聚乙二醇修饰的重组人睫状神经营养因子对非酒精性脂肪肝的药效学作用

目的 研究聚乙二醇（PEG）修饰的重组人睫状神经营养因子（recombinant human ciliary neurotrophic factor,rhCNTF）对ob/ob小鼠非酒精性脂肪肝（non-alcoholic fatty liver disease,NAFLD）的治疗作用,为PEG-rhCNTF的临床应用提供参考。方法 以14只4～10周龄雌性野生型C57BL/6J小鼠为正常对照组,83只4～10周龄雌性ob/ob小鼠分为赋形剂组、利拉鲁肽0.1 mg/kg组（每天给药2次）和0.2 mg/kg组（每4 d给药1次）、rhCNTF 0.1 mg/kg组（每天给药1次）以及PEG-rhCNTF 0.05、0.1和0.2 mg/kg组（每4 d给药1次）,每组11～12只,均经皮下注射给药。给药期间测定小鼠体质量、24 h饲料消耗量、连续血糖和随机血糖水平及口服葡萄糖耐量;实验终点时,先测定小鼠空腹血糖水平,再测定空腹血清胰岛素（fasting serum insulin,FINS）水平,并计算胰岛素抵抗指数（homeostasis model assessment of i...     

重组人睫状神经营养因子突变体及其长效分子对饮食诱导性肥胖伴胰岛素抵抗大鼠的治疗作用

目的探讨重组人睫状神经营养因子(recombinant human ciliaryneurotrophic factor,rhCNTF)突变体rhCNTF(R6315)及其长效分子P-rhCNTF(R6315)对饮食诱导性肥胖(diet induced obesity,DIO)伴胰岛素抵抗(insulin resistance,IR)模型大鼠的治疗作用。方法利用基因工程重组表达技术制备rhCNTF突变体rhCNTF(R6315)及其长效分子P-rhCNTF(R6315)。通过饮食诱导建立肥胖伴胰岛素抵抗大鼠模型(DIO-IR),经背部皮下分别注射rhCNTF(R6315)0. 025、0. 05及0. 1 mg/kg(每天1次)及P-rhCNTF(R6315)0. 025、0. 05、0. 1、0. 2及0. 4 mg/kg(每7 d1次),共治疗28 d,评价rhCNTF(R6315)及P-rhCNTF(R6315)对DIO-IR大鼠的治疗作用。结果 DIO-IR大鼠治疗28 d后,rhCNTF(R6315)0. 1 mg/kg剂量组高血清胰岛素水平显著改善(P均<0. 01)...     

重组人睫状神经营养因子的纯化

目的纯化重组人睫状神经营养因子(rhCNTF)。方法破菌液上清经硫酸铵分级沉淀和G-25脱盐后,用QSepharose FF阴离子交换层析初纯,再经Superdex 75 prepgrade凝胶过滤精制,并对纯化产物进行各项检测。结果纯化的rhC-NTF纯度达95%以上,蛋白浓度约为2mg/ml,收率约30%,相对分子质量21600,等电点为6.15,与理论值相符合。Western blot检测证明纯化蛋白能够与特异性抗体结合,并能够明显刺激鸡胚背根神经节突触生长。结论采用盐析、离子交换、凝胶过滤等方法有机组合,成功分离纯化了rhCNTF蛋白。     

重组人睫状神经营养因子纯化工艺的建立

目的建立适于生产应用的重组人睫状神经营养因子(recombinant human ciliary neurotrophic factor,rh CNTF)纯化工艺,并对纯化产物进行鉴定。方法将CNTFnew CC22突变体工程菌BL21(DE3)菌体破碎上清经25%饱和度硫酸铵沉淀后,沉淀上清先后经Butyl HP疏水层析和Q HP阴离子交换层析进行纯化。通过非还原SDS-PAGE和反相高效液相色谱分析(RP-HPLC)检测样品纯度;质谱法测定rh CNTF的相对分子质量;Edman化学降解法测定rh CNTF的N-端氨基酸序列;TF-1细胞/CCK-8比色法和对饮食诱导性肥胖(diet-induced obesity,DDIO)大鼠的减肥作用分别测定rh CNTF的体外和体内生物学活性。结果纯化后rh CNTF的纯度可达99.6%,纯化回收率为34.5%;相对分子质量为21 153;N-端15个氨基酸序列为Ala-Phe-Thr-Glu-His-Ser-Pro-Leu-Thr-Pro-His-Arg-Arg-Asp-Leu;生物学比活性高于2×106 IU/mg,并能有效降低DIO大鼠体重。结论经3步纯化成功获得了高纯度的rh CNTF,为进一步对其性质、功能和生产应用研究奠定了基础。     

睫状神经营养因子突变体的克隆、表达、纯化及生物学活性

目的设计具有更高生物学活性的睫状神经营养因子突变体,并对其进行表达、纯化及生物学活性检测。方法以计算机分子模拟系统设计突变体,重叠延伸PCR方法获得突变体的编码区DNA序列,克隆入表达载体pThioHisA,转化E.coliBL21,以IPTG诱导表达。复性纯化后,用鸡胚背根神经节无血清培养法和小鼠减重法进行生物学活性测定。结果目的蛋白以包涵体形式存在,表达量在35%以上,纯化后的纯度达95%以上,能有效地促进鸡胚背根神经节的生长,并能使正常小鼠的体重降低,脂肪指数下降。结论所设计的突变体经表达及纯化后,具有良好的生物学活性,为进一步研究突变体蛋白的促神经生长和减肥作用奠定了基础。     

聚乙二醇化人睫状神经营养因子蛋白含量反相高效液相色谱检测方法的建立及验证

目的建立聚乙二醇化重组人睫状神经营养因子(PEGylated ciliary neurotrophic factor,PEG-CNTF)蛋白含量反相高效液相色谱(reversed-phase high performance liquid chromatography,RP-HPLC)检测方法,并进行验证。方法以未修饰的人CNTF原型蛋白为对照品,采用反相高效液相色谱法(reversed-phase high performance liquid chromatography,RP-HPLC)测定PEG-CNTF的蛋白含量。色谱柱:Jupiter 5u C4 300A(150 mm×4.6 mm);流动相:0.1%三氟乙酸的水溶液、0.1%三氟乙酸的乙腈;检测波长:280 nm;流速:1 mL/min;柱温:室温;进样体积:20μL。对该方法进行专属性、线性、精密性、准确性、耐用性验证。结果空白溶剂在CNTF、PEG-CNTF样品设定积分范围内无特异峰出现;建立的方法在PEG-CNTF含量为0.125～4 mg/mL浓度范围内线性关系良好(R2> 0.99);供试品重复性检测的相...     

人睫状神经营养因子基因克隆、表达、纯化及其生物活性

［摘 要］目的 克隆人睫状神经营养因子（hCNTF）基因，在大肠杆菌宿主系统中高效表达并纯化，获得具有生物活性的hCNTF蛋白。方法 以人染色体DNA为模板，经PCR扩增到编码CNTF成熟蛋白的cDNA序列，并克隆进原核表达载体 pBV220，在大肠杆菌 BL21（Gold）中进行表达，产物经 CM-Sepharose FF柱纯化后，以体外培养的鸡胚背根神经节（DRG）测定生物活性。结果 重组表达质粒pBV22Z- CNTF在大肠杆菌BL21（Gold）中获得了非融合的稳定、高效表达，目的蛋白占细胞总蛋白的45％左右，以可溶性和包涵体两种形式存在，表达上清及包涵体复性后经CM柱一步纯化纯度达90％以上；表达的重组hCNTF能促进培养的鸡胚背根神经节神经突起的生长。结论 基因工程rhCNTF的获得为进一步研究其结构功能关系和临床应用奠定基础。     

乳糖诱导重组人睫状神经营养因子在大肠杆菌中的可溶性表达

目的利用乳糖替代IPTG,诱导重组人睫状神经营养因子(Recombinanthumanciliaryneurotrophicfactor,rhC-NTF)在原核工程菌BL-TCS中可溶性表达,并选择最适的诱导表达条件。方法在不同的温度下,利用不同浓度的乳糖及0·5mmol/L的IPTG,诱导含有人CNTF基因的工程菌9h,比较菌体生长、乳糖消耗以及目标蛋白的表达规律。结果乳糖组的菌体A600值均高于IPTG组。乳糖诱导产生的rhCNTF主要以可溶形式表达,各浓度乳糖的诱导效果均能接近或超过IPTG的效果。以2%乳糖在25℃下诱导6h,可以达到比较好的诱导效果。结论乳糖可取代IPTG诱导人CNTF基因表达,并获得良好效果。     

重组人睫状神经营养因子生物学活性检测方法的建立

目的建立重组人睫状神经营养因子(Recombinant human ciliary neurotrophic factor,rhCNTF)的生物学活性检测方法 ,在其研制和生产中进行有效的质量控制。方法应用鸡胚睫状神经节细胞培养法和鸡胚背根神经节法,对rhCNTF进行定性和半定量检测;应用TF-1.CN5a.1细胞增殖法检测rhCNTF的活性;应用正常小鼠减重法,对rhCNTF的减肥生物学活性进行检测。并探索4种方法的实验条件及优缺点。结果上述4种方法均能用于rhCNTF的生物学活性测定,以TF-1.CN5a.1细胞增殖法及正常小鼠减重法结合使用最为有效。结论可联合使用TF-1.CN5a.1细胞增殖法和正常小鼠减重法,对重组rhCNTF进行质量控制。     

Effects of maleic anhydride grafted polyethylene on rheological,thermal, and mechanical properties of ultra high molecular weight polyethylene/poly(ethylene glycol) blends

Ultra‐high molecular weight polyethylene (UHMWPE) has gained considerable fame due to its excellent wear and mechanical properties, though the inferior processability has restricted its further extensive applications. In this study, a combination of UHMWPE and poly(ethylene glycol) (PEG) was considered based on the recent reports, and aiming to further exploit the potential of PEG that acts as processing aid, and also to obtain greater enhanced processability along with other properties, the effects of incorporating maleic anhydride grafted polyethylene (MAPE) was thoroughly investigated. Rheological tests revealed a further significant reduction in melt viscosity of UHMWPE/PEG blends after MAPE introduced, showing a potential of better processability, while the flexural strength and toughness of UHMWPE blends experienced a satisfying increase without any obvious compromises in other mechanical properties. A slight improvement of thermal stability in UHMWPE ternary blends along with an increase of vicat softening temperature were characterized by thermal tests, while the crystallinity of UHMWPE was diminished after the introduction of MAPE. Morphology analysis indicated that better dispersion and decreased size of PEG particles were achieved in UHMWPE matrix when MAPE was incorporated, which confirmed the improved interfacial interactions and other reinforcements obtained in UHMWPE/PEG/MAPE blends. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42701.     

Combined effect of isophthalic acid and polyethylene glycol in polyethylene terephthalate polymer on thermal,mechanical, and gas transport properties

The objective of this article is to study the combined effect of isophthalic acid (IPA) and polyethylene glycol (PEG‐400) in PET polymer and film on thermal, mechanical, and gas transport properties. The purpose of developing this material is to reduce the melting point, improve mechanical, thermal, and gas barrier properties. The chosen raw materials, namely, IPA and PEG for copolyester synthesis will replace partially the acid and diol monomers of PET. The molar concentration of comonomers (IPA and PEG‐400) were varied from 2 to 50% and the result shows that the gas barrier properties (namely O₂, CO₂, N₂, and water vapor transmission rate), mechanical, and thermal properties were lesser than that of PET polymer. On improving the crystallinity of PET‐isophthalate‐PEG (PET‐IP) copolymer, barrier properties are improved than that of PET polymer. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Effects of polymer molecular weight,concentration, and role of polyethylene glycol as additive on polyacrylonitrile homopolymer membranes

Polyacrylonitrile (PAN), is an important base polymer to cast porous membranes. In this work, a detailed study of the effects of molecular weight of PAN homopolymer and its concentration on the cast membrane has been undertaken. The effects of molecular weight of additive polyethylene glycol (PEG) and its concentration have also been investigated. The membranes are cast using dimethyl formamide as solvent using phase inversion technique. They are characterized in terms of porosity, water permeability, molecular weight cut off, average pore diameter, contact angle, and tensile strength at breaking point. Fourier transform infrared spectroscopy and scanning electron microscopy are used to characterize the chemical and morphological changes of the membrane surface. Further studies have been considered to evaluate the antifouling property of various membranes using filtration of bovine serum albumin solution. Membrane modified by PEG 400 membrane shows an improved separation performance and antifouling characteristics. POLYM. ENG. SCI., 54:2375–2391, 2014. © 2013 Society of Plastics Engineers     

Preparation and electrical conductivity of graphene/ultrahigh molecular weight polyethylene composites with a segregated structure

Graphene-coated ultrahigh molecular weight polyethylene (UHMWPE) powders were prepared by a two-step process. The first step is to coat UHMWPE polymers with graphene oxide (GO) sheets. The second step is to reduce GO on the powders to graphene. The two-step process can effectively prevent the aggregation of graphene during reduction. The resultant graphene/UHMWPE mixtures were hot pressed at 200 °C to obtain the composites with a segregated structure. The composites exhibit high electrical conductivity at a very low percolation threshold (0.028 vol.%). Our method provides a new route for preparing electrical conductive graphene/polymer composites with low percolation threshold.     

Preparation,characterization, and properties of silicate/polyurethaneurea composites based on dipropylene glycol dibenzoate

The silicate/polyurethaneurea composites based on dipropylene glycol dibenzoate were prepared via a room‐temperature‐cured process. Characterization of the composites was accomplished using scanning electron microscope, X‐ray diffraction, Fourier transformation infrared spectrum, and Raman spectroscopy, and the mechanism, mechanical properties and stability were discussed in detail. The obtained results indicated that a durable silicate/polyurethaneurea composite with inorganic‐organic network structure had been successfully prepared. The composites were thermally stable below 210°C and the compressive and flexural strength of the material could reach 42.6 and 29.2 MPa after curing for 6 h, respectively. POLYM. COMPOS., 37–43, 2016. © 2014 Society of Plastics Engineers     

Effects of mixing on morphology,rheology, and mechanical properties of blends of ultra-high molecular weight polyethylene with linear low-density polyethylene

Various blends of ultra-high molecular weight polyethylene (UHMWPE) with linear low-density polyethylene (LLDPE) were prepared in an internal (Banbury type) mixer, a static mixer, and by solvent blending. Two mixing techniques, namely simultaneous and sequential loading methods, were employed with the internal mixer. In the former case, the two polymer components were simultaneously loaded at 180°C and mixed. The latter method allowed the UHMWPE component to diffuse at 250°C and cooled it down to 180°C, then the LLDPE component was added subsequently and mixed. Rheological and mechanical properties of these blends are profoundly affected by the mixing techniques used. Rheological results shows yield characteristics of UHMWPE/LLDPE blends, in particular in blends of high UHMWPE contents. Tensile properties of sequentially loaded blends vary more or less linearly with blend compositions. However, negative or positive deviations are seen in the simultaneously prepared blends. Differential scanning calorimetry (DSC) studies indicate that co-crystallization takes place between UHMWPE and LLDPE components in sequentially mixed blends. DSC and small-angle light scattering (SALS) studies show that separate crystallization takes place in simultaneously blended compounds as a result of poor mixing. It seems that the sequential loading method provides more homogeneous compounds than those of simultaneous blending.     

High density polyethylene/ultra high molecular weight polyethylene blend. II. Effect of hydroxyapatite on processing,thermal, and mechanical properties

Hydroxyapatite (HA) is part of bone mineral composition. Several attempts have been made to incorporate HA into high density polyethylene (HDPE) to produce bone replacement biomaterials since neat HDPE is not suitable as bone replacement. The blending of HDPE with ultra high molecular weight polyethylene (UHMWPE) up to 50% by weight was performed with the aim of improving the toughness of composites. Reinforcement of blend with HA of up to 50% by weight was carried out. Methods of characterizing the composites included density, differential scanning calorimetry, thermal gravimetric analysis, ash content, and morphological examination using scanning electron microscope. For the mechanical properties of the composites, tensile, flexural, and impact tests were carried out. Incorporation of HA into HDPE has resulted in the brittleness of the composites. Blending of HDPE with UHMWPE in the presence of HA was found to improve the mechanical properties and promote a ductile failure of the resulting composites. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 3931–3942, 2006