The metabolic profilings study of serum and spinal cord from acute spinal cord injury rats ^1H NMR spectroscopy

目的：采用-1H NMR核磁共振代谢组学的方法研究急性脊髓损伤模型大鼠的代谢组学特征及生物标志物,探讨核磁共振代谢组学应用于脊髓损伤研究的可行性。方法：取8周龄清洁级雄性SD大鼠20只,体重（200±10）g,按照随机数字法分为假手术组和模型组,每组10只,模型组采用改良的Allens法制作急性脊髓不完全损伤模型,假手术组不损伤脊髓,术后第1、5、7天采用BBB运动功能评分法进行行为学观察,术后第7天收集脊髓组织作病理学观察,核磁共振代谢组学对两组大鼠血清和脊髓样本进行代谢组学分析。结果：BBB评分显示假手术组术后后肢运动无明显改变,各时间点差异无统计学意义,模型组大鼠术后双下肢呈迟缓性瘫痪,BBB运动评分较低,各时间点差异存在统计学意义,两组运动功能评分在各时间点的差异均有统计学意义;病理切片显示假手术脊髓结构正常,神经分布均匀,模型组脊髓组织结构紊乱,神经元数目减少,存在炎性细胞浸润和空腔坏死组织。代谢组学分析表明,血清中极低密度脂蛋白（VLDL）、低密度脂蛋白（LDL）、谷氨酰胺（glutamine）、柠檬酸（citrate）、二甲基甘氨酸（DMG）等物质和脊髓中谷胱甘肽（glutathione）、3-羟基丁酸（3-OH-butyrate）、N-乙酰天冬氨酸（NAA）、磷酸胆碱（GPC）、谷氨酸（glutamate）、抗坏血酸（ascorbate）等物质浓度有明显变化（P〈0.05）。结论 ：通过对假手术组和模型组大鼠血清和脊髓样本进行代谢组学检测和分析得到了两组样本的差异性代谢物质,有助于解释急性脊髓损伤后血清和脊髓组织中的特异性小分子物质的变化规律,为后期针对性地研究这些代谢标记物在急性脊髓损伤中的作用提供研究基础。Objective： To establish the rat model of acute spinal cord injury,followed by aprimary study on this model with 1H NMR based on metabonomics and to explore the metabonomics and biomarkers of spinal cord injury rat.Methods： Twenty eight-week-old adult male SD rats of clean grade,with body weight of （200±10） g,were divided into sham operation group and model group in accordance with the law of random numbers,and every group had 10 rats. The rats of sham operation group were operated without damaging the spinal cord,and rats of model group were made an animal model of spinal cord incomplete injury according to the modified Allen＇s method. According to BBB score to observate the motor function of rats on the 1th,5th,and 7th days after surgery. Postoperative spinal cord tissue was collected in order to pathologic observation at the 7th day,and the metabolic profilings of serum and spinal cord from spinal cord injury rats were studied by 1H NMR spectroscopy.Results： The hindlimb motion of rats did not obviously change in sham operation group,there was no significant difference at each time point;and rats of model group occurred flaccid paralysis of both lower extremities,there was a significant difference at each time; there was significant differences between two groups at each time. Pathological results showed the spinal cord structure was normal with uniform innervation in shame group,while in model group,the spinal cord structure was mussy,and the neurons were decreased,with inflammatory cells and necrotic tissue. Analysis of metabonomics showed that concentration of very low density fat protein （VLDL）,low density fat protein （LDL）,glutamine,citric acid,dimethylglycine （DMG） in the serum and glutathione,3-OH-butyrate,N-Acetyl-L-aspartic acid （NAA）,glycerophosphocholine （GPC）,glutamic acid,and ascorbate in spinal cord had significant changes（P〈0.05）.Conclusion： There are significant differences in metabolic profile from serum and spinal cord sample between model group and sha浙江省自然基金（编号：LY15H270003）;浙江省中医药科技计划项目（编号：2015ZZ017

Purification and Properties of β-glucosidase from Aspergillus glaucus EU7-22

目的:利用灰绿曲霉Eu7-22发酵产纤维素酶,从中分离到β-葡萄糖苷酶,分析其理化特性,确定其最佳活性条件。方法:灰绿曲霉Eu7-22发酵液离心后,上清液经硫酸铵沉淀、PHEnyl 6 fAST flOW(HIgHSub)疏水层析和SEPHACryl S-200凝胶层析,获得纯化的β-葡萄糖苷酶。结果:纯酶的比活性为5.1 Iu/Mg,得率为13.89%。SdS-PAgE凝胶电泳分析表明该酶是单亚基蛋白,其分子量为56.2 kdA。在PH4.0--6.0范围内,β-葡萄糖苷酶具有较高的稳定性,该酶的最适酶促反应PH为5.0。当β-葡萄糖苷酶在温度低于60℃的缓冲液中温育1 H后,酶活损失不大,表现了较好的稳定性;当该酶在温度高于60℃的缓冲液中温育1 H后,酶活迅速丧失。β-葡萄糖苷酶在70℃时具有最大催化活性。结论:灰绿曲霉Eu7-22发酵产生的β-葡萄糖苷酶具有较高活性,具有分子量较小、最佳催化温度较高的特点。Objective:β-glucosidase produced by Aspergillus glaucus EU7-22 was purified and characterized.Method:The β-glucosidase of Aspergillus glaucus EU722 was purified from ferment supernatant liquid by three steps of purification,ammonium sulfate precipitation(80%,W/V),Phenyl 6 Fast Flow(high sub) column chromatography and Sephacryl S-200 column chromatography,with a specific activity of 5.1 IU/mg and a yield of 13.89%.Result:The purified β-glucosidase was determined as a monomeric protein with a molecular weight of 56.2 kDa.The enzyme exhibited high stability when it was kept in the buffer at pH 4.0～6.0 and at the temperature below 60℃.β-glucosidase exhibited its optimal activity at 70℃.Conclusion: β-glucosidase produced by Aspergillus glaucus EU7-22 with small molecular showed high activity under the optimal conditions.国家“973”计划项目(2010CB732201);国际科技合作重点项目(2009DFA60930)资

不同群落类型的长苞铁杉林的根际微生物研究

对长苞铁杉纯林、长苞铁杉猴头杜鹃混交林和长苞铁杉毛竹混交林3个不同群落类型的根际微生物进行研究,发现3个不同群落的微生物数量及组成各不相同,其具体大小关系为(单位:cfu/g·(drymass)×104):长苞铁杉纯林:根际细菌(20.00)>非根际细菌(12.00)>根际真菌(9.37)>非根际真菌(6.74)>根际放线菌(2.69)>非根际放线菌(1.83);长猴混交林:根际细菌(34.90)>非根际细菌(18.00)>根际真菌(6.54)>非根际真菌(3.78)>根际放线菌(2.96)>非根际放线菌(1.76);长毛混交林:非根际细菌(88.00)>根际细菌(68.60)>非根际放线菌(13.40)>根际放线菌(12.60)>根际真菌(4.64)>非根际真菌(3.46).通过比较根际与非根际土壤微生物得出不同群落类型长苞铁杉的根际效应,3个群落类型的总微生物的根际效应大小为:长猴混交林(1.87)>长苞铁杉纯林(1.56)>长毛混交林(0.82)

一株高效抑藻放线菌的分离筛选及鉴定

从福建云霄国家红树林自然保护区滩涂沉积物样品中,共分离获得521株纯培养物.通过检测球形棕囊藻(Pha-eocystis globosa)荧光强度计算抑藻率,从521株菌中筛选到27株具有抑藻活性的菌株.在27株抑藻菌中,菌株O3-26对球形棕囊藻具有最高的抑藻率(高达96.71%).菌株O3-26的抑藻谱实验显示,该菌株抑藻活性表现出一定的种属特异性,对硅藻门(Bacillariophyta)的三角褐指藻(Phaeodactylum tricornutum)和中肋骨条藻(Skeletonema costatum)2株测试藻株没有抑制作用,而对绿藻门(Chlorophyta)盐生杜氏藻(Dunaliella salina)和自养小球藻(Chlorella autotrophi-ca)2株藻株具有较强抑藻作用.扫描电镜观察显示,该菌株孢子丝直至螺旋状且孢子表面带刺.生理生化实验显示,该菌株在所得到的大多数培养基上生长良好,在营养琼脂培养基中可以产生水溶性色素;不能在棉子糖作为唯一碳源的培养基上生长.16SrRNA基因相似性分析表明,菌株O3-26属于链霉菌属(Streptomyces),并与灭癌素链霉菌(Streptomycesgancidicus)15412菌株具有最高的同源性(99%).生理生化实验表明,二者之间生理特征存在一定差异.综合形态特征、生理特征以及系统发育分析的结果,鉴定该菌株为灭癌素链霉菌

长苞铁杉幼苗在林窗不同位置的建立

通过2003年12月到2005年1月对福建省天宝岩国家级自然保护区长苞铁杉林内椭圆形林窗(面积118m2)中心、中部、边缘和林下样地进行种子埋藏及幼苗定位观测实验,研究了长苞铁杉幼苗在林窗不同位置中的建立。结果表明:在林窗内不同位置对长苞铁杉幼苗建立有显著影响。林窗中心样地、林窗中部样地、林窗边缘和林下样地内长苞铁杉幼苗发生率分别为10%,10.7%,6%和6%。从林冠下到林窗中心,长苞铁杉种子的幼苗发生率略有增高趋势。在林窗中心样地和林窗中部样地中雨水冲刷是幼苗死亡的最主要原因,而在林窗边缘样地和林下样地中昆虫的取食是幼苗死亡的最主要原因。林窗位置对幼苗的存活率有显著影响,林窗中部样地幼苗存活率最高(11.4%),林窗中心样地幼苗存活率次之(6.7%),而林窗边缘样地和林下样地幼苗则均全部死亡。种子营养消耗完后,在林窗中心、林窗中部、林窗边缘和林下等4个位置样地中,林窗中心样地幼苗平均高度最高。经过一个生长季后,林窗中心样地中幼苗的根生物量、茎生物量和总生物量均略高于林窗中部样地的幼苗,但差异并不显著。林窗中心样地幼苗叶生物量、叶重比、叶/地上等指标显著高于林窗中部样地的幼苗,而茎重比则低于林窗中部样地的幼苗

灰绿曲霉产纤维素酶的研究

灰绿曲霉(Aspergillus glaucus)发酵液通过硫酸铵盐析、Sephadex G-100分子筛、DEAE Sepharose Fast Flow离子交换柱和Phenyl Sepharose Fast Flow疏水层析,分离纯化一种外切葡聚糖酶(CBH)和一种内切葡聚糖酶(EG).通过SDS-PAGE和凝胶柱层析法测定分子质量表明:CBH全酶分子质量为71 ku,由两个分子质量为35 ku的同型亚基组成;EG为单体蛋白,全酶分子质量为32 ku.酶学性质研究表明:CBH催化pNPC的最适pH为6.0,最适温度为55℃,酶活在pH 5.0~8.0区间和温度低于55℃时稳定;EG催化CMC-Na的最适pH为4.0,最适温度为50℃,酶活在pH3.5~7.5区间和温度低于65℃时稳定.Na+、K+、Ba2+、Mg2+以及NO3-和SO42-对CBH和EG酶活均无影响;Ca2+和Mn2+对CBH有激活作用,Fe2+和Mn2+对EG有激活作用,而Zn2+、Cd2+和Cu2+对CBH和EG均有不同程度的抑制效应.酶动力学分析表明:CBH催化pNPC水解的米氏常数Km值为1.4 mmol/L(pH 6.0,55℃),EG催化CMC-Na水解的米氏常数Km值为5.0 mg/mL(pH 4.0,50℃)

光照条件对长苞铁杉种子萌发与幼苗生长的影响

2004-2006年研究了在不同光照下(100%,50%,25%,10%全日照)长苞铁杉(Tsuga longibracteata)种子萌发率、幼苗存活率、幼苗生物量及其分配比例和幼苗根与叶、生理特性。结果表明,50%全日照条件下,种子萌发率和幼苗存活率最高;幼苗根、茎、叶及总生物量最高;光照的增强促使生物量往地下分配以加强根部吸收水分的能力,并促使地上部分的生物量更多的分配到叶片生长上。随着光强的提高,幼苗叶片叶绿素a、叶绿素b、总叶绿素和类胡萝卜素含量均呈下降的趋势,叶绿素a与叶绿素b比值和类胡萝卜素与总叶绿素比值呈上升趋势。在光强不超过50%时,随着光强的提高,幼苗叶片和细根的MDA含量、SOD和POD活性呈升高趋势;光照强度达到全日照时,叶片MDA含量、SOD和POD活性呈下降趋势。幼苗叶片和细根Pro含量在25%全日照时最低。50%全日照附近是长苞铁杉育苗的合适光照强度

Application of Accelerated Solvent Extraction Technique for Analysis of Active Components in Traditional Chinese Medicinal Herbs

以两种药材为研究实例,对加速溶剂萃取法(ASE)在中药材有效成分提取研究中的应用进行了简要介绍。采用正交试验法考察了提取丹参中丹酚酸B的提取条件(萃取温度、静态萃取时间、萃取溶剂以及料液比),确定了较好的实验条件。比较了ASE、水蒸气蒸馏法、超声波提取法及索氏提取法对木香挥发油的提取效果,结果表明ASE对木香挥发油的提取效果最好。The application of accelerated solvent extraction(ASE) technique for the Analysis of active components in traditional Chinese medicinal herbs was introduced by using two kinds of herbs as examples.The conditions including extraction temperature,static extraction time,the ratio of material to solvent and solvent of ASE for extraction of salvianolic acid B in Salvia miltiorrhiza were optimized by orthogonal experiments,and the optimal conditions were obtained.Different extraction methods(ASE,steam distillation,ultrasonic wave and Soxhlet extraction) were used to extract volatile oil in Aucklandia lappa Decne.Results of the comparative experiments indicated that ASE was the most effective method in this case.All the results from these studies demonstrate that ASE is indeed a powerful tool in the preparation of herbal extracts for downstream chromatographic analysis.青岛“2004将才计划”(04-3-JJ-11);; 共建生物医药研发测试中心(LS-05-KJZX-76)资

Analysis of Alkaloids in Coptis chinensis Franch by High Performance Capillary Electrophoresis-Electrospray Ionization Time of Flight Mass Spectrometry

建立了高效毛细管电泳-电喷雾飞行时间质谱联用(HPCE-ESI-TOF/MS)快速定性分析黄连中生物碱类化合物的分析方法.使用未涂层石英毛细管,以50mmol/L乙酸铵-0.5%甲醇溶液(用氨水调至pH=7.2)作为运行缓冲液,分离电压为25kV;鞘液组成为50%甲醇-49.5%水-0.5%乙酸,鞘液流速为4μL/min;质谱选用正离子模式,碰撞电压(Fragmentor)为100V.结果表明,通过各色谱峰紫外光谱和质谱测得精确分子量结果,结合文献,对黄连中7种生物碱进行了鉴定.表明本方法简便、快速,是黄连中生物碱类化合物快速分离、鉴别的有效方法.A new method for the analysis of alkaloids in Coptis chinensis Franch was established by high performance capillary electrophoresis-electrospray ionization time of flight mass spectrometry (HPCE-ESI-TOF/MS). The real samples were separated by an uncoated capillary. 50 mmol/L ammonium acetate containing 0.5% methanol (pH=7.2) was used as the running buffer, and separation voltage was 25 kV. A coaxial sheath flow interface was used as the CE-MS interface, and a 50% methanol-49.5% water- 0.5% acetic acid mixture was used as the sheath liquid with a flow rate of 4 μL/min. The lens voltages in a positive ion mode with a collision induced dissociation (CID) voltage of 100 V were used for ESI-TOF/MS analysis. Seven alkaloids in Coptis chinensis Franch methanol extracts were separated and identified by CE-DAD and CE-ESI-TOF/MS. The coupling of HPCE separation with accurate mass measurement capability of ESI-TOF/MS provides an attractive tool for the identification of alkaloid compounds in Coptis chinensis Franch.国家自然科学基金重点项目(No.20235020);; 青岛“2004将才计划”(No.04-3-JJ-11);; 共建生物医药研发测试中心(No.LS-05-KJZX-76)资助项目