反之明胶碳粒在基质中沉淀,基质不变色,经接种标准菌株和临床常见菌２０３株,试验结果表明,该明胶碳粒使用简便,容易判定,特异性强,灵敏度高,质量稳定,可以代替国外同类产品。有助于提高细菌鉴定工作效率和质量,降低检测成本,具有很好的应用价值。     

Impact of legume seed extracts on degradation and functional properties of gelatin from unicorn leatherjacket skin

Trypsin inhibitor was extracted from the seed flour of soybean (SB; Glycine max), mung bean (MB; Vigna radiata), cowpea bean (CP; Vigna unguiculata) and adzuki bean (AB; Vigna angularis) using 0.15 M NaCl, followed by heat precipitation at 70 °C. The extract from SB showed the highest specific trypsin inhibitory activity, followed by those from MB, CP and AB, respectively. Based on inhibitory activity staining, molecular weights (MWs) of trypsin inhibitor from SB, MB, CP and AB were 20.1, 14, 10 and 13 kDa, respectively. The SB extract powder (SBEP) containing trypsin inhibitor in the range of 10–100 TIU/g effectively prevented the degradation of γ-, β- and α-chains of collagenolytic proteins of leatherjacket skin subjected to incubation at 50 °C for 30 min. The impact of SBEP on the extraction yield, physical and functional properties of gelatin from leatherjacket skin was investigated. The gelatin extracted in the presence of SBEP contained α₁ and α₂ chains as the predominant components with some degradation peptides. FTIR spectra indicated the significant loss of molecular order of triple helix and higher degradation was found in gelatin extracted in the absence of SBEP. Gelatin extracted in the presence of SBEP had the higher gel strength (232.8–268.5 g) than that extracted in the absence of SBEP (90.4 g). Higher foam stability (FS) but lower emulsion stability index (ESI) was observed in the former. Therefore, the addition of SBEP effectively prevented the degradation of gelatin from the skin of unicorn leatherjacket, thereby yielding the gelatin with improved gel strength and foam stability.     

Fingerprinting of gelatinase subtypes for different topographic regions on non-retaining placenta of Holstein cows

The contribution of matrix metalloproteinases (MMP) to timely discharge of the placenta from bovine uterus at parturition is yet inconclusive, partly because of the presence of multiple MMP forms in situ. In the current study, the expression of different gelatinase subtypes on non-retaining placentas of Holstein cows was fingerprinted by using gelatin zymography. Different topographic regions on the placenta were measured separately, including the placentome-like structure and the fetal and maternal sides of interplacentomal placenta, all sampled from the central and peripheral areas of the placenta, respectively. The spontaneously ruptured umbilical cords were cross-sectioned as fetus end, middle and placenta end also for separate measurement. Body fluids including blood samples from the parturient cows, their neonatal calves and umbilical cord, as well as fetal fluids and the first colostrum were measured concomitantly. Results showed multiple forms of gelatinases subtypes in the placenta tissues and body fluids, including neutrophil gelatinase-associated lipocalin (NGAL)-MMP-9 complex, both the latent and active forms of MMP-2 and MMP-9; of them, the latent forms were much more abundantly and frequently expressed than the active forms. NGAL-MMP-9 complex was more prevalently present in the body fluids than in the placenta tissues. No distinguishable pattern of the expression of any gelatinase subtype was observed among the placentome-like structure, interplacentomal placenta and umbilical cord, or between fetal and maternal sides. Nonetheless, for interplacentomal placenta, proMMP-9 expression was higher in the central than in the peripheral area. In addition, proMMP-2 expression was higher in the rupture end (fetus end) than the placenta end of the umbilical cord. In conclusion, the current validated gelatin zymography detected a gradient proMMP-9 expression on the non-retaining placenta of cows in reverse to the proximity to the umbilical insertion point, and a gradient proMMP-2 expression on a section of the umbilical cord in reverse to the proximity to the rupture site, suggesting roles played by gelatinases in normal discharge of the placenta at term.     

肌间隙移植明胶海绵获取成体干细胞

通过移植生物材料到小鼠体内而建立一种新的干细胞获取方法.移植明胶海绵到小鼠后肢的大腿内侧肌间隙,12天后分离明胶海绵中的迁移细胞.海绵来源干细胞形态、增殖能力、多向分化能力与骨髓源干细胞相似,但海绵中成纤维细胞集落形成单位的比例(82.2±10.6/105)远远高于骨髓中的比例(43.7±7.4/106)(P＜0.05...     

纤溶活性蛋白检测方法研究进展

迄今为止,对纤溶活性蛋白(fibrinolytic protein)的检测主要有4种方法:纤维平板法(fibrin plate method)、显色底物法(colorimetric assay using chromogenic substrates)、反相纤维蛋白自显影法(reverse fibrin autography)和纤维蛋白酶谱法(fibrin zymography)。纤维平板法可以用来快速判断样品是否具有纤溶活性,同时,纤维平板法和显色底物法也可以用来对纤溶活性蛋白进行半定量分析。反相自显影法和纤维蛋白酶谱法则是两种较新的技术,主要用来对纤溶活性蛋白进行定性分析。详细阐述了这几种技术的发展过程、原理、优缺点和应用范围。