HBV DNA疫苗阳离子脂质体复合物转染小鼠实验研究
DNA疫苗,,阳离子脂质体,；DNA疫苗；,基因输送,HBVDNA疫苗阳离子脂质体复合物转染小鼠实验研究,1材料与方法,2结果,3讨论,参考文献
摘 要 目的：评价阳离子脂质体作为基因输送载体的效果及初步探讨其作用机理。方法：将二油酰基磷脂酰乙醇胺(DOPE)与1,2-二油酰基-3-三甲基氨基内烷(DOTAP)用超声法或挤压法制成阳离子脂质体多片层囊泡(MLV)或小单层囊泡(SLV)。MLV或SLV与DNA(pS2.S/pFP,1∶1)直接混合形成正负电荷比为3∶1或1∶3的阳离子脂质体/DNA复合物MLV-DNA或SLV-DNA。25只BALB/c平均小鼠分成五组：A，B，C，D，E。分别用4种阳离子脂质体/DNA复合物(A，B，D，E组)和10 μg裸DNA(C组)一次性肌肉注射免疫接种小鼠。裸DNA、MLV-DNA(3∶1)和SLV-DNA(3∶1)分别与DNase I反应30 min后，1%琼脂糖凝胶110V电泳2 h分离鉴定DNA。结果：免疫接种后第2周，A组和B组血清转阳率均为40%；免疫接种后第4周，A组和B组血清转阳率均达到100%，并保持至第8周以后。免疫接种后第4周，A组血清平均抗-HBs滴度比对照的C组血清平均抗-HBs滴度高27倍 (2.3706，0.9370，P<0.02)。免疫接种8周后，A组和B组小鼠血清抗-HBs水平仍呈上升趋势，平均抗体滴度分别是25687，1.9533。D组与E组小鼠血清未见转阳。与DNase温育30 min后，裸DNA (C组) 被完全降解，而MLV-DNA(3∶1)和SLV-DNA(3∶1)中DNA基本保持完整。结论： 阳离子脂质体是一种高效的非病毒基因输送介质；免疫接种后抗体持续高水平，可能是阳离子脂质体/DNA复合物可保护DNA免受体液中脱氧核糖核酸酶的破坏，缓慢释放DNA有关。关键词 阳离子脂质体 ；DNA疫苗； 基因输送
Experimental study of transfection by HBV DNA vaccine cationic liposome complex in mice
Abstract Objective: To evaluate the efficiency of cationic liposome as gene transfer vehicle and its delivery mechanism. Method: Cationic liposome MLV or SLV were made from DOPE/DODAP by sonication or extrusion respectively.Then the MLV or SLV were mixed with plasmid DNA (pS2·S/pFP,1∶1) to form MLV-DNA or SLV-DNA complexes at charge ratio 3∶1 (+/-). 25 BALB/c mice were equally assigned to five groups, namely Group A, B, C,D and E. 10 μg naked DNA (pS2·S/pFP, 1∶1 ) for Group C and 10 μg DNA in MLV-DNA. SLV-DNA complexes,were intramuscular administrated respectively, Naked DNA, MLV-DNA (3∶1) and SLV-DNA (3∶1) were incubated with DNase I for 30 minutes, 1% agarose gel and electric field of 110V were used to examine the integrity of DNA.Results: Seroconversion ratio was 40% at the end of 2nd week for both Groups A and B, then rised to 100% at the end of 4th week for both groups till the end of 8th week. Mean anti-HBs titers of mice for group A was 27 folds higher than that of group C at the end of 4th week ( 2.3706, 0.9370, P<0.02 ). Mean anti-HBs of Group A and Group B rised to 2.5687, 1.9533 respectively at the end of 8th week. There was no seroconvertion in Group D and Group E. After incubation with DNase I for 30 minutes, naked DNA was completely degraded while DNA in complexes MLV-DNA (3∶1) and SLV-DNA (3∶1) was almost intact. Conclusion: Cationic liposome is a non-viral gene delivery system with high efficiency. Mean titers sustained high level in BALB/c mice immunized with cationic liposome/DNA complexes. Cationic liposome/DNA complexes may protect DNA from deoxyribonuclease attack and acted as a sustained release system forDNA. ......
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