脂质体包裹反义核酸对调控效果和降解时间的影响

目的 观察脂质体Dotap包裹反义脱氧寡核苷酸对酪氨酸酶(TYR)的调控效果和降解时间的影响,寻找适合于临床应用的高效转染、低降解率的载体和应用方法。方法 应用DNA合成仪合成针对TYR的反义脱氧寡核苷酸,32P标记、脂质体包埋,加入黑色素细胞培养体系进行点杂交,行黑素含量、酪氨酸酶mRNA表达量检测以及降解时间的同位素液闪检测。结果 经脂质体包埋的反义脱氧寡核苷酸使细胞黑素含量由(64±2.3)pg/cell下降至(23±1.5)pg/cell,并可使基因表达水平由160%降低至82%;同位素液闪显示经脂质体包埋的反义脱氧寡核苷酸能使其半衰期由8 h延长至12 h。结论 脂质体可以明显增强反义核酸的调控效果,并可延长其作用半衰期。     

Priming with a very low dose of DNA complexed with cationic block copolymers followed by protein boost elicits broad and long-lasting antigen-specific humoral and cellular responses in mice

Cationic block copolymers spontaneously assemble via electrostatic interactions with DNA molecules in aqueous solution giving rise to micellar structures that protect the DNA from enzymatic degradation both in vitro and in vivo. In addition, we have previously shown that they are safe, not immunogenic and greatly increased antigen-specific CTL responses following six intramuscular inoculations of a very low dose (1 μg) of the vaccine DNA as compared to naked DNA. Nevertheless, they failed to elicit detectable humoral responses against the antigen. To gain further insight in the potential application of this technology, here we show that a shorter immunization protocol based on two DNA intramuscular inoculations of 1 μg of DNA delivered by these copolymers and a protein boost elicits in mice broad (both humoral and cellular) and long-lasting responses and increases the antigen-specific Th1-type T cell responses and CTLs as compared to priming with naked DNA. These results indicate that cationic block copolymers represent a promising adjuvant and delivery technology for DNA vaccination strategies aimed at combating intracellular pathogens.     

The Experiment of Gene Therapy for Gallbladder Carcinoma Cell in Vitro with Herpes Simplex Virus Thymidine Kinase/Ganciclovir

Objective To evaluate preliminarily the effect of HSV - tk/GCV system on gallhladder carcinoma cells in vitro.Methods Recombinant retroviral vector PLtkSN containing tk suicide gene was transfacted into gallbladder carcinoma cells, mediated by LipofectAMINETM 2000 liposome, and the growth - inhibiting rotes of GBC, SD/tk cells in the different GCV concentrations were measured by MTr methods; GBC - SD cells were mixed in the different proportions, and death rotes of the mixed cells treated with GCV were detected to verify by stander effect. Resuts GCV could lead GBC - SD/tk cells to significant death, and there was striking difference compared with the control cells （p〈0.01）. When the concentration of GCV was 1,10, 50,100,500ug/ml, the killing rate of GBC - SD/tk cells was respecfively 6.8%, 2.5.2%, 54.5%, 66.3%, 89.3%, indicating dose - dependent phenomenon; With GBC - SD/tk cells in the proportion of 0, 10%, 20% ,50%, 70%, 100%, the killing rate of the mixed cells treated with GCV was 0, 19.7%, 40.3%, 77.7%, 88.0%, 93.5%, respectively. It was apparent that bystander effect was observed in the experiment. Condusion HSV - tk/GCV system could be a potential tool for the treatment of carcinoma.     

Cholera Toxin B-Mediated Targeting of Lipid Vesicles Containing Ganglioside GM1 to Mucosal Epithelial Cells

Purpose. To determine whether the non-toxic pentameric B subunit of Cholera toxin (CTB) binding to ganglioside GM1 on both the lipid vesicles and epithelial cells may provide a means to target lipid vesicles to mucosal cells expressing surface GM1. Methods. Sonicated lipid vesicles containing ganglioside GM1 were prepared. Inter-vesicle cross-linking due to pentameric CTB binding to these GM1 vesicles was determined with a sub-micron particle analyzer. Association of CTB to GM1 vesicles was analyzed with continuous sucrose gradient centrifugation. CTB-mediated binding of GM1 vesicles to human mucosal epithelial cells (Caco-2 and HT-29), mucous membranes of mouse trachea, and nasal tissues were detected with fluorescent labeled vesicles. Results. An increase in lipid particle size due to binding of CTB to lipid vesicles and inter-vesicles cross-linking was detected. At a 30-to-1 mole ratio of membrane-bound GMl-to-CTB, optimum increase in GM1 vesicle aggregation, was detected. Under such conditions, all the added CTB molecules were associated with GM1 vesicles. Time course analysis showed that inter-vesicles cross linking by CTB was detectable within 10 min. and reached a maximum value at 60 min. CTB associated GM1-vesicles bind to mucosal epithelial cells HT-29 and Caco-2 with similar affinity [K_d = 7.8 × 10^–4 M lipid (Caco-2) and 7.6 × 10^–4 M lipid (HT-29)]. GM1 mediated binding specificity was demonstrated by blocking with anti-GMl antibody and the insignificant degree of CTB-associated GM1 vesicle binding to GM1 deficient C6 cells. Conclusions. The CTB-mediated GM1 binding to multiple membrane surfaces provides selective localization of GM1 vesicles to GM1 expressing mucosal cells and tissues. The strategy may be useful in localizing drugs and proteins to gut and respiratory tract mucosa.     

脂质体携载SOD的实验研究

本文采用去垢剂透析法成功制备出携载ＳＯＤ的脂质体（Ｌ－ＳＯＤ），其直径为１０２ｎｍ，对ＳＯＤ的包裹率为２１％，９０％以上酶活性存在于脂质体内部。将Ｌ－ＳＯＤ注射到大鼠静脉血中，其半衰期超过４ｈ，明显长于天然ＳＯＤ（８ｍｉｎ）。表明Ｌ－ＳＯＤ优于天然ＳＯＤ，具有临床应用前景。     

维生素E脂质体的制备

用超声波制备了维生素Ｅ脂质体，测定了脂质体的包封率及放置后的稳定性；观察了脂质体的形态，粒度分布。０．２％维生素Ｅ脂质体用于治疗老年性角化斑，试用２０例总有效率达９５％，优于１例维生素Ｅ乳剂。     

表面活性剂在硝酸士的宁注射液酸性染料比色法中的应用

表面活性剂在光度分析中已经有了广泛的应用,不论是在水溶液中测定的胶束增溶光度法还是在有机溶剂中测定的胶束萃取光度法中,表面活性剂的加入都具有明显的增溶、增敏和增稳作用,它能直接影响显色反应的速度、平衡位置以及配合物的构型,能改变溶剂的     

Comparison of anticonvulsant effects of valproic acid entrapped in positively and negatively charged liposomes in amygdaloid-kindled rats

Intraperitoneal injection of free valproic acid (VPA) suppressed amygdaloid-kindled seizure 1 h after injection in rats, but had no effect at 24 h. VPA entrapped in positively charged liposomes showed a prolonged anticonvulsant effect lasting for 2 days, while the effect evaluated at 1 h was not different from that with free VPA. VPA entrapped in negatively charged liposomes exerted a significantly stronger effect at 1 h than did free VPA, while it had no significant effect at 24 h. These results suggest that surface charges on liposomes play an important role in modifying the anticonvulsant effect of VPA.     

AlEtCl_2／t－BuCl引发α－蒎烯阳离子聚合反应研究

考察了在ＡｌＥｔＣｌ２／ｔ－ＢｕＣｌ引发体系作用下的α－蒎烯阳离子聚合行为．结果表明，ｔ－ＢｕＣｌ与ＡｌＥｔＣｌ２复合后，由于生成（ＣＨ３）Ｃ正离子而使引发活性增大．但ｔ－ＢｕＣｌ的引人对产物分子量及其分布影响不大，产物主要由二、三聚体组成．聚合过程的动力学研究表明，α－蒎烯在进行阳离子聚合时容易发生链终止反应而导致单体转化率和产物分子量较低．α－蒎烯在聚合反应的同时伴随着异构化反应，用制备ＧＰＣ分离出异构化产物，然后经１ＨＮＭＲ测定其结构．