重组腺病毒载体转染Apo^(E-/-)小鼠的安全性评价和在主动脉血管组织的表达情况  

Evaluation of Transfection Efficiency and Safety of Recombinant Adenovirus Vector in ApoE^(-/-) Mice

作　　者：王雪梅[1] 魏琴[1] 段明军[1] 张春[1] 姜涛[1] 杨毅宁[1] 

机构地区：[1]新疆医学动物模型研究重点实验室新疆医科大学第一附属医院临床医学研究院,乌鲁木齐830011

出　　处：《生物医学工程研究》2016年第2期98-103,108,共7页Journal Of Biomedical Engineering Research

基　　金：新疆维吾尔自治区自然科学基金;青年基金资助项目(2015211C095)

摘　　要：腺病毒介导的脂联素过表达对Apo E-/-小鼠动物的安全性检测。将48只普通饮食饲养的12周龄雄性Apo E-/-小鼠分为对照组和脂联素干预组,每组24只。对照组给予一次性尾静脉注射生理盐水100μl;干预组则同法注射100μl重组腺病毒(Ad-APN-e GFP,3.0×108p.f.u)。分别在注射后0天、1周、2周、及4周处死小鼠,取全血和主动脉血管组织。用ELISA法测定血清中脂联素浓度;用WB法检测血管组织中标识绿色荧光蛋白(GFP)以确定重组腺病毒的转染率。全自动生化仪检测血常规指标、肝功能(ALT、AST)、肾功能指标(CR、BUN)、心肌酶(CK、CKMB)、以确定重组腺病毒转染的安全性。腺病毒对血常规指标和心肌酶的表达没有显著影响,对肝功能指标谷丙转氨酶(ALT)(1周时组间差异P<0.01和2周时组间差异P<0.05)、肾功能指标尿素氮BUN(1周时组间差异P<0.01)有一过性影响。重组腺病毒可在体内高效介导外源基因脂联素的表达,血清中的脂联素浓度在病毒转染后1周达到了最高值,约是正常对照组的4倍(78.28±15.02 vs 23.42±5.35)μg/ml,2周内稳定表达。1周时GFP蛋白在血管组织中表达的量最高,1周时GFP/GAPDH为83.63±1.50%,随后逐渐降低,2周时GFP/GAPDH为54.00±4.00%,4周时GFP/GAPDH为22.00±3.99%。重组腺病毒注射存在一过性的肝毒性和肾毒性,但是对动物的生理状态没有显著影响。重组腺病毒对Apo E-/-小鼠的血管组织有高效转染、2周内安全、稳定表达的特性。To assess the transfection efficiency and the safety of the adenovirus in 48 male Apo E- /-mice aged 12- week. They were randomly assigned into two groups（ 24 mice per group）,and were injected tail- intravenously with either 100 μl of saline（ control group） or Ad- APN- e GFP virus（ 3. 0 × 108 p. f. u,APN group）. At 0 d,1,2,and 4 weeks of post- injection,blood samples and aortic vascular tissues were executed from the 2 groups of mice. Enzyme- linked immunosorbent assay（ ELISA） was used to detect serum APN concentration; Western blot（ WB） was conducted to assess the transfection efficiency of Ad- APN- e GFP virus,as indicated by the expression of e GFP in vascular tissues; Full automatic biochemical analyzer（ FABA） was used to analyze blood conventional indexes,cardiac enzymes,liver and kidney function indexes. Adenovirus had no significant effect on blood routine indexes andmyocardial enzyme expression,had significant effect on the liver function of ALT（ P 〈 0. 01）,renal function indexes BUN（ P 〈0. 01）. r Ad- APN- e GFP vector in vivo efficiently mediated exogenous APN gene expression,serum APN concentration in serum reached the highest value was 78. 28 ± 15. 02 vs 23. 42 ± 5. 35（ μg / ml） after 1 week transfection,2 weeks stable expression. At 1week,the expression of GFP in vascular tissue was the highest,GFP / GAPDH was 83. 63 ± 1. 50%,then decreased gradually,and GFP / GAPDH was 54. 00 ± 4. 00% in 2 weeks,GFP / GAPDH was 22. 00 ± 3. 99% in 4 weeks. Adenovirus vector has the characteristics of high efficiency transfection and stable expression and safety in 2 weeks. It is an ideal gene therapy vector for vascular disease.Adenovirus injection has a transient liver toxicity and renal toxicity,but has no significant effect on the physiological state of the animal. r Ad- APN- e GFP is a high efficiency,stable expression and safety vector in vascular in 2 weeks.

关 键 词：重组腺病毒 脂联素 APOE-/-小鼠 基因治疗 转染效率和安全性 

分 类 号：R341[医药卫生—基础医学] R318