腺相关病毒介导的狨猴P53基因沉默

目的在细胞和整体动物水平,利用RNA干扰技术下调绒猴p53基因表达。方法对狨猴p53基因做生物信息学分析,针对靶序列设计shRNA干扰序列,构建在腺相关病毒载体上,转染非洲绿猴肾细胞(cos-7),在细胞水平用荧光定量PCR检测p53mRNA抑制效果,以Western blot方法检测p53蛋白水平表达变化;优选shRNA干扰序列,包装含shRNA干扰序列的8型腺相关病毒,静脉注射感染狨猴;手术取少量肝脏组织,用Western blot和免疫组化方法检测p53蛋白水平的变化。结果细胞水平研究发现2个有效RNA干扰靶点,mRNA干扰效率分别为(82.7±8.1)%和(80.7±7.5)%(P0.05);蛋白表达下调(77.3±11.5)%和(73.7±10.7)%(P0.05);2只绒猴感染病毒后,经活体荧光成像分析可见病毒在肝脏、睾丸、颈部等位置分布,狨猴肝脏P53蛋白经Western blot、免疫组化分析未见明显变化。结论本研究在细胞水平实现绒猴P53基因表达下调,但整体动物水平狨猴肝脏P53蛋白表达未发现明显变化;今后需在感染方式等方面做进一步优化。

Adeno-associated virus mediated p53 gene silence in marmosets

Objective To decrease the p53 gene expression at cellular and animal levels in marmoset using RNA interference technique. Methods The shRNA interference sequences were designed and inserted into the adeno-associated virus vector plasmid after bioinformatics analysis. The plasmids were transfected into African green monkey kidney cos-7 cells. The suppression of p53 mRNA was detected by real-time PCR, and the changes of p53 protein expression were detected by Western bolt. The adeno-associated virus-8 was injected through the hind leg vein. The changes of p53 protein expression in the liver tissue was detected by Western blot and immunohistochemistry. Results We screened two RNA interference effective arget sequences. The expression of p53 mRNA was suppressed (82.7±8.1)% and (80.7±7.5)%, respectively (P<0.05), and the expression of p53 protein was decreased (77.3±11.5)% and (73.7±10.7)%, respectively (P<0.05). The two marmosets after virus infection showed that there were virus distributions in the liver, testes, and neck detected by in vivo fluorescence imaging. The expression of p53 in the marmoset liver was detected by western blot, immunohistochemistry analysis showing no obvious changes. Conclusions In the present study, the decrease of P53 gene expression at cellular level is achieved, however, the liver P53 protein in the marmoset liver is not significantly changes. Further optimization of the way of infection is needed in the future.