口服草原兔尾鼠卵透明带3纳米乳剂DNA疫苗免疫效果研究

用免疫不育疫苗控制有害动物的数量成为动物种群数量控制的一个有效措施,对于大多数动物来说．口服饵料发送系统是最好的选择。但是口服疫苗容易产生耐受,且生物利用度低,利用多聚物包裹口服疫苗是一种打破口服耐受．提高其生物利用度的新途径。本文以纳米乳剂作为草原兔尾鼠卵透明带3（Lugurus zone pellucida 3,LZP3）DNA疫苗的发送载体,探讨通过口服途径增强小鼠的免疫效果和抗生育率。用纳米乳剂包裹lzp3 DNA疫苗后,采用强阴离子交换色谱法测定纳米乳剂的包封率．将制备好的质粒纳米乳剂lzp3 DNA疫苗通过口服饵料免疫小鼠,检测到免疫后小鼠的体内产生了特异性的抗LZP3的IgG和IgA。对免疫后小鼠进行抗生育实验,分析生育后免疫小鼠的卵巢病理切片。研究结果表明,用质粒纳米乳剂如p3DNA疫苗通过口服途径免疫．在小鼠的血清和粘液中检测到了特异性的抗LZP3的IgG和IgA,同时也产生了相应的抗生育作用．表明纳米乳剂作为口服不育疫苗的发送载体是可行的,为草原兔尾鼠鼠害的防治提供了理论基础．     

姜黄素共聚物胶束的制备与体外细胞毒评价

目的:制备一种姜黄素共聚物胶束以提高姜黄素的水溶性及其抗肿瘤活性。方法:采用乳化溶剂挥发法制备了载姜黄素的共聚物胶束(Cur/PTL1胶束),对其粒径、载药量、包封率和体外药物释放行为进行了考察;并采用MTT法考察了PTL1空白胶束和Cur/PTL1胶束的体外细胞毒作用。结果:制备了粒径在40 nm左右的载姜黄素共聚物胶束,载药量为9.78±0.29%,包封率为97.24±2.68%。体外药物释放实验表明,游离姜黄素在24 h内的药物累积释放率达到90%以上,而Cur/PTL1胶束在24 h内药物累积释放率为23.8%,能够持续释放14天,14天内累积释放率为85.9%,具有一定的缓释能力。MTT实验结果表明,当PTL1空白胶束浓度达到1 mg/mL时,细胞的存活率仍在90%以上;Cur/PTL1胶束组IC50为4.73±0.23μg/mL,游离姜黄素组IC50为6.42±0.35μg/mL。结论:实验结果表明,Cur/PTL1胶束可以作为一种有前景的纳米药物输送系统。     

新型人工红细胞的制备

目的:接枝淀粉包裹血红蛋白制备新型人造红细胞的代替品。方法:利用油酸接枝淀粉,在超声条件下下自组装,包裹天然牛血红蛋白,并鉴定其物理化学及生物学性能。测定包封率、红外光谱分析(FTIR)、电镜观察形态学及粒径,测定P50和Hill系数。结果:人工红细胞呈圆球形,平均粒径250nm,包封率高,具有良好的携氧、释氧能力。结论:成功制备了人工纳米红细胞,为进一步临床应用提供了基础。     

壳聚糖-有机累托石/海藻酸钠复合纳米粒的制备

目的:以BSA作为模型药物,制备壳聚糖季铵盐-OREC复合物纳米微粒,建立一种安全有效的药物控释传递系统。方法:超声条件下,制备不同质量比的具有壳聚糖硅酸盐插层结构的复合物纳米微粒,观察其形态学特征、进行红外光谱分析。同时,测定OREC对BSA包封率和载药量的影响。结果:成功制备了不同质量比的OREC-HTCC纳米粒子。电镜结果显示纳米粒呈圆球形,均匀,平均粒径约为30nm。红外图谱分析证实,HTCC插入了OREC插层中,BSA成功地包裹入HTCC-ALG/OREC混合材料制备的纳米微粒。加入OREC后,纳米粒子的包封率及载药量均明显提高,但随着加入量的增加,包封率及载药量逐渐减少。结论:OREC-HTCC纳米粒子是良好的蛋白药物载体,具有粒径小、包封率高、缓释效果好等优点,为CS-OREC作为潜在的药物给药系统的进一步应用提供科学依据。     

胆甾醇基γ-聚谷氨酸负载阿霉素纳米胶束的制备与体内外释药性能评价

笔者制备了胆甾醇基γ-聚谷氨酸负载阿霉素纳米胶束(DOX/NPs),并考察了该载药纳米胶束体系的形态与粒径、载药量、包封率以及体内外释药的特性。结果表明:DOX/NPs的最佳载药量为22.4%,包封率为90.2%,平均粒径为(312.3±7.2)nm,电镜下观察呈现明显的核壳结构。体外释药结果显示,DOX/NPs能延缓阿霉素的释放,并具有p H敏感的释药特性。小鼠体内释药结果表明:阿霉素经包埋后其消除半衰期(t1/2)、药时曲线下面积(AUC)、平均滞留时间(MRT)均明显大于游离阿霉素,达到了药物缓释的目的。     

阿霉素脂质体的制备及其体外抗肿瘤活性的初步研究

目的:应用超声波分散法制备脂质体阿霉素,并比较脂质体阿霉素与游离性阿霉素抗肿瘤活性。方法:以卵磷脂和胆固醇为原料,将阿霉素包封于脂质体中,采用超声分散法制备脂质体阿霉素,对其在290-700nm范围内进行紫外扫描,用SephedexG-50柱分离脂质体阿霉素并计算其包封率。以昆明种小鼠为载体建立肿瘤模型(S180型肉瘤)和细胞荧光染色法研究脂质体阿霉素的抗肿瘤活性,以ZITA SIZER3000型表面电位与粒度测定仪测定其粒径分布。结果:脂质体阿霉素在480nm处有最大吸收峰值,包封率达91.3%,细胞荧光染色显示,脂质体及游离型阿霉素均对S180细胞有明显的抑制作用。结论:此法制备的脂质体阿霉素包封率高,粒径分布集中,脂质体阿霉素较游离型阿霉素有较强的抗肿瘤活性剂及较低的细胞毒作用,对阿霉素的临床应用有一定的参考价值。     

靶向肿瘤新生血管纳米粒的构建和质量控制

目的：制备F56多肽修饰的长春新碱纳米粒（F56-VCR-NP）,并建立其质量控制方法。方法：乳化－溶剂挥发法优化制备F56．VCR-NP：HPLC法测定其载药量、包封率,透射电镜下观察其形态,激光粒度分析仪测定其粒径和Zeta电位,CBQCA试剂盒测定纳米粒表面多肽密度,XPS进行表面元素分析。结果：优化制备的F56－VCR-NP粒径约为153nm,Zeta电位为－20．8mv,包封率为21．4％,载药量为1．9％,多肽连接效率为26．3％。结论：以聚乙二醇－聚乳酸（PEG-PLA）为原料,长春新碱为模型药物,成功制备出纳米粒子,并建立起有效的质量控制方法,对该实验样品进行了表征。结果表明此类纳米粒子尺寸均匀,表面多价连接F56多肽,载药量和包封率稳定可控,工艺成熟。     

新型"脂质-HAP-DNA复合体"的制备和用于真核细胞基因转染研究

为寻找一种简单、经济、有效的DNA递送系统用于基因转染和基因治疗,制备了表面电荷为正电的纳米HAP,与表面电荷为负电的DNA结合形成DNA-HAP复合物,采用逆向蒸发法,用卵磷脂、DOPE和胆固醇制备成脂质体包封DNA-HAP复合物形成脂质-HAP-DNA复合体,脂质体和HAP对照,对所形成的脂质-HAP-DNA复合体(LHD)的特性、包封率、转染Hela细胞的效果进行初步检测研究。所获得的脂质-HAP-DNA复合体呈球形、平均粒径为643nm;平均包封率达11.67%,为中性脂质体;能有效转染真核细胞。该方法可作为提高基因转染效果的简单、经济、有效的手段之一,也为进一步提高非病毒载体的转染效率提供了一个思路。     

盐酸洛拉曲克脂质体的制备及其质量考察

目的：制备盐酸洛拉曲克脂质体并考察其理化特性。方法：采用薄膜挤压-硫酸铵梯度法制备盐酸洛拉曲克脂质体,透射电镜及激光粒度分析仪分别观察和检测其粒径大小及分布,通过紫外分光光度法测定包封率及评估体外释药试验。结果：制备的盐酸洛拉曲克脂质体包封率达83.6%±2.37%,粒径103.5±26nm且分布均匀。24h体外释放实验结果提示约有66.5%的盐酸洛拉曲克从脂质体释放出来。结论：新制备的盐酸洛拉曲克脂质体粒径大小均匀,包封率尚有提高空间,具有体外缓慢释药的特性。     

氢氧化铝佐剂制备工艺的建立及其在鼠疫疫苗中的免疫效果评价

目的建立氢氧化铝佐剂制备工艺,并吸附抗原配制鼠疫疫苗,对其进行免疫效果评价。方法采用氯化铝与氢氧化钠反应生成氢氧化铝胶体,通过优化制备工艺,制备出纳米级铝佐剂,并对其进行连续4批质量检测,将其与丹麦ALH铝佐剂在吸附率、沉降率、粒径、配制鼠疫疫苗安全性及免疫原性方面进行比较。结果 4批次氢氧化铝含量平均值为18.67 mg/mL,氯化钠含量平均值为29.60 mg/mL,平均pH为5.66;各批次氢氧化铝佐剂沉降率及吸附率检测结果均符合《中华人民共和国药典》2020版(四部)的检测要求;粒径控制已达到纳米级,平均值为100～600 nm;配制鼠疫疫苗安全性检测结果合格,20200404批铝佐剂与ALH铝佐剂分别配制鼠疫疫苗免疫NIH小鼠,20200404疫苗组V抗体IgG、IgG1抗体效价均高于ALH疫苗组,差异均有统计学意义(P0.000 5);20200404疫苗组V抗体IgG2a抗体效价与ALH疫苗组差异无统计学意义(P0.05);20200404疫苗组与ALH疫苗组F1抗体IgG、IgG1、IgG2a抗体效价差异均无统计学意义(P0.05)。结论成功建立了氢氧化铝佐剂制备工艺,为制备鼠疫疫苗用铝佐剂奠定了基础。     

人角质化细胞生长因子-2基因的克隆、表达及产物的纯化、鉴定

用高表达菌株BL21codon plus compentent cells表达重组人角质化细胞生长因子(Hkgf-2)蛋白并初步纯化和检测其活性。通过RTPCR从流产胎儿肺组织中钓取hKGF-2cDNA,将其克隆入pBV220载体质粒。在大肠杆菌BL-21codon plus compent cells中表达hKGF-2蛋白。采用亲和层析和离子交换层析分离纯化,以细胞增殖实验测定表达蛋白的生物活性。结果显示,hKGF-2蛋白在BL21中得到高效表达;hKGF-2蛋白能刺激NIH3T3细胞的增殖,具有显著的促有丝分裂活性。     

Separation of plasmid DNA from protein and bacterial lipopolysaccharides using displacement chromatography

The preparation of plasmid DNA at large scale constitutes a pressing problem in bioseparation. This paper describes a first investigation of displacement chromatography as a means to separate plasmid DNA (4.7 kb) from E. coli lipopolysaccharides and protein (holo transferrin), respectively. Displacement chromatography has advantages in this regard, since the substance mixture is resolved into rectangular zones of the individual components rather than into peaks. Thus a higher total concentration can be maintained in the pooled product fractions. Hydroxyapatite (type I and II) and anion exchange stationary phases were included in the experiments. In addition to a conventional anion exchange column packed with porous particles, the recently introduced continuous bed UNOTM anion exchange column was investigated. No DNA purification was possible with either hydroxyapatite material. Conventional particle based columns in general were not suited to the separation of any two substances varying considerably in molecular mass, e.g. plasmid DNA and standard protein. Presumably, the direct competition for the binding sites, which is essential in displacement chromatography, was restricted by the size dependency of the accessible stationary phase surface area in this case. Better results were obtained with the continuous bed column, in which the adsorptive surface coincides with the walls of the flow through pores. As a result the accessible surface does not vary as much with the size of the interacting molecules as for the conventional stationary phase materials. Sharper transitions were also observed between substance zones recovered from the UNOTM column. The steric mass action model was used to aid method development in case of the anion exchange approach. While further research in obviously necessary, displacement chromatography on continuous bed columns has been shown to be capable of separating plasmid DNA from typical impurities. This revised version was published online in July 2006 with corrections to the Cover Date.     

一种安全高效的血红蛋白纯化方法

目的:建立一种适用于大量制备的,安全、高效的血红蛋白纯化方法。方法: 将压积红细胞装入透析袋,以含有还原剂的Tris缓冲液透析破碎,破碎的上清经两级硫酸铵沉淀后透析至上样缓冲体系,离心后取上清即得血红蛋白提取液;红细胞提取液通过阴离子交换柱层析进一步分离,计算回收率。纯化产物浓缩后以SDS-PAGE及HPLC鉴定纯度,进行紫外-可见光谱扫描并以ABL800血气分析仪分析血气指标,以鲎试剂测定内毒素含量,以磷测定法测定脂质含量。结果: 血红蛋白提取液中脂质去除率98%,容易通过0.45μm滤膜;经阴离子交换层析纯化的血红蛋白经SDS-PAGE(银染法)及WB分析没有杂蛋白条带,HPLC分析纯度>99%、总回收率>85%;内毒素含量<2 EU,高铁血红蛋白含量<5%。结论: 该血红蛋白纯化方法安全高效、成本低廉、易于放大生产,具有较好的应用前景。     

分子佐剂C3d增强草原兔尾鼠卵透明带3 DNA疫苗的免疫应答

为增强免疫不育的效果,探讨分子佐剂C3d对草原兔尾鼠透明带3(LaguruslagurusZonaPellucida3,LZP3)基因免疫的调节作用。构建了含3个拷贝C3d的lzp3基因真核细胞表达质粒pcDNA3-LZP3-C3d3(pcD-LC),以不含C3d的质粒pcDNA3-LZP3(pcD-L)为对照,体外转染HeLa细胞,RT-PCR和Westernblot分别检测到lzp3mRNA和蛋白的表达。对NIH雌性小鼠肌肉进行基因免疫注射,ELISA结果表明pcD-LC免疫诱导的特异性抗LZP3-IgG水平明显高于pcD-L对照组(P<0.05),且有长期免疫应答效应;MTT结果显示pcD-LC能够诱导淋巴细胞增殖活性的提高。抗生育实验表明pcD-LC免疫组显著降低窝产仔数(P<0.05),且卵巢切片正常。研究结果证实C3d能够增强lzp3DNA疫苗的特异性免疫应答并达到抗生育效果,为DNA疫苗控制草原兔尾鼠的深入研究提供了基础。     

Rapid determination of PEGylated liposomal doxorubicin and its major metabolite in human plasma by ultraviolet-visible high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the quantification of doxorubicin derived from PEGylated liposomal doxorubicin (Doxil) and its major metabolite in human plasma. This method utilizes Triton X-100 to disperse the liposome, followed by a protein precipitation step with 5-sulfosalicylic acid. Analytes in the resultant supernatant are separated on a Discovery RP amide C(16) column (250 x 3 mm I.D., 5 microm) using an isocratic elution with a mobile phase consisting of 0.05 M sodium acetate (pH 4.0) and acetonitrile (72:28). The retention times for doxorubicin and the internal standard daunorubicin were 4.8 and 10.1 min, respectively. The column eluate was monitored by UV-visible detection at 487 nm. The determination of doxorubicin was found to be linear in the range of 1.0 ng/mL to 25 microg/mL, with intra-day and inter-day coefficients of variation and percent error < or =10%. The recovery of doxorubicin from plasma was >69.3%, with a liposomal dispersion efficiency of >95.7%. Our analytical method for free and PEGylated doxorubicin in human plasma is rapid, avoids organic extractions, and maintains sensitivity for the parent compound and its major metabolite, doxorubicinol.     

Determination of the specific activity of NaB3H4

A method for the rapid determination of the specific activity of NaB3H4 is presented. NaB3H4 is used to reduce NAD+ to [3H]NADH, which is then isolated by anion exchange chromatography. The specific activity of the NaB3H4 is calculated from measurements of radioactivity and absorbance (340 nm) in the [3H]NADH fractions.     

固相萃取-高效液相色谱法测定复合薯片中丙烯酰胺

建立了一种利用固相萃取小柱与高效液相色谱联用,采用光电二极管阵列检测器测定复合薯片中丙烯酰胺的方法。以0．1％的甲酸水溶液为提取试剂,采用Waters Oasis HLB固相萃取小柱（200mg／6cc）对提取液净化处理,然后以流动相为甲醇／水（5：95,V：V）,流速为0．6mL／min,检测波长为210nm,利用AgilentC18反相色谱柱进样分析。在该条件下丙烯酰胺的回收率达80％以上,最低检测限小于10ng／mL。该方法操作简便、重复性好、稳定性高,可用于油炸复合薯片中丙烯酰胺的快速检测。     

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection

A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively.     

苦玄参药材中苦玄参苷ⅠA和ⅠB的含量分析

采用HPLC法对不同产地的苦玄参药材主成分苦玄参苷ⅠA和ⅠB的含量进行分析,发现苦玄参苷ⅠB含量高于苦玄参苷ⅠA;色谱条件:Waters C18(4.6mmi.d.×250mm,5μm)色谱柱,流动相∶乙睛∶水=36?64(体积比),流速为0.8mL/min,检测波长为264nm。苦玄参苷ⅠA、ⅠB分别在0.05～0.50mg/mL和0.072～0.720mg/mL与峰面积有良好的线性关系。苦玄参苷ⅠA的线性回归方程Y=13658X-92.72(r=0.9993),平均回收率为100.58%(n=5),相对标准偏差(RSD)为1.86%;苦玄参苷ⅠB的线性回归方程Y=5258.9X-41.01(r=0.9994),平均回收率为101.15%(n=5),相对标准偏差(RSD)为1.31%。该研究为制定苦玄参药材质量检测标准提供了简便、快速、准确的方法。