银杏叶提取物对糖尿病大鼠学习、记忆及海马血红素加氧酶1表达的影响

目的探讨银杏叶提取物(EGb)对糖尿病大鼠学习记忆及海马血红素加氧酶1(HO-1)表达的影响。方法大鼠腹腔注射链脲佐菌素建立糖尿病模型,6个月后以Morris水迷宫观察其学习记忆能力,以RT-PCR观察大鼠海马HO-1 mRNA的表达,以免疫组织化学法观察海马HO-1蛋白表达。结果大鼠糖尿病6个月后Morris水迷宫成绩下降,海马的HO-1 mRNA表达和蛋白表达水平升高(分别为1．635±0．326 vs 0．978±0．214,7．2±1．7 vs 1．9±0．5,均P＜0．01)；糖尿病EGb干预组(100、50 mg/kg)大鼠较糖尿病大鼠Morris水迷宫成绩提高,HO-1 mRNA表达和蛋白表达水平降低(100 mg/kg：0．954±0．144,2．0±0．8；50 mg/kg：0．988±0．154,2．5±0．6,均P＜0．01)。结论EGb可抑制海马的HO-1表达,提高糖尿病大鼠的学习记忆能力。     

白纹伊蚊β-肌动蛋白基因片段的克隆及其

目的获取白纹伊蚊β-肌动蛋白基因序列并探讨其作为基因表达内参照的作用。方法根据昆虫β-肌动蛋白核苷酸序列的高度保守区设计引物，通过PCR的方法从白纹伊蚊C6／36细胞中扩增获得白纹伊蚊β-肌动蛋白基因片段，进一步通过RT—PCR的方法验证其在稳定转染空载体和40S核糖体蛋白S4（RPS4）基因的白纹伊蚊C6／36细胞中的表达。结果获得白纹伊蚊β-肌动蛋白基因片段，长911bp，与其他几种蚊β-肌动蛋白基因对应序列的相似性在89％以上。在稳定转染空载体和RPS4基因的C6／36细胞中，均可稳定地扩增出目的基因。结论成功获得了白纹伊蚊β-肌动蛋白基因片段，并且该片段完全可以用作基因表达差异分析时的内参照基因。     

Identifying the origin of local flexibility in a carbohydrate polymer

Correlating the structures and properties of a polymer to its monomer sequence is key to understanding how its higher hierarchy structures are formed and how its macroscopic material properties emerge. Carbohydrate polymers, such as cellulose and chitin, are the most abundant materials found in nature whose structures and properties have been characterized only at the submicrometer level. Here, by imaging single-cellulose chains at the nanoscale, we determine the structure and local flexibility of cellulose as a function of its sequence (primary structure) and conformation (secondary structure). Changing the primary structure by chemical substitutions and geometrical variations in the secondary structure allow the chain flexibility to be engineered at the single-linkage level. Tuning local flexibility opens opportunities for the bottom-up design of carbohydrate materials.

Natural polymers adopt a multitude of three-dimensional structures that enable a wide range of functions (1). Polynucleotides store and transfer genetic information; polypeptides function as catalysts and structural materials; and polysaccharides play important roles in cellular structure (2–6), recognition (5), and energy storage (7). The properties of these polymers depend on their structures at various hierarchies: sequence (primary structure), local conformation (secondary structure), and global conformation (tertiary structure).Automated solid-phase techniques provide access to these polymers with full sequence control (8–12). The correlation between the sequence, the higher hierarchy structures, and the resulting properties is relatively well established for polynucleotides (13, 14) and polypeptides (15, 16), while comparatively little is known for polysaccharides (17). Unlike polypeptides and polynucleotides, polysaccharides are based on monosaccharide building blocks that can form multiple linkages with different configurations (e.g., α- or β-linkages) leading to extremely diverse linear or branched polymers. This complexity is exacerbated by the flexibility of polysaccharides that renders structural characterization by ensemble-averaged techniques challenging (17). Imaging single-polysaccharide molecules using atomic force microscopy has revealed the morphology and properties of polysaccharides at mesoscopic, submicrometer scale (18–22). However, imaging at such length scales precludes the observation of individual monosaccharide subunits required to correlate the polysaccharide sequence to its molecular structure and flexibility, the key determinants of its macroscopic functions and properties (23).Imaging polysaccharides at subnanometer resolution by combining scanning tunnelling microscopy (STM) and electrospray ion-beam deposition (ES-IBD) (24, 25) allows for the observation of their monosaccharide subunits to reveal their connectivity (26–28) and conformation space (29). Here, we use this technique to correlate the local flexibility of an oligosaccharide chain to its sequence and conformation, the lowest two structural hierarchies. By examining the local freedom of the chain as a function of its primary and secondary structures, we address how low-hierarchy structural motifs affect local oligosaccharide flexibility—an insight critical to the bottom-up design of carbohydrate materials (30).We elucidate the origin of local flexibility in cellulose, the most abundant polymer in nature, composed of glucose (Glc) units linked by β-1,4–linkages (31–33). Unveiling what affects the flexibility of cellulose chains is important because it gives rise to amorphous domains in cellulose materials (34–37) that change the mechanical performance and the enzyme digestibility of cellulose (38). Cellohexaose, a Glc hexasaccharide (Fig. 1A), was used as a model for a single-cellulose chain as it has been shown to resemble the cellulose polymer behavior (12). Modified analogs prepared by Automated Glycan Assembly (AGA) (11, 12) were designed to manipulate particular intramolecular interactions responsible for cellulose flexibility. Cellohexaose, ionized as a singly deprotonated ion in the gas phase ([M-H]⁻¹) was deposited on a Cu(100) surface held at 120 K by ES-IBD (24) (Materials and Methods). The ions were landed with 5-eV energy, well suited to access diverse conformation states of the molecule without inducing any chemical change in the molecule (29). The resulting cellohexaose observed in various conformation states allowed its mechanical flexibility (defined by the variance in the geometrical bending between two residues) to be quantified for every intermonomer linkage. The observed dependence of local flexibility on the oligosaccharide sequence and conformation thus exemplifies how primary and secondary structures tune the local mechanical flexibility of a carbohydrate polymer.Open in a separate windowFig. 1.STM images of cellohexaose (AAAAAA) and its analogs (AXAAXA). Structures and STM images of cellohexaose (A) and its substituted analogs (B–E). Cellohexaose contains six Glcs (labeled as A; colored black) linked via β-1,4–glycosidic bonds. The cellohexaose analogs contain two substituted Glcs, as the second and the fifth residues from the nonreducing end, that have a single methoxy (–OCH₃) at C(3) (labeled as B; colored red), two methoxy groups at C(3) and C(6) (labeled as C; colored green), a single carboxymethoxy (–OCH₂COOH) at C(3) (labeled as D; colored blue), and a single fluorine (–F) at C(3) (labeled as F; colored purple).The effect of the primary structure on the chain flexibility was explored using sequence-defined cellohexaose analogs (Fig. 1). Cellohexaose, AAAAAA (Fig. 1A), was compared with its substituted analogs, ABAABA, ACAACA, ADAADA, and AFAAFA (written from the nonreducing end) (Fig. 1 B–E), where A is Glc, B is Glc methylated at OH(3), C is Glc methylated at OH(3) and OH(6), D is Glc carboxymethylated at OH(3), and F is Glc deoxyfluorinated at C(3). These substitutions are designed to alter the intramolecular hydrogen bonding between the first and the second as well as between the fourth and fifth Glc units (Fig. 1). These functional groups also affect the local steric environment (i.e., the bulky carboxymethyl group) (Fig. 1D) and the local electronic properties (i.e., the electronegative fluorine group) (Fig. 1E). When compared with the unsubstituted parent cellohexaose, these modified cellohexaoses exhibit different aggregation behavior and are more water soluble (12).All cellohexaose derivatives adsorbed on the surface were imaged with STM at 11 K (Fig. 1). The oligosaccharides were deposited as singly deprotonated species and were computed to adsorb on the surface via a single covalent RO–Cu bond, except for ADAADA which was deposited as doubly deprotonated species and was computed to adsorb on the surface via two covalent RCOO–Cu bonds (R = sugar chain). All cellohexaoses appear as chains containing six protrusions corresponding to the six constituent Glcs. The unmodified cellohexaose chains (Fig. 1A) mainly adopt a straight geometry, while the substituted cellohexaoses (Fig. 1 B–E) adopt both straight- and bent-chain geometries. Chemical substitution thus increases the geometrical freedom of the cellulose chain, consistent with the reported macroscopic properties (12).Large-chain bending between neighboring Glc units is observed exclusively for the substituted cellohexaose (Fig. 1). The large, localized bending reveals the substitution site and allows for the nonreducing and the reducing ends of the chain to be identified. These chains are understood to bend along the surface plane via the glycosidic linkage without significant tilting of the pyranose ring that remains parallel to the surface (illustrated in SI Appendix, Fig. S1), as indicated by the ∼2.0-Å height of every Glc (29).The bending angle measured for AA and AX linkages (Fig. 2; Materials and Methods has analysis details) shows that, while both AA and AX prefer the straight, unbent geometry, AX displays a greater variation of bending angles than AA. AX angular distribution is consistently ∼10° wider than that for AA, indicating that AX has a greater conformational freedom than AA. This increased bending flexibility results from the absence of the intramolecular hydrogen bonding between OH(3) and O(5) of the neighboring residue. Methylation of OH(6), in addition to methylation of OH(3), results in similar flexibility (Fig. 2 B and C), suggesting the greater importance of OH(3) in determining the bending flexibility. Steric effects were found to be negligible since AD displayed similar flexibility to other less bulky AX linkages.Open in a separate windowFig. 2.Bending flexibility of AA linkage and substituted AX linkages. Chain bending (Fig. 1) is quantified as an angle formed between two neighboring Glcs (Materials and Methods). The results are given in A for AA, in B for AB, in C for AC, in D for AD, and in E for AF, showing that AX (where X = B, C, D, F) has a higher conformational freedom than AA. The angle distributions (bin size: 10°) are fitted with a Gaussian (solid line) shown with its half-width half-maximum. The computed potential energy curves are shown with the half-width at 0.4 eV and fitted with a parabola to estimate its stiffness (k; in millielectronvolts per degree²).Density functional theory (DFT) calculations support the observations, showing that substitution of OH(3) decreases the linkage stiffness by up to ∼40% (Fig. 2). Replacing OH(3) with other functional groups weakens the interglucose interactions by replacing the OH(3)··O(5) hydrogen bond with weak Van der Waals interactions. The similar flexibility between AB and AC linkages is attributed to the similar strength of the interglucose OH(2)··OH(6) hydrogen bond in AB (Fig. 2B) and the OH(2)··OMe(6) hydrogen bond in AC (Fig. 2C). The negligible steric effect in AD is attributed to the positional and rotational freedom of the bulky moiety that prevents any “steric clashes” and diminishes the contribution of steric repulsion in the potential energy curve. Comparing the potential landscape in the gas phase and on the surface shows that the stiffness of the adsorbed cellohexaoses is primarily dictated by their intramolecular interactions instead of molecule–surface interactions (SI Appendix, Fig. S2). Primary structure alteration by chemical substitution modifies the interglucose hydrogen bonds and enables chain flexibility to be locally engineered at the single-linkage level.We subsequently investigate how molecular conformation (secondary structure) affects the local bending flexibility. We define the local secondary structure as the geometry formed between two Glcs, here exemplified by the local twisting of the chain (Fig. 3). The global secondary structure is defined as the overall geometry formed by all Glcs in the chain, here exemplified by the linear and cyclic topologies of the chain (Fig. 4).Open in a separate windowFig. 3.Bending flexibility of untwisted and twisted AA linkages. (A) STM image of a cellohexaose containing two types of AA linkages: untwisted (HH and VV) and twisted (HV and VH; from the nonreducing end). The measured bending angles and the computed potential curve are given in B for HH, in C for HV, and in D for VV, showing that the twisted linkage (HV) is more flexible than the untwisted ones (HH and VV). In the molecular structures, interunit hydrogen bonds are given as dotted blue lines, and the pyranose rings are colored red for the horizontal ring (H) and green for vertical (V). The angle distributions (bin size: 10°) are fitted with a Gaussian distribution (solid line) labeled with its peak and half-width half-maximum. The computed potential curves are labeled with its half-width at 0.4 eV and fitted with a parabola to estimate its stiffness (k; in millielectronvolts per degree²).Open in a separate windowFig. 4.Bending flexibility of AA linkage in linear (LIN) and cyclic (CYC) chains. STM image, measured bending angle distribution, and computed potential of AA linkage are given in A for a linear cellohexaose conformer and in B for a cyclic cellohexaose conformer, showing that chain flexibility is reduced in conformations with cyclic topology. The same data are given in C for α-cyclodextrin that is locked in a conformation with cyclic topology. The measured angles (bin size: 10°) are each fitted with a Gaussian distribution (solid line) labeled with its peak and half-width half-maximum. The computed potentials are each labeled with its half-width at 0.4 eV and fitted with a parabola to estimate its stiffness (k; in millielectronvolts per degree²).The effect of local secondary structure on chain flexibility is exemplified by the bending flexibility of twisted and untwisted linkages in a cellohexaose chain (Fig. 3A). The untwisted and twisted linkages are present due to the Glc units observed in two geometries, H or V (Fig. 3), distinguished by their heights (h). H (h ∼ 2.0 Å) is a Glc with its pyranose ring parallel to the surface, while V (h ∼ 2.5 Å) has its ring perpendicular to the surface (29). These lead to HH and VV as untwisted linkages and HV and VH (written from nonreducing end) as twisted linkages.The twisted linkage is more flexible than the untwisted one, as shown by the unimodal bending angles for the untwisted linkage (HH and VV in Fig. 3 B and D, respectively) and the multimodal distribution for the twisted linkage (HV in Fig. 3C). DFT calculations attribute the increased bending flexibility to the reduction of accessible interunit hydrogen bonds from two to one. Linkage twisting increases the distance between the hydrogen-bonded pair, which weakens the interaction between Glc units and increases the flexibility at the twisting point. The increase in local chain flexibility conferred by chain twisting shows how local secondary structures affect chain flexibility.The effect of the global secondary structure on the local chain flexibility was examined by comparing the local bending flexibility of cellohexaose chains possessing different topologies. Cellohexaose can adopt either linear (Figs. 3A and ​and4A)4A) or cyclic topology (Fig. 4B), the latter characterized by the presence of a circular, head-to-tail hydrogen bond network (29). The cyclic conformation of cellohexaose is enabled by the head-to-tail chain folding from the 60° chain bending of the VV linkage. The VV segment in the cyclic chain is stiffer than in the linear chain since the bending angle distribution for the cyclic chain is 6° narrower than that for the linear chain. The observation is corroborated by DFT calculations that show that the VV linkage in the cyclic chain is about three times stiffer than that in the linear chain.To characterize the degree of chain stiffening due to the linear-to-cyclic chain folding, we compare the flexibility of the cyclic cellohexaose and α-cyclodextrin (an α-1,4–linked hexaglucose covalently locked in cyclic conformation). The α-cyclodextrin provides the referential local flexibility for a cyclic oligosaccharide chain. Strikingly, the local flexibility in α-cyclodextrin was found to be identical to that in the cyclic cellohexaose, as evidenced by the similar width of the bending angle distribution and the computed potentials (Fig. 4 B and C). The similar stiffness indicates that the folding-induced stiffening in cellohexaose is a general topological effect unaffected by the type of the interactions that give the cyclic conformation (noncovalent hydrogen bond in cellohexaose vs. covalent bond in α-cyclodextrin). The folding-induced stiffening is the result of the creation of a circular spring network that restricts the motion of Glc units and reduces their conformational freedom. The folding-induced stiffening reported here provides a mechanism by which carbohydrate structures can be made rigid. The dependence of the local chain flexibility on the chain topology shows how global secondary structures modify local flexibility.Using cellulose as an example, we have quantified the local flexibility of a carbohydrate polymer and identified structural factors that determine its flexibility. Modification of the carbohydrate primary structure by chemical substitution alters the mechanical flexibility at the single-linkage level. Changing secondary structure by chain twisting and folding provides additional means to modify the flexibility of each linkage. Control of these structural variables enables tuning of polysaccharide flexibility at every linkage as a basis for designing and engineering carbohydrate materials (30). Our general approach to identify structural factors affecting the flexibility of a specific molecular degrees of freedom in a supramolecular system should aid the design of materials and molecular machines (39) and the understanding of biomolecular dynamics.     

1H-MRS检测帕金森病伴轻度认知障碍患者后扣带回代谢物

目的 探讨¹H-MRS波谱技术联合线性拟合模型（LCmodel）软件在帕金森病（PD）伴认知障碍中的诊断价值。方法 选取PD患者35例（PD组）和健康体检者22名（对照组）,并根据是否伴有认知功能障碍,将PD组分为PDN亚组和PDMCI亚组。采用¹H-MRS波谱技术联合LCmodel软件获取PD组与对照组后扣带回（PCG）区域的波谱及代谢物的绝对浓度。比较2组各代谢物绝对浓度,并分析各代谢物绝对浓度与认知功能评分的相关性。结果 PDN亚组各代谢物绝对浓度与对照组差异均无统计学意义（P均>0.05）,PDMCI亚组总肌酸（tCr）、N-乙酰天门冬氨酸（NAA）、肌醇（mI）和胆碱复合物（tCho）的绝对浓度均较对照组降低（P均<0.05）;PDMCI亚组tCr绝对浓度较PDN亚组降低（P<0.05）。tCr（r=0.444,P=0.01）、谷胱甘肽（GSH;r=0.393,P=0.024）绝对浓度与MMSE评分存在相关性;tCr（r=0.367,P=0.035）、GSH（r=0.376,P=0.031）及tCho（r=0.375,P=0.031）绝对浓度与MoCA评分存在相关性。结论 ¹H-MRS技术联合LCmodel软件可定量分析PCG区域代谢物变化,有助于评估PD伴认知障碍。     

闭环靶控下不同镇静麻醉深度对肌松药用量的影响

目的分析闭环靶控下不同镇静麻醉深度对肌松药用量的影响。方法随机选取我院接收救治的60例ASA I-II级择期行腹腔镜胆囊切除术患者,随机分成观察组和对照组,各30例。两组患者均给予闭环靶控下维持镇静麻醉深度,观察组脑电双频谱字数(BIS)控制在50,对照组BIS控制在60,两组行全麻诱导插管,并给予同等剂量的诱导药,肌松药在同等肌松闭环注射系统条件下进行维持给予,对两组肌松药用量、复苏时间等围术期情况进行回顾性统计学分析。结果观察组麻醉时间、手术时间、复苏时间、拔管时间与对照组相比,数据差异无统计学意义(P均>0.05),观察组术中流血量、输液量、尿量与对照组相比,数据差异无统计学意义(P均>0.05);观察组顺苯阿曲库铵每分钟用量与对照组相比,数据差异无统计学意义(P均>0.05),观察组顺苯阿曲库铵每小时每公斤体质量用量显著低于对照组(P<0.05)。结论闭环靶控下不同镇静麻醉深度对肌松药用量存在一定程度的影响,闭环靶控下静脉麻醉深度较浅时,BIS 60值患者顺苯阿曲库铵用量较大。     

血浆D-二聚体检测在急性主动脉夹层中临床应用观察

目的探讨血浆D-二聚体检测在急性主动脉夹层(AAD)临床诊断及预后判断中应用价值。方法回顾2012年1月~2014年5月共30例确诊AAD患者(AAD组),以同期住院30例急性心肌梗死(AMI)患者为对照组(AMI组),检测所有患者入院时血浆D-二聚体水平(D-二聚体>0.3mg/L为阳性)。结果 AAD组29例血浆D-二聚体升高,检测阳性率达97%,AMI组4例D-二聚体升高,检测阳性率达13.3%,AAD组D-二聚体检测水平明显高于AMI组,差异有统计学意义(P<0.05)。AAD组中Stanford A型患者D-二聚体水平显著高于Stanford B型患者,差异具有统计学意义(P<0.05),7 d内死亡的AAD患者血浆D-二聚体水平显著高于存活的患者,差异具有统计学意义(P<0.05)。结论 D-二聚体阴性有助于排除AAD的诊断,D-二聚体的水平与AAD夹层撕裂的程度和范围相关,并可能是AAD患者不良预后的参考指标。     

替代对照品法在镇静安神类药物HPLC快速检验方法中的应用

目的 采用替代对照品法建立镇静安神类药物氯氮卓、马来酸咪达唑仑、硝西泮、艾司唑仑、奥沙西泮、劳拉西泮和阿普唑仑的高效液相色谱快速检验方法。方法 采用Agilent ZORBAX Eclipse Plus C₈色谱柱(4.6 mm×150 mm,5 μm),流动相：0.01 mol·L^-1磷酸二氢钾溶液(pH 2.5)-(甲醇-乙腈1∶1)(57∶43),流速：1.0 mL·min^-1,柱温：30 ℃。采用相对容量因子和紫外光谱相似度双指标进行定性;采用相对校正因子法进行定量分析。结果 在确定的色谱条件下,氯氮?、咪达唑仑、硝西泮、艾司唑仑、奥沙西泮、劳拉西泮和阿普唑仑完全分离;采用紫外光谱相似度和相对容量因子进行定性,结果准确可靠;采用替代对照品法,计算药物的相对校正因子进行含量测定,能有效减少对照品的使用,加快高效液相色谱分析速度。结论 该方法快速、简便、可靠,适用于快速检验镇静安神类药物。     

以脾虚模型小鼠胃肠激素含量为依据优化枳术颗粒提取工艺

目的探索枳术颗粒分离部位及其组合对脾虚模型小鼠胃肠激素含量的影响,优化枳术颗粒的提取工艺。方法用优化的工艺制备枳术颗粒的不同分离部位。72只小鼠随机分为对照组、脾虚模型组、多潘立酮组、枳实总黄酮组、枳实总生物碱组、枳实组、枳实白术组、枳术方组和枳术颗粒组。大黄水浸煎液灌胃制作小鼠脾虚模型,建模的同时给药,对照组则给予等量0.5%羧甲基纤维素钠溶液。酶联免疫法检测各组小鼠血清胃泌素(gastrin,GAS)、十二指肠胃动素(motilin,MTL)和胃窦组织中血管活性肠肽(vasoactive intestinal peptide,VIP)水平的变化。结果与对照组比较,模型组小鼠血清GAS、十二指肠组织MTL含量降低,胃窦组织VIP含量升高。枳术颗粒不同分离部位均能不同程度调节脾虚小鼠胃肠激素水平,各部位合用与枳术颗粒效果相当。结论枳术颗粒分离部位合用能有效调节脾虚小鼠胃肠激素水平,优化后的工艺可行。     

微波辅助提取蓝莓果渣中花青素的工艺研究

目的:确定微波辅助萃取蓝莓果渣中花青素的最佳试验条件。方法:以家用微波炉为微波萃取装置,采用正交试验确定微波萃取蓝莓果渣中花青素的最佳试验条件。结果:微波萃取蓝莓果渣中花青素的最佳试验条件为酸性乙醇作为提取剂、萃取时间70s、微波功率560W、料液比1:20。在此条件下蓝莓果渣花青素萃取量为2.98mg/g。结论:此工艺可有效提取蓝莓果渣中的花青素,达到了合理利用蓝莓资源获得高附加值产品的目的。     

人脐带间充质干细胞定向诱导分化成类神经元的实验研究

目的:采用不同细胞因子定向诱导人脐带间充质干细胞分化成类神经元,为脐带间充质干细胞移植治疗脊髓损伤提供理论依据。方法:采用Ⅱ型胶原酶消化法分离培养人脐带间充质干细胞,采用流式细胞术鉴定细胞。取第5代细胞随机分成A、B、C、D 4组,在A组基础培养基中加入b FGF,B组中加入b FGF和BDNF,C组中加入b FGF、BDNF和BHA诱导培养,D组为对照组,使用含有10%胎牛血清的DMEM-F12培养基培养。诱导2周后采用实时荧光定量PCR检测细胞的nestin、NEFH和GFAP mRNA表达情况,并通过原子力显微镜观察细胞超微结构的的变化。结果:Ⅱ型胶原酶消化法能成功分离人脐带间充质干细胞。通过流式细胞术检测第3代细胞发现,细胞均强表达CD29、CD44和CD105,而CD34、CD45和HLA-DR均未见表达。经定向诱导分化成类神经元后,原子力显微镜观察细胞表面突起增多,实时荧光定量PCR检测显示nestin在A、B、C组均呈阳性表达,NEFH在A、B组呈阳性表达,而GFAP在A、B、C、D 4组均不表达。A、B组n EFH和nestin表达具有显著差异(P﹤0.05)。结论:本实验成功分离培养人脐带间充质干细胞,所培养的细胞扩增迅速,生物学性状稳定;与b FGF单独处理相比,b FGF联合BDNF更能有效诱导人脐带间充质干细胞分化成类神经元。