摘要
通过干扰血管生成素(ANG)mRNA的表达,揭示ANG在糖尿病视网膜病变过程中的作用,本研究根据NCBI中查询的ANG序列设计阴性对照序列(NC)和3条ANG-shRNA序列(ANG-shRNA-1,ANG-shRNA-2,ANG-shRNA-3)构建至慢病毒载体GV248中,并进行病毒包装,转染至293T细胞进行滴度的测定。将包装好的慢病毒感染大鼠肝癌细胞(RH-35),测定感染效率,提取RNA,通过实时荧光定量PCR(qPCR)技术筛选出干扰效率最高的ANG-shRNA序列。结果显示,通过qPCR对3条序列慢病毒干扰效率的筛选发现,NC组ANG的表达高于空白对照组(p<0.01),ANG-shRNA-1、ANG-shRNA-2和ANG-shRNA-3组分别与NC组相比ANG表达均下降至99%、95%和94%。最终结果表明,在mRNA水平上3条ANG-shRNA序列干扰效率均高于90%,其中ANG-shRNA-1序列的干扰效率最高达到99%,因此3条干扰序列均可用到活体实验中。
To reveal the role of ANG in diabetic retinopathy by interfering with the expression of angiopoietin(ANG) mRNA, we designed the negative control sequence(NC) according to the ANG sequence of NCBI, 3 sequences of ANG-shRNA(ANG-shRNA-1, ANG-shRNA-2, ANG-shRNA-3) were constructed into lentiviral vector GV248. These 4 recombinant vectors were packed into lentivirus and transfected into 293 T cells for evaluating titers, then transfected into RH-35 cells for measuring interferential efficiency. The total RNA was isolated for detecting the interferential efficiency by Real-time PCR(RT-qPCR). Result shows that the expression of ANG in NC group was significantly higher than in normal group, whereas significantly decreased in ANG-shRNA-1,ANG-shRNA-2 and ANG-shRNA-3(p〈0.01) about 99%, 95% and 94%, respectively. Final results indicated that the interferential efficiency of three recombinant ANG-shRNAs were higher than 90% at mRNA level, of which ANG-shRNA-1 was the highest to 99%. Therefore, three interference sequences can be used in vivo experiments.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2017年第10期3943-3947,共5页
Genomics and Applied Biology
基金
河北省科技厅科技支撑计划项目(14277727D)资助