摘要
为了探讨miRNA-93-5p对梅花鹿血管内皮生长因子(VEGF)的转录调控作用及其与鹿茸细胞生长的关系,分离了鹿茸顶端软骨组织细胞,利用Trizol试剂法提取细胞总RNA,反转录合成c DNA。根据Gen Bank已发表的相关序列设计梅花鹿VEGF基因的3'端非编码区部分序列(3'UTR)特异引物并进行克隆,构建VEGF基因的3'UTR野生型及其突变体序列双荧光素酶报告基因载体并进行荧光素酶活性检测。再将人工合成的miRNA-93-5p模拟物转染鹿茸软骨细胞,MTT法检测鹿茸细胞体外增殖的变化;Western blotting分析VEGF蛋白的表达丰度。结果表明:成功获得了鹿茸组织VEGF基因的3'UTR序列,野生型序列长度为356 bp,突变体长度为336bp。荧光素酶活性检测结果表明,转染野生型质粒组细胞荧光素酶活性降低,而转染突变体组细胞荧光素酶活性无明显变化。MTT法和Western blotting结果显示,鹿茸细胞的体外增殖受到抑制,VEGF蛋白的表达水平下降,且呈时间依赖性。
We isolated the cartilage cells from antler tip and extracted cells total RNA by Trizol reagent to explore the effect tran-scriptional regulation of miRNA-93-5p on vascular endothelial growth factor （ VEGF） and the relationship with the growth of antler cells, and synthesized cDNA by reverse transcription.With the published relative sequence in GenBank, we de-signed and cloned the specially primers of 3’ UTR of sika deer VEGF gene, constructed dual luciferase reporter gene vec-tors of VEGF gene 3’ UTR containing wild-type or mutant sequence, and detected the relative activity of luciferase.We de-tected the changes of the in vitro proliferation of cartilage cells by MTT after transfected cartilage cells using miRNA-93-5p mimics, and analyzed the expression abundance of VEGF protein by western blotting.We obtained 3’ UTR sequence of VEGF from antler tissue.The length of wild-type sequence is 356 bp and the length of mutant sequence is 336 bp.Lucifer-ase activity detection indicates that the luciferase activity of the wild-type plasmid transfected cells is reduced, whereas mu-tant cells have no obvious change of luciferase activity .By MTT assay and western blotting, antler cells are inhibited in vitro and the expression level of VEGF protein decrease along with the time increase.
出处
《东北林业大学学报》
CAS
CSCD
北大核心
2014年第11期146-149,共4页
Journal of Northeast Forestry University
基金
国家自然科学基金(30972083)
吉林省科技发展计划项目(20090574)
