目的 构建幽门螺旋杆菌尿素酶纳米抗体UreNb重组质粒,在大肠杆菌系统中通过诱导表达目的蛋白并予以纯化,再对表达产物及纯化产物进行鉴定.方法 利用DNA重组技术,先后使用两种原核表达载体pET22b和pSumo-mut表达目的蛋白UreNb,构建幽门螺旋杆菌尿素酶纳米抗体UreNb,重组质粒.将构建成功的纳米抗体UreNb重组质粒分别转化至大肠杆菌BL-21、Arctic Express、Rosetta菌株中,采用不同温度经IPTG诱导表达目的蛋白.将成功表达的重组蛋白扩大培养,通过SDS-PAGE法分析其表达蛋白,表达蛋白在沉淀物中即确定为包涵体蛋白.将包涵体蛋白变、复性后,通过镍柱亲和层析法纯化蛋白并用 Western Blot法鉴定.结果 纳米抗体UreNb目的序列成功插入质粒中,在大肠杆菌表达系统BL-21 中以包涵体形式表达(经SDS-PAGE法验证).经过包涵体复性后纯化,获得大量高纯度目的蛋白pSumo-mut-UreNb,纯化蛋白的分子量约为 27 kDa(经Western Blot鉴定).结论 使用pSumo-mut质粒构建的幽门螺旋杆菌尿素酶纳米抗体UreNb能够在原核系统中得到高效表达,通过纯化获得具有活性的纯化蛋白.经鉴定,其大小与理论大小基本相符,可用于后续研究.

Potentilla anserina L., a well-known perennial herb, is widely used in traditional Tibetan medicine and used as a delicious food in humans. The present investigation reports on the activity of P. anserina phenols (PAP) in regulating glycolipid metabolism in 3T3-L1 adipocytes. Insulin sensitivity tests showed that PAP improved insulin-stimulated glucose uptake by promoting the phosphorylation of serine/threonine kinase Akt. Moreover, an assay involving the differentiation of 3T3-L1 preadipocytes demonstrated that PAP also decreased the accumulation of lipid droplets by suppressing the expression of adipokines during the differentiation process. In addition, the underlying mechanism from the aspects of energy metabolism and oxidative stress is also discussed. The improvement in energy metabolism was supported by an increase in mitochondrial membrane potential (MMP) and intracellular ATP. Amelioration of oxidative stress was supported by decreased levels of intracellular reactive oxygen species (ROS). In summary, our findings suggest that PAP can ameliorate the disorder of glycolipid metabolism in insulin resistant 3T3-L1 adipocytes by improving energy metabolism and oxidative stress and might be an attractive candidate for the treatment of diabetes.

In this study, supercritical fluid extraction (SFE), ultrasonic-assisted extraction (UAE), and microwave-assisted extraction (MAE) were applied to explore the most suitable extraction method for fatty acids of Potentilla anseris L. from 12 different producing areas of the Qinghai-Tibetan Plateau. Meanwhile, the important experimental parameters that influence the extraction process were investigated and optimized via a Box-Behnken design (BBD) for response surface methodology (RSM). Under optimal extraction conditions, 16 fatty acids of Potentilla anserina L. were analyzed via high-performance liquid chromatography (HPLC) with fluorescence detection, using 2-(4-amino)-phenyl-1-hydrogen-phenanthrene [9,10-d] imidazole as the fluorescence reagent. The results showed that the amounts of total fatty acids in sample 6 by applying SFE, UAE, and MAE were, respectively, 16.58 ± 0.14 mg/g, 18.11 ± 0.13 mg/g, and 15.09 ± 0.11 mg/g. As an environmental protection technology, SFE removed higher amounts of fatty acids than did MAE, but lower amounts of fatty acids than did UAE. In addition, the contents of the 16 fatty acids of Potentilla anserina L. from the 12 different producing areas Qinghai-Tibetan Plateau were significantly different. The differences were closely related to local altitudes and to climatic factors that corresponded to different altitudes (e.g., annual mean temperature, annual mean precipitation, annual evaporation, annual sunshine duration, annual solar radiation.). The temperature indices, photosynthetic radiation, ultraviolet radiation, soil factors, and other factors were different due to the different altitudes in the growing areas of Potentilla anserina L., which resulted in different nutrient contents.

目的 观察藏药十味乳香散对高尿酸血症动物模型的降尿酸作用并探讨机制.方法 观察药物预处理高尿酸血症动物模型后动物血尿酸、尿尿酸、血肌酐、尿肌酐水平及肝脏黄嘌呤氧化酶(XOD)的活性.结果 藏药十味乳香散可使正常健康大鼠血尿酸水平显著降低(P＜0.05)、高尿酸血症大鼠在显著性降低血尿酸(P＜0.05)同时伴有尿尿酸水平的降低(P＜0.05);可使高尿酸血症小鼠血尿酸水平降低(P＜0.05)、肝脏XOD活性抑制(P＜0.05).结论 藏药十味乳香散可以降低正常健康动物的血尿酸,也可以降低高尿酸血症动物模型的血尿酸;可能机制为通过抑制肝脏XOD的活性实现降血尿酸作用.

目的:研究藏药五鹏丸的抗炎作用及机制.方法:采用二甲苯致小鼠耳肿胀法、小鼠腹腔毛细血管通透性法、角叉菜胶致大鼠踝关节肿胀法观察五鹏丸的抗炎作用,测定炎症组织中前列腺素E2(PGE2),环氧酶-2(COx-2),白三烯B4 (LTB4)的含量.结果:与对照组比较,五鹏丸高剂量能显著减少小鼠耳肿胀度,降低小鼠腹腔毛细血管通透性及大鼠踝关节肿胀度和炎症组织中LTB4含量(P＜0.05或P＜0.01).结论:藏药五鹏丸有显著的抗炎作用.

目的:通过观察十五味乳鹏散对尿酸钠(MSU)致急性痛风性关节炎大鼠膝关节液及滑膜组织中环氧酶2(COX-2),前列腺素E2(PGE2)蛋白及mRNA水平的影响,探讨十五味乳鹏散治疗急性痛风性关节炎的机制.方法:大鼠分为正常组,模型组,十五味乳鹏散低、中、高剂量(0.4,0.8,1.2 g·kg-1)组,吲哚美辛(3 mg·kg-1)组.正常组和模型组ig生理盐水;各给药组按剂量ig给药,连续7d.末次给药1h后,正常组双侧膝关节腔注射50 μL无菌1％磷酸盐缓冲液(PBS),其余各组注射50 μL尿酸钠溶液,于造模后24 h用酶联免疫吸附试验(ELISA)和实时荧光定量PCR (QPCR)检测关节滑液和滑膜组织COX-2,PGE2蛋白及mRNA水平.结果:与正常组比较,模型组MSU注射进大鼠膝关节腔24 h后,COX-2,PGE2在大鼠关节滑液中的含量及在滑膜组织中的mRNA表达量均明显增加(P＜0.01);与模型组比较,十五味乳鹏散低、中、高剂量组及吲哚美辛组均显著降低MSU致大鼠痛风性关节炎关节滑液COX-2和PGE2的蛋白水平及滑膜组织COX-2 mRNA水平(P＜0.05,P＜0.01),但滑膜组织PGE2mRNA水平在各组间并无显著性差异.结论:MSU可以增加关节滑液及滑膜组织中COX-2的表达,同时伴有COX-2,PGE2水平的增加;十五昧乳鹏散可能通过降低COX-2的表达,降低COX-2,PGE2的水平产生抗MSU痛风性关节炎的作用.