四环素调控复制缺陷性疱疹病毒载体介导LacZ基因在原代培养神经元中的表达

Tetracycline-controlled LacZ gene expression in primary cultured neurons mediated by a tetracycline regulated replication-defective HSV-1 vector system

目的利用四环素调控复制缺陷性单纯疱疹病毒I型重组载体(QR9TO-LacZ)感染体外原代培养的大鼠皮层神经元,探讨其介导半乳糖苷酶(LacZ)基因的表达情况及基因调控效能;并检测该载体对神经元活力的影响。方法用RUL9-8细胞系扩增QR9TO-LacZ病毒株,收集后采用噬斑法测定病毒滴度;体外原代培养大鼠皮层神经元。将培养的原代神经元分为强力霉素(DOX)组和对照组。两组细胞均用QR9TO-LacZ转染,强力霉素组加入四环素衍生物强力霉素(0.5μg/ml),对照组加入等剂量空白对照液。同时根据感染复数(MOI),即MOI=3、5、10、30 PFU/cell,将两组培养细胞进一步分为亚组。病毒转染48h后行X-gal染色;光镜下观察神经元形态,随机选取10个高倍镜视野(×400),计算每高倍镜视野中平均蓝染细胞率,比较不同MOI病毒感染神经元的效率。采用四甲基偶氮唑盐微量酶反应比色法(MTT法)检测感染病毒48 h(MOI同上)后神经元的活性。结果在含有DOX的培养环境中,不同MOI亚组(3、5、10、30 PFU/cell)QR9TO-LacZ感染神经元出现蓝染细胞百分比分别为5.35%±0.84%、10.64%±1.92%、19.73%±2.87%、34.41%±2.58%;在无DOX的培养环境中,各亚组均未观察到蓝染细胞。上述MOI感染的神经元细胞存活率分别为:90.24%±8.16%、88.00%±5.69%、83.95%±3.82%、78.90%±5.49%。结论 QR9TO-LacZ载体能有效地将目的基因导入原代培养神经元中并表达;目的基因表达的开启受强力霉素调控;QR9TO-LacZ对体外原代培养神经元毒性较低。

Objective To observe the regulative effectiveness of transferred gene expression in primary cultured neurons in vitro and the gene expression in neurons infected with a tetracycline regulated, replication - defective herpes simplex virus type I （ HSV - 1 ） recombinant vector （QPOTO -LacZ） with LacZ gene as reporter. Methods Amplified QR9TO -LacZ virus stock and determined the titer with standard plaque as- say; infected primary cultured neurons with different multiplicity of infection （multiplicity of infection, MOI = 3, 5, 10, 30 PFU/cell）, in the presence （0.5 μg/ml） or absence of doxycycline （ doxyeycline, DOX, tetracycline derivative） ; stained the neurons with X - gal 48h post infec- tion （48hpi） ; examined the neuronal morphology and X - gal staining positive rate with optical microscope in 10 random high - power view （ x 400）. Cell viability was tested with MTY assay 48 hpi. Results X - gal staining showed that the positive rates of blue stained cells were 5.35 % ± 0.84%, 10.64% ± 19.73%, and 34.41% at MOI values of 3, 5, 10 and 30 PFU/cell subgroups, respectively in the presence of DOX; but no blue stained cells was observed in the groups without DOX. Survival rates of infected neurons were 90.24 ± 8.16%, 88.00 ± 5.69%, 83.95 ± 3.82%, and 78.90 ± 5.49% at MOI values of 3,5, 10, and 30 PFU/cell, respectively. Conclusion LacZ gene may be transferred to primary cultured neurons with QR9TO - LacZ viral vector without significant toxicity and the gene expression can be controlled with DOX.