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10μg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2.5687,1.9533。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?
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. 10μg 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. Calionic liposome/DNA complexes may protect DNA from deoxyribonuclease attack and acted as a sustained release system forDNA.
出处
《广东药学院学报》
CAS
2002年第4期294-297,共4页
Academic Journal of Guangdong College of Pharmacy
