两种方法制备的水痘-带状疱疹病毒(Oka株)毒种感染2BS细胞的敏感性

目的用现有方法及新方法分别制备水痘-带状疱疹病毒(varicella-zoster virus,VZV)(Oka株)毒种,比较两种方法制备的毒种对2BS细胞的感染复数及増殖动态差异。方法用现有方法(用含2%小牛血清的病毒维持液于-70℃条件下冻存Oka株毒种)和新方法(用含90%小牛血清的病毒冻存液于-196℃条件下冻存Oka株毒种)制备第47代Oka株VZV毒种,采用不同的MOI(0.000 5、0.001、0.005、0.01、0.05、0.1)感染第37代2BS细胞,观察细胞病变(CPE)情况,收获不同MOI接种和培养不同时间点病毒制备疫苗,采用噬斑法检测疫苗原液、成品滴度以及37℃稳定性,确定两种方法制备的毒种对2BS细胞感染的最适MOI。结果现有方法制备毒种以0.01~0.1 MOI感染2BS细胞72 h后,病变达到高峰期;新方法制备毒种以0.001~0.01 MOI感染2BS细胞72 h后,病变达到高峰期。两种方法收获的病毒液制备的成品疫苗的病毒滴度为4.4~4.6 lg PFU/ml,37℃放置1周,病毒滴度3.8~4.0 lg PFU/ml;疫苗原液、成品滴度以及37℃稳定性无差异,均达到本公司水痘减毒活疫苗制造与检定规程制定的相关标准。结论现有方法和新方法制备的毒种最适MOI分别为0.01~0.1和0.001~0.01,新方法制备的毒种更适合水痘疫苗的生产。     

不同生产工艺制备水痘减毒活疫苗(VZV 84-7株)的比较

目的采用细胞工厂替代传统转瓶工艺制备水痘减毒活疫苗(VZV 84-7株)。方法分别用细胞工厂和3 L转瓶培养2BS细胞,制备水痘减毒活疫苗(VZV 84-7株)各6批,采用Countstar全自动细胞计数仪进行细胞计数,并以计数结果中的单位面积细胞数、成活率及消化后细胞的平均直径为指标,考察细胞质量,同时采用蚀斑法检测原液、成品及成品37℃放置7 d后(热稳定性)的病毒滴度。结果采用细胞工厂连续制备的6批2BS细胞的平均单位面积细胞数、细胞平均成活率均略优于3 L转瓶培养工艺,平均直径变动范围小于3 L转瓶;细胞工厂制备的6批疫苗,平均原液滴度为5.58 lg PFU/ml,平均成品滴度为4.78 lg PFU/0.5ml,37℃放置7 d后的平均病毒滴度为4.13 lg PFU/0.5ml,优于3 L转瓶。结论利用细胞工厂制备水痘减毒活疫苗(VZV 84-7株),可获得更高滴度病毒液,降低疫苗生产过程中的劳动强度及污染风险,并提高了疫苗产量及质量的稳定性,适合于水痘减毒活疫苗(VZV 84-7株)的规模化生产。     

水痘-带状疱疹病毒疫苗株在Vero细胞中的传代适应

目的探索水痘-带状疱疹病毒(VZV)疫苗株在Vero细胞中的传代适应及大量制备抗原的可行性。方法以VZV疫苗株在Vero细胞中传代适应,并检测适应株的生物学性状,采用15L旋转培养瓶培养,大量制备VZV ELISA抗原,并检测抗原的特异性。结果VZV疫苗株在Vero细胞中传代至第9代时,CPE明显增多,病毒滴度从2～3logPFU/ml提高到不低于4·2logPFU/ml。所制备毒种的适宜MOI为0·01,在72h左右收获的病毒滴度较高。毒种在-70℃保存1年后,病毒滴度未显著降低。抗VZV阳性血清检测证实制备的抗原具有较好特异性,抗原最适稀释浓度为1:4000。结论VZV疫苗株在Vero细胞中能传代适应,并可用15L瓶旋转培养大量制备VZV抗原。     

一次性培养瓶在水痘减毒活疫苗生产中的应用

目的比较一次性培养瓶与方瓶培养人二倍体细胞2BS株的效果。方法接种相同浓度的人二倍体细胞2BS株至一次性培养瓶和小方瓶中,(37±0.5)℃静置培养3 d,于显微镜下观察细胞生长状态,并进行细胞计数。用0.025～0.05 MOI水痘-带状疱疹病毒Oka株感染2BS细胞,(35±0.5)℃静置培养3 d,待细胞病变达70%以上时,收获病变细胞,制备冻干水痘减毒活疫苗,并参考水痘减毒活疫苗注册标准及《中国药典》(三部)2010版,进行各项指标检定。结果在接种2BS细胞浓度相同的情况下,与方瓶相比,一次性培养瓶可增加一定的细胞培养面积,细胞总数相对较高;一次性培养瓶培养的2BS细胞贴壁速度较快,呈梭形,密度高,方向性好,轮廓清晰,分布均匀,胞质饱满,而方瓶培养的2BS细胞贴壁速度较慢,生长不均匀;使用一次性培养瓶及方瓶培养2BS细胞生产的水痘减毒活疫苗,各项检定指标结果均符合《中国药典》(三部)2010版相关要求,在病毒滴度无明显差异的情况下,使用一次性培养瓶可使水痘疫苗产量大幅度增加。结论使用一次性培养瓶培养人二倍体细胞2BS株生产的水痘减毒活疫苗疫苗产量较方瓶有较大幅度的提高。     

水痘-带状疱疹病毒gE糖蛋白基因扩增法鉴别水痘减毒活疫苗

目的利用水痘-带状疱疹病毒(VZV)gE糖蛋白基因鉴别水痘减毒活疫苗。方法以提取的病毒DNA为模板,PCR扩增gE糖蛋白基因。经琼脂糖凝胶电泳鉴定。结果水痘减毒活疫苗Oka株VZV(病毒滴度≥2.0lgPFU/ml)通过琼脂糖电泳均可见特异性的条带。使用同样的方法检测麻疹减毒活疫苗、麻疹-腮腺炎联合疫苗、风疹减毒活疫苗以及Ⅰ、Ⅱ型单纯疱疹病毒(病毒滴度≥4.2lgCCID50/ml),结果均为阴性。结论gE糖蛋白基因可以作为VZV的特异性指标,进行病毒鉴别试验。     

水痘-带状疱疹减毒活疫苗毒种(Oka株)制备方法的改进

目的改进水痘-带状疱疹减毒活疫苗毒种(Oka株)的制备方法,提高毒种的病毒滴度及稳定性,延长保存时限。方法分别采用原法(病毒吸附感染法)和改进的方法(病毒混合感染法)制备水痘-带状疱疹病毒Oka株工作种子批,采用蚀斑法检测病毒滴度及-70℃保存0、1、6、12个月的稳定性,并各制备出3批疫苗进行全面检定。结果采用病毒混合感染法和病毒吸附感染法制备的毒种病毒滴度分别为6.6和6.5 lgPFU/ml,病毒混合感染法制备的毒种在-70℃以下保存12个月,病毒滴度无明显下降;病毒吸附感染法制备的毒种-70℃以下保存6个月,病毒滴度下降明显。两中方法制备的水痘减毒活疫苗成品的各项检定结果均符合要求,病毒滴度及37℃稳定性无明显差异。结论采用病毒混合感染法制备的毒种提高了病毒滴度和-70℃保存的稳定性。     

人二倍体细胞(2BS株)细胞库的建立及生物学特性鉴定

目的建立人二倍体细胞(2BS株)主细胞库和工作细胞库。方法将人二倍体细胞(2BS株)扩大培养后冻存,建立主细胞库和工作细胞库,采用台盼蓝染色计数细胞,计算细胞活力,并进行染色体、外源病毒因子、微生物污染、致瘤性的检测及鉴别试验。将全面检定合格的工作细胞库细胞分别接种至T75一次性培养瓶、3 L玻璃转瓶和10层细胞工厂,观察细胞形态;将水痘病毒分别按0.010 5、0.012 8、0.018 8 MOI接种至T75一次性培养瓶、3 L玻璃转瓶和10层细胞工厂上的单层细胞,收获病毒原液,检测病毒滴度。结果主细胞库细胞活力为94.1%,工作细胞库细胞活力为94.4%;1 000个处于分裂相时期的细胞的核型分析结果均在《中国药典》三部(2010版)人二倍体细胞染色体分析标准的规定范围内;细胞上清液和裂解液中人外源病毒因子检测结果均为阴性,符合《中国药典》三部(2010版)规程要求;细胞均贴壁,成纤维细胞形态,种属鉴别为人细胞B型;细胞库未被细菌、真菌、病毒及支原体污染,也未出现致瘤性。建立的细胞库在3种培养器皿中均可在工艺要求时间内生长为单层,单位面积活细胞浓度在(2.01~2.59)×105个/cm2之间,收获的病毒滴度在5.0~5.25 lg PFU/ml之间,符合成都生物制品研究所有限责任公司《水痘减毒活疫苗制造及检定规程》要求。结论建立了人二倍体细胞(2BS株)主细胞库和工作细胞库,为2BS细胞应用于水痘减毒活疫苗的研发和生产提供细胞资源和理论依据。     

微载体无血清培养水痘-带状疱疹病毒的效果

目的探讨利用生物反应器微载体无血清培养水痘-带状疱疹病毒(varicella-zoster virus,VZV)的效果。方法利用生物反应器,将人二倍体细胞2BS培养至在微载体上形成单层后,分别采用BD008无血清培养基及含有2%新生牛血清的MEM维持液按MOI为0. 004～0. 008感染VZV毒种;当细胞呈现70%细胞病变效应(cytopatho-genic effect,CPE)时,无血清培养的细胞沉淀经PBS洗涤1次,含血清培养基培养的细胞洗涤1～3次后收获,分别制备3批VZV原液;噬斑法检测病毒滴度,ELSIA法检测牛血清白蛋白残留量,并对制备的水痘减毒活疫苗(live attenuated varicella vaccine,VarV)进行热稳定性检测。结果洗涤1～3次获得的3批含血清培养VZV原液滴度从5. 00 lgPFU/mL降至4. 12 lgPFU/mL;牛血清白蛋白残留量分别为203. 2、120. 3和81. 2 ng/mL,前2批超出合格范围(≤100 ng/mL)。洗涤1次获得的3批无血清培养VZV原液滴度分别为4. 92、5. 00和5. 30 lgPFU/mL;牛血清白蛋白残留量分别为75. 2、82. 3和71. 9 ng/mL,均值低于洗涤1及2次含血清培养VZV原液(P <0. 05)。3批无血清培养的VarV热稳定性均符合要求。结论获得的无血清培养的VZV原液滴度及牛血清白蛋白残留量均符合《中国药典》三部(2015版)标准,本研究为提高VarV质量及生物反应器微载体工艺改进提供了参考。     

北京株水痘活疫苗的研制

选择处于对数生长期的人胎肺二倍体细胞（2BS），接种北京-7株水痘-带状疱疹病毒．待受染病毒的细胞出现CPE时，将维持液换为不含牛血清的MEM。接种后的2M细胞，培育36～38小时即可收获较高满度的病毒。采用pH7．2～7．4含25％山梨醇作保护剂、以6000r／min离心30分钟除去病毒液中的细胞碎片，制成减毒活疫苗。     

带状疱疹疫苗生产中2BS细胞细胞工厂培养工艺的建立

目的建立带状疱疹疫苗生产中2BS细胞在细胞工厂中的培养工艺。方法将2BS细胞复苏(28代)后,连续传代至35、36代,再分别按1∶4、1∶2比例传至10层细胞工厂中,采用不同的加液量(200、300、400 ml/层)进行培养,显微镜下观察细胞形态,并进行细胞计数。取细胞工厂,按MOI 0.008接种水痘-带状疱疹病毒,制备成带状疱疹减毒活疫苗,检测疫苗原液及成品的滴度;疫苗成品进行37℃稳定性试验;采用酶联免疫法检测抗生素残留量和牛血清白蛋白残留量;凝胶法检测内毒素含量;水分容量滴定法检测水分含量。结果 2BS细胞按1∶2比例传代,细胞工厂单层加液量为300 ml,可制备单层致密、优质的细胞,细胞工厂收获时细胞病变达80%以上,且病变细胞圆润、典型、广泛,细胞形态保持较好;传代比例为1∶4的细胞制备的疫苗原液及成品的滴度明显低于传代比例为1∶2的细胞制备的疫苗,且1∶4传代的细胞制备的疫苗成品37℃稳定性滴度不符合带状疱疹疫苗放置1周病毒滴度不小于4.6 lg PFU/ml的质量标准;其他各项质量指标均符合要求。结论建立了带状疱疹疫苗生产中2BS细胞在细胞工厂中的培养工艺,利用细胞工厂可制备出质量均一、致密优质的2BS细胞。     

Dependence of bulk viscosity of polypropylene on strain,strain rate,and melt temperature

The relations between bulk viscosity, strain, strain rate, and melt temperature of polypropylene are investigated in uniaxial compression similar to the measurement of PVT diagram of a polymer. A new and more practical bulk viscosity model for the simulation of injection molding is proposed. The bulk viscosity increased with the increase of compression deformation and decreased when the compression speed and the melt temperature are higher. This dependency is attributed to the orientation and occupancy of the molecule within the free interstitial space. In addition, the magnitude of the bulk viscosity was found to be significantly greater than the shear viscosity at high compression deformation and low strain rate. Thus, not only shear viscosity but also the bulk viscosity should be considered in the simulation of the packing‐holding stage of the plastic injection molding process. POLYM. ENG. SCI., 57:830–837, 2017. © 2016 Society of Plastics Engineers     

Creep strain versus residual strain of a concrete loaded under various levels of compressive stress

An experimental study of the impact of microcracking of creep behaviour of concrete is presented in this article. Creep and residual strain results (after unloading) observed on specimens loaded in compression at various stress levels (30, 50, and 70% of the compression strength of the studied concrete) are presented for both sealed and unsealed (drying) specimens.The principal conclusion of this experimental study is the following: the residual strain is a linear function of the creep strain. This function is independent of hygral exchange conditions, sealed or unsealed specimens.This conclusion is explained by the assumption of a physical mechanism related to the presence of microcracking during creep.     

Graphene-based transparent strain sensor

Transparent strain sensors based on graphene were fabricated in a form of rosette on a flexible plastic or stretchable rubber substrate by using reactive ion etching and stamping techniques. Their piezoresistive properties were investigated under a tensile strain up to 7.1%. We demonstrated this sensor on a transparent glove and measured magnitudes and directions of the principal strains on the glove induced by the motion of fingers.     

Recovery strain of ceramics during sintering

A recovery strain after compressive loading was observed for bulk ceramic specimens during sintering. For pure alumina and low temperature co-firing ceramic (LTCC), a transient expansion occurs only in the loading direction after rapid removal of the load. This effect increases with stress magnitude and loading time.     

聚异氰氨酸酯唑烷酮的高应变动态响应研究

通过基本力学性能测试和Kolsky杆(SHPB)方法,测试聚异氰氨酸脂唑烷酮(POZD)在高应变率下的非线性力学行为。结果表明,防爆新材料POZD应力应变关系能够较好地用Mooney-Rivlin模型来表示,该模型采用左柯西张量不变量表示的应变能函数来描述POZD材料的力学响应特性,以便对其防爆性能进行进一步设计和优化。     

A new development of the strain energy function for hyperelastic materials using a logarithmic strain approach

The development of a new strain energy function for hyperelastic solids based on the logarithmic strain measure is the objective of the present article. For all possible types of deformation it was shown that the proposed energy function is based on three independent material parameters. Using available experimental data for rubber‐like materials from the literature, one may determined the materials parameters by a nonlinear fitting. The available domain of the strain energy function can be determined by plotting the third invariant of logarithmic strain vs the second one. The numerical integration of the experimental data of true stress as a function of the logarithmic strain for various types of deformation yields the strain energy function W, for rubber‐like solids. The proposed model involves only one parameter that must be determined by fitting with the experimental data. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 660–672, 2000     

Temperature and strain registration by fibre-optic strain sensor in the polymer composite materials manufacturing

ABSTRACT

The presence of process-induced strains induced by various manufacturing and operational factors is one of the characteristics of polymer composite materials (PCM). Conventional methods of registration and evaluation of process-induced strains can be laborious, time-consuming and demanding in terms of technical applications. The employment of embedded fibre-optic strain sensors (FOSS) offers a real prospect of measuring residual strains. This paper demonstrates the potential for using embedded FOSS for recording technological strains in a PCM plate. The PCM plate is manufactured from prepreg, using the direct compression-moulding method. In this method, the prepared reinforcing package is placed inside a mould, heated, and then exposed to compaction pressure. The examined technology can be used for positioning FOSS between the layers of the composite material. Fibre-optic sensors, interacting with the material of the examined object, make it possible to register the evolution of the strain process during all stages of polymer-composite formation. FOSS data were recorded with interrogator ASTRO X 327. The obtained data were processed using specially developed algorithms.     

Effect of strain rate and temperature on stress-strain properties of EVA-LDPE blends and the mechanism of strain hardening

Stress-strain behaviour of different blends of poly(ethylene-co-vinyl acetate) (EVA) (28 wt.-% VA content) and low density polyethylene (LDPE) is studied under various strain rates and temperatures. It is found that stress-strain plots of such semicrystalline polymer blends consist of three parts, namely, elastic or Hookeian region, region of chain slippage and region of strain hardening. Decrease in strain rate has an increasing effect on the strain hardening region. Increase in measurement temperature adversely affects the whole stress-strain plot. It is apparent from the study that at an elevated temperature the process of strain hardening is dependent on the crystalline melting point of the major component in the blend. The X-ray and DSC studies reveal that the process of strain hardening is mainly due to a change in internal order of crystallites in LDPE and LDPE-rich blends, whereas in EVA and EVA-rich blends it is due to induced crystallization in the amorphous phase.