论文部分内容阅读
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 effect of cationic liposomes as a gene delivery vector and to explore its mechanism. Methods: DOPE and 1,2-dioleoyl-3-trimethylaminotane (DOTAP) were made into cationic liposome multilamellar vesicles by ultrasonication or extrusion MLV) or small unilamellar vesicles (SLV). MLV or SLV was directly mixed with DNA (pS2.S / pFP, 1: 1) to form a cationic liposome / DNA complex MLV-DNA or SLV-DNA with a positive / negative charge ratio of 3: 1 or 1: Twenty-five BALB / c mice were divided into five groups: A, B, C, D and E. Mice were immunized with a single intramuscular injection of four cationic liposomes / DNA complexes (Groups A, B, D and E) and 10 μg of naked DNA (Group C) respectively. After reaction of naked DNA, MLV-DNA (3:1) and SLV-DNA (3:1) with DNase I respectively for 30 min, the DNA was separated and identified by 1% agarose gel electrophoresis at 110 V for 2 h. Results: In the second week after immunization, the seroconversion rates in both groups A and B were 40%. In the fourth week after immunization, the seroconversion rates in both groups A and B reached 100% after. At 4 weeks after immunization, the average serum anti-HBs titer in group A was 27-fold (2.3706, 0.9370, P <0.02) higher than the control group C serum anti-HBs titer. After 8 weeks of immunization, serum anti-HBs levels of mice in groups A and B still showed an upward trend, with mean antibody titers of 2.5687 and 1.9533, respectively. D group and E group mice serum did not turn the sun. After incubation with DNase for 30 min, the naked DNA (group C) was completely degraded whereas the DNA in MLV-DNA (3: 1) and SLV-DNA (3: 1) remained essentially intact. CONCLUSION: Cationic liposomes are an efficient non-viral gene delivery mediator. Antibodies remain high after immunization and may be positive