重组1和2型腺相关病毒转导增强绿色荧光蛋白在大鼠脑中的表达

目的　探讨重组1、2型腺相关病毒(rAAV1和rAAV2)载体经不同途径向大鼠脑转入增强绿色荧光蛋白(EGFP)报告基因的表达情况,选择更加适于中枢神经系统(CNS)转基因治疗的rAAV血清型和可行的基因转入途径。方法　将24只雄性健康成年SD大鼠随机分为4组:rAAV1海马注射组、rAAV1脑室注射组、rAAV2海马注射组和rAAV2脑室注射组,每组6只大鼠。对每组大鼠立体定向注射剂量和滴度相匹配的rAAV1 EGFP和rAAV2- EGFP载体,分别于注射后2周和4周处死大鼠取脑,行序列冰冻切片并以荧光显微镜观察EGFP在脑内的表达情况,计算EGFP在脑的表达体积。结果　2周时各组小鼠EGFP表达量少或无表达,差异无统计学意义; 4周时rAAV1海马注射组表达体积为(7 .00±0. 98)mm3,rAAV2海马注射表达体积为(0. 81±0 .28)mm3 (P<0 .01); 4周时AAV1脑室注射组表达体积为(12 .72±0 .28)mm3, AAV2脑室注射组表达体积为(0 .24±0. 13)mm3(P<0 .001)。结论　在CNS中rAAV1是一种比rAAV2更为优越的转基因载体,rAAV1可通过脑室内注射途径在CNS高表达,并可直接转导胶质细胞。

Expression of enhanced green fluorescent protein in rat brain transduced by recombinant adeno-associated viruses type 1 and type 2

OBJECTIVE: To explore the expression of enhanced green fluorescent protein (EGFP) transduced into the brain via recombinant adeno-associated virus (rAAV) type 1 and rAAV type 2 vectors so as to select the better rAAV serotype and feasible gene transfer route to central nervous system (CNS). METHODS: Twenty-four SD male adult rats were randomly divided into 4 equal groups: rAAV1 intra-hippocampus injection group, rAAV1 intra-ventricular injection group, rAAV2 intra-hippocampus injection group, and rAAV2 intra-ventricular injection group to be injected stereotactically with titer and volume matched rAAV1-EGFP and rAAV2-EGFP vectors respectively. The rats were sacrificed respectively 2 and 4 weeks after injection and their brains were removed to be made into serial frozen coronal sections. Fluorescence microscopy was used to observe the expression of EGFP in the brain and to calculate the expression volume of EGFP in different parts of the brain. RESULTS: Two weeks after injection EGFP was expressed in a small amount or not expressed in all groups. Four weeks after injection the EGFP expression volume were (7.00 +/- 0.98) mm(3) and (0.81 +/- 0.28) mm(3) in the rAAV1 and rAAV2 intra-hippocampus injection groups respectively (P < 0.01), and were (12.72 +/- 0.28) mm(3) and (0.24 +/- 0.13) mm(3) in the rAAV1 and rAAV2 intra-ventricular injection groups respectively (P < 0.001). CONCLUSION: As gene-transducing vector in CNS rAAV1 is superior to rAAV2. High expression can be achieved by intra-ventricular injection with rAAV1 vectors.