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新型佐剂dc-chol对不同来源HBsAg促细胞免疫比较研究

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中国生物工程杂志 2006年第3期26卷 22-25页

作者：易学瑞袁有成祖萍施理孔祥平解放军第四五八医院全军肝病中心全军肝病重点实验室广州510600

目的:筛选治疗性乙肝疫苗的候选抗原。方法:分别用国产CHO乙肝表面抗原和酵母乙肝表面抗原结合dcchol脂质体佐剂免疫BALB/c小鼠,1周后检测其脾细胞产生的IL2和IFNγ的水平。ELISPOT法评价铝佐剂和dcchol脂质体佐剂结合HBsAg促小鼠细胞... 详细信息

目的:筛选治疗性乙肝疫苗的候选抗原。方法:分别用国产CHO乙肝表面抗原和酵母乙肝表面抗原结合dcchol脂质体佐剂免疫BALB/c小鼠,1周后检测其脾细胞产生的IL2和IFNγ的水平。ELISPOT法评价铝佐剂和dcchol脂质体佐剂结合HBsAg促小鼠细胞免疫反应水平。结果:酵母乙肝表面抗原结合dcchol脂质体所诱导的IL2和IFNγ的水平分别为CHO乙肝表面抗原结合dcchol脂质体的20倍和119倍。酵母乙肝表面抗原结合dcchol脂质体所诱导的IL4和IFNγ的斑点数分别为酵母乙肝表面抗原结合铝佐剂的2.8倍和46.3倍。结论:就dcchol脂质体佐剂而言,酵母表达的乙肝表面抗原细胞免疫原性强,是适合用于治疗性乙肝疫苗的候选疫苗。

关键词： dc-chol CHO 酵母 IL-2 IFN-γ ELISPOT

dc-chol诱生的免疫能克服对乙型肝炎疫苗的无应答性

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国际生物制品学杂志 2000年第1期23卷 32-33页

作者：侯秀霞谢东生

dc-chol阳离子脂质体佐剂对流感疫苗免疫效果的影响

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昆明医科大学学报 2021年第3期42卷 1-4页

作者：刘艳红赵祥月谢青昕普梦笛鲁卫东昆明医科大学药学院暨云南省天然药物药理重点实验室云南昆明650500

目的观察阳离子脂质体dc-chol修饰的四价流感疫苗的免疫原性。方法采用薄膜分散结合冻融-冻干法制备dc-chol脂质体,检测其包封率。将小鼠分为七组,为dc-chol脂质体疫苗组(250μg/只、500μg/只、750μg/只、900μg/只)、PBS组、中性脂... 详细信息

目的观察阳离子脂质体dc-chol修饰的四价流感疫苗的免疫原性。方法采用薄膜分散结合冻融-冻干法制备dc-chol脂质体,检测其包封率。将小鼠分为七组,为dc-chol脂质体疫苗组(250μg/只、500μg/只、750μg/只、900μg/只)、PBS组、中性脂质体组、疫苗原液组(n=3),通过MTT法确定dc-chol脂质体的最佳剂量。将小鼠分为四组,dc-chol脂质体组、疫苗原液组、中性脂质体组以及PBS对照组,腹腔免疫小鼠,于第7天、14天、28天处死,MTT法和T淋巴细胞表面标记实验检测小鼠淋巴细胞增殖情况观察免疫原性。结果MTT实验表明,dc-chol修饰脂质体的最佳用量为500μg/鼠(Pdc-chol阳离子脂质体能明显增强脾淋巴细胞数(Pdc-chol阳离子脂质体作为四价流感疫苗的佐剂具有良好的免疫增强作用。

关键词：脂质体 dc-chol 流感疫苗细胞免疫

新型佐剂dc-chol对不同来源HBsAg促细胞免疫比较研究

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新型佐剂DC-Chol对不同来源HBsAg促细胞免疫比较研究

Ionization and transfection activity of n-methyl-substituted carbamoyl-cholesterol derivatives

2006年慢性乙型肝炎治疗进展研讨会

作者：易学瑞袁有成祖萍施理孔祥平解放军第四五八医院、全军肝病中心

目的:筛选治疗性乙肝疫苗的候选抗原。方法:分别用国产CHO乙肝表面抗原和酵母乙肝表面抗原结合dc-chol脂质体佐剂免疫BALB/c小鼠,l周后检测其脾细胞产生的IL-2和IFN-γ的水平。ELISPOT法评价铝佐剂和dc-chol脂质体佐剂结合HBsAg促小鼠... 详细信息

目的:筛选治疗性乙肝疫苗的候选抗原。方法:分别用国产CHO乙肝表面抗原和酵母乙肝表面抗原结合dc-chol脂质体佐剂免疫BALB/c小鼠,l周后检测其脾细胞产生的IL-2和IFN-γ的水平。ELISPOT法评价铝佐剂和dc-chol脂质体佐剂结合HBsAg促小鼠细胞免疫反应水平。结果:酵母乙肝表面抗原结合dc-chol脂质体所诱导的IL-2和IFN-γ的水平分别为CHO乙肝表面抗原结合dc-chol脂质体的20倍和119倍。酵母乙肝表面抗原结合dc-chol脂质体所诱导的IL-4和IFN-γ的斑点数分别为酵母乙肝表面抗原结合铝佐剂的2.8倍和46.3倍。结论:就dc-chol脂质体佐剂而言,酵母表达的乙肝表面抗原细胞免疫原性强,是适合用于治疗性乙肝疫苗的候选疫苗。

关键词： dc-chol CHO 酵母 IL-2 IFN-γ ELISPOT

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Journal of Biophysical Chemistry 2011年第2期2卷 53-62页

作者： Samuel Acheampong Michalakis Savva Division of pharmaceuticals Sciences Arnold & Marie Schwartz College of Pharmacy & Health Sciences Long Island University New York United States

Five novel cationic lipids, the polar head group of which was attached to the cholesterol backbone via a tertiary carbamate linker, were synthesized and their physicochemical properties were compared to their transfection efficiencies. Transfection activity of the primary amine analog was highest among the series, while the quaternary ammonium iodide salt was essentially transfection incompetent. Contrary to dc-chol, methyl and ethyl carbamoyl derivatives of dc-chol mediated high levels of transfection in the absence of DOPE. Ionization of the cationic assemblies in 40 mM Tris buffer pH 7.2 exactly correlated with the competitive nature of the inductive and steric effects of the methyl groups on the aliphatic nitrogen of the lipids’ polar moiety. Interestingly, the pH interaction zone of all lipid dispersions at 25°C was extended by ± 2 pH units from the pKa, while the pKa of the cationic lipids determined in mixed vesicles composed of 90 % DOPC and cholesterol was approximately 1.3 to 1.5 times higher than that of pure cationic assemblies. The interaction of cationic lipids with plasmid DNA was correlated with pKa, but not the transfection activity.

关键词： Cationic Lipids dc-chol Transfection Pka Plasmid DNA Monolayer Surface Pressure.

阳离子修饰的流感疫苗脂质体冻干粉的制备及免疫原性研究

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阳离子修饰的流感疫苗脂质体冻干粉的制备及免疫原性研究

作者：赵祥月昆明医科大学

学位级别：硕士

[目的]考察dc-chol和DOTAP在脂质体成膜前或成膜后不同阶段加入所制备的阳离子脂质体的理化性质及免疫原性,筛选dc-chol和DOTAP修饰脂质体疫苗的最佳制备方法,筛选修饰脂质体的dc-chol和DOTAP的最佳用量;筛选SA在在脂质体成膜前加入所... 详细信息

[目的]考察dc-chol和DOTAP在脂质体成膜前或成膜后不同阶段加入所制备的阳离子脂质体的理化性质及免疫原性,筛选dc-chol和DOTAP修饰脂质体疫苗的最佳制备方法,筛选修饰脂质体的dc-chol和DOTAP的最佳用量;筛选SA在在脂质体成膜前加入所制备的阳离子脂质体的理化性质及免疫原性,筛选SA修饰脂质体的最佳用量。[方法]采用薄膜分散联合冻融-冻干法分别制备中性脂质体、dc-chol成膜前加入脂质体(dc-chol剂量分别为250μg/鼠、500μg/鼠、750μg/鼠、900μg/鼠)、DOTAP成膜前加入脂质体(DOTAP剂量分别为450μg/鼠、600 μg/鼠、900 μg/鼠),腹腔免疫小鼠,以MTT法为指标,以单因素方差分析确定两种不同阳离子所修饰脂质体的最佳修饰用量;在最佳修饰用量的基础上,分别制备dc-chol成膜前加入脂质体、dc-chol成膜后加入脂质体、DOTAP成膜前加入脂质体、DOTAP成膜后加入脂质体,腹腔免疫小鼠,进行MTT实验,以单因素方差分析确定两种不同阳离子所修饰脂质体的最佳制备方法;以SA为电荷修饰物,采用薄膜分散联合冻融-冻干法分别制备SA与大豆磷脂摩尔比为2%、3%、4%、5%、10%的脂质体冻干粉,进行MTT实验,以单因素方差分析确定SA修饰流感疫苗脂质体冻干粉的最佳用量;以激光粒度仪分别测定两种阳离子最佳制备方法和最佳修饰用量制备的脂质体疫苗的粒径和电位。以筛选出的最佳修饰用量和最佳制备方法制备脂质体,设计实验,分别免疫小鼠,于第7d、14d、28d通过MTT实验检测脾淋巴细胞增殖能力,流式细胞仪检测T淋巴细胞表面标记实验来考察其免疫原性。[结果]通过MTT实验,采用单因素方差分析确定了两种阳离子修饰脂质体疫苗的最佳制备方法为成膜后加入法,dc-chol修饰脂质体的最佳用量为50Oμg/鼠,成膜后加入dc-chol修饰的脂质体平均粒径约为2.2μm,zeta电位约为18.9mV;DOTAP修饰脂质体的最佳用量为600μg/鼠,成膜后加入DOTAP修饰的脂质体平均粒径约为2.2μm,zeta电位约为17.9mV;脂质体成膜前加入SA修饰脂质体的最佳用量为2%(摩尔比),所制备的脂质体平均粒径约为733.9nm,zeta电位约为13.2mV。免疫相同周期时,脂质体成膜后加DOTAP和dc-chol组分别与中性脂质体组、非脂质体疫苗原液组、PBS阴性对照组比较,Pdc-chol制得的阳离子脂质体疫苗免疫原性强于中性脂质体疫苗;脂质体成膜水化后加入DOTAP组与脂质体成膜水化后加入dc-chol组进行组间比较,P>0.05,无显著性差异,说明两种阳离子脂质体免疫原性一致。[结论]成膜前或成膜后加入阳离子对两种阳离子修饰的流感疫苗脂质体冻干粉的免疫原性并无显著性影响,但为加强实验过程的可操作性和简易性,选择成膜后加入阳离子作为制备阳离子修饰脂质体的最佳方法;dc-chol修饰脂质体的最佳用量为500 μg/鼠;DOTAP修饰脂质体的最佳用量为600μg/鼠;在7d、14d、28d的免疫周期内,7d时疫苗对小鼠脾淋巴细胞的刺激指数最高;在14d、28d时,阳离子修饰的脂质体疫苗对小鼠脾淋巴细胞的刺激指数稍有降低,但与中性脂质体7d时的免疫原性一致,仍能诱导较强的细胞免疫。

关键词：脂质体 DOTAP dc-chol SA 流感疫苗细胞免疫

In vitro investigation of RGD-modified stabilized cationic liposomes as non-viral vehicle for siRNA delivery

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Journal of Chinese Pharmaceutical Sciences 2008年第2期17卷 97-105页

作者：杨世进杨丽娟姜娟王坚成张强北京大学药学院药剂学系北京100083

In this study, the novel RGD-modified stabilized cationic liposomes were developed as the delivery vehicle for siRNA targeting human MDR1 gene. The complex of cationic liposomes and siRNA, RGD-Lipo-siRNA, was prepared with a narrow size distribution below 200 nm. It was shown that the encapsulated siRNA in the liposomes could be effectively protected from serum degradation. Also, enhanced cell binding and intracellular uptake of siRNA in the doxorubicin-resistant human ova- rian cancer cell lines SKOV3/A were found in RGD-Lipo-siRNA preparation as compared to that of unmodified cationic lipsomes （Lipo-siRNA）. Using the post-insertion method for RGD modification, lysosome release of siRNA in pRGD-Lipo-siRNA was improved. From flow cytometry, significant increase of doxorubicin accumulation was observed in the SKOV3/A cells treated with pRGD-Lipo-siRNA targeting human MDR1 gene. In vitro cytotoxicity assay showed that the significant cell growth inhibition was achieved in the SKOV3/A cells after treating with the combined use of siRNA and doxorubicin. In conclusions, postinserted RGD modified lipoplex, pRGD-Lipo-siRNA, was successfully used for siRNA transfection and achieved drug resistance reversal in human ovarian cancer SKOV3/A （doxorubicin-resistant） cells. It suggested that this liposomes might be a potential vehicle for siRNA delivery in vivo.

关键词： siRNA Cationic liposomes dc-chol RGD modification

Effects of pegylated cationic liposomes on siRNA transfection

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Journal of Chinese Pharmaceutical Sciences 2009年第3期18卷 267-272页

作者：杨丽娟杨婷姜娟徐振中王坚成张强北京大学药学院药剂学系北京100191

This study aimed to investigate the effects of cationic liposomes containing different cationic lipids （dc-chol and DOTAP） and different pegylation ratios on siRNA transfection in human U251 glioma cells. The data showed that the transfection efficiency of DOTAP was much higher than that of dc-chol and PEG at 2 mol% enhanced cellular uptake of siRNA. Cationic liposome-siRNA complexes with particle size around 100 nm were prepared. PEG modification could efficiently stabilize the liposome in the presence of serum, which might protect the siRNA from serum degradation and prolong the circulation time in vivo. Efficient intracellular uptake and lysosome release of siRNA in human U251 glioma cells were observed for pegylated DOTAP-based lipososomes compared with the control transfection reagent lipofectamine 2000. The results demonstrated that this cationic liposome might be a potential vehicle for the in vivo delivery of siRNA.

关键词： siRNA Cationic liposomes dc-chol DOTAP PEG